Spliceosome assembly and regulation: insights from analysis of highly reduced spliceosomes

  1. Stephen D. Rader1
  1. 1Department of Chemistry and Biochemistry, University of Northern British Columbia, Prince George, British Columbia, Canada V2N 4Z9
  2. 2Department of Anatomy and Cell Biology, McGill University, Montréal, Quebec, Canada H3A 0C7
  3. 3Biodiversity Research Center and Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4
  4. 4Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
  1. Corresponding authors: rader{at}unbc.ca, amacmill{at}ualberta.ca, nfast{at}mail.ubc.ca
 
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Abstract

Premessenger RNA splicing is catalyzed by the spliceosome, a multimegadalton RNA–protein complex that assembles in a highly regulated process on each intronic substrate. Most studies of splicing and spliceosomes have been carried out in human or S. cerevisiae model systems. There exists, however, a large diversity of spliceosomes, particularly in organisms with reduced genomes, that suggests a means of analyzing the essential elements of spliceosome assembly and regulation. In this review, we characterize changes in spliceosome composition across phyla, describing those that are most frequently observed and highlighting an analysis of the reduced spliceosome of the red alga Cyanidioschyzon merolae. We used homology modeling to predict what effect splicing protein loss would have on the spliceosome, based on currently available cryo-EM structures. We observe strongly correlated loss of proteins that function in the same process, for example, in interacting with the U1 snRNP (which is absent in C. merolae), regulation of Brr2, or coupling transcription and splicing. Based on our observations, we predict splicing in C. merolae to be inefficient, inaccurate, and post-transcriptional, consistent with the apparent trend toward its elimination in this lineage. This work highlights the striking flexibility of the splicing pathway and the spliceosome when viewed in the context of eukaryotic diversity.

Keywords

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INTRODUCTION

Pre-mRNA splicing

Pre-mRNA splicing is an essential step of gene expression in eukaryotes. It consists of the removal of introns and ligation of protein-coding exons via two transesterification reactions. Splicing proceeds via a complex process involving assembly of 80–200 proteins and RNA splicing factors that recognize conserved sequences in each pre-mRNA, principally the 5′ splice site (5′SS), 3′ splice site (3′SS), and branchpoint sequence (BPS). The complex formed by these factors is known as the spliceosome, and assembles anew for each round of splicing (Cheng and Abelson 1987; Hoskins et al. 2011; Cordin and Beggs 2013).

Spliceosome assembly on pre-mRNA substrates in both HeLa and yeast cell extracts has been viewed as proceeding in a stepwise fashion through discrete complexes. Historically, the progression from the prespliceosomal complex A through the spliceosomal B and C complexes was characterized by native gel electrophoresis as well as sedimentation analysis (Konarska and Sharp 1986, 1987; Cheng and Abelson 1987). This assembly pathway included the early or commitment complex (E/CC) as a precursor to the A complex (Seraphin and Rosbash 1989; Das and Reed 1999). Subsequent biochemical, genetic, and mass spectrometric analyses have elaborated this description (for review, see Will and Lührmann 2011), which has now been detailed in multiple high-resolution structural analyses by Cryo-EM as described below.

In Saccharomyces cerevisiae (yeast), the 5′SS is initially recognized by the U1 small nuclear RNA (snRNA) and its associated proteins, which are assembled into a small nuclear ribonucleoprotein particle (snRNP; Cheng and Abelson 1987). Meanwhile, proteins BBP/Msl5 and Mud2 interact with the BPS and polypyrimidine tract (Berglund et al. 1997; Chen and Cheng 2012). At this stage, the assembling particle is referred to as the E complex. BBP/Msl5 is subsequently replaced at the BPS by a stable interaction with the U2 snRNP to form the A complex or prespliceosome (Yan and Ares 1996). The remaining snRNPs, U4, U5, and U6, preassociate in the tri-snRNP and bind to the A complex to yield the precatalytic B complex (Cheng and Abelson 1987). The A complex undergoes a series of rearrangements catalyzed by ATPases Prp28 and Brr2 (Raghunathan and Guthrie 1998a,b) resulting in replacement of U1 snRNA by U6 snRNA at the 5′SS and destabilization and release of the U4 snRNP to yield the activated B complex (Bact; Strauss and Guthrie 1991; Staley and Guthrie 1998). The ATPase Prp2 converts Bact to B*, which carries out the first transesterification reaction to generate the C complex (Liu and Cheng 2012; Ohrt et al. 2012). The ATPase Prp16 displaces the first-step factors Cwc25 and Yju2 (Schwer 2008; Chiang and Cheng 2013; Tseng et al. 2017), allowing the second-step factors Slu7 (Frank and Guthrie 1992) and Prp18 (Brys and Schwer 1996; James et al. 2002) to bind and form the C* complex, which carries out the second catalytic step resulting in the post-catalytic P complex (Chen et al. 2013). Finally, the spliceosome is disassembled through the action of Prp22, a DEAH-box helicase that releases the mRNA (Schwer and Gross 1998; Schwer 2008), and the Ninteteen-Related (NTR) Complex, which disassembles the spliceosome (Chen et al. 2013). The assembly and disassembly steps in metazoan splicing are substantially similar (Will and Lührmann 2011).

For several decades following the discovery of splicing in 1977, central goals of the splicing field were to define the composition of the spliceosome, obtain a general sense of its three-dimensional organization (Chow et al. 1977), and elucidate its mechanism of action (Berget et al. 1977). With the publication of high-resolution cryo-EM structures of the spliceosome in 2015 (Nguyen et al. 2015; Yan et al. 2015), the field has seen a dramatic shift yielding essentially complete views of nearly every step in the splicing cycle including the location of many splicing factors (Agafonov et al. 2016; Abelson 2017; Bai et al. 2017, 2021; Bertram et al. 2017a,b; Zhan et al. 2018a,b; Fica et al. 2019; Zhang et al. 2019; Charenton et al. 2020; Townsend et al. 2020; Wilkinson et al. 2021; Tholen et al. 2022). The focus of splicing research has consequently shifted more toward the mechanism and evolution of splicing.

Our understanding of pre-mRNA splicing comes largely from studies on two organisms: S. cerevisiae and humans. Splicing evolved early in the evolutionary history of eukaryotes (Jurica and Moore 2003; Koonin 2006; Roy and Gilbert 2006) and as a result the general splicing machinery consisting of five snRNAs and their associated proteins appears similar across eukaryotes. However, examination of lesser-studied eukaryotes reveals unexpected plasticity in the splicing process and spliceosome composition. An excellent example of this plasticity is the phenomenon of trans splicing in which a Splice Leader sequence is attached onto all mRNA transcripts via the spliceosome (Günzl 2010). Trans splicing was discovered in trypanosomes (Boothroyd and Cross 1982), and has since been observed in a number of kinetoplastids, nematodes, and other lineages including chordates (Günzl 2010). Splicing in T. brucei mainly occurs through the trans pathway although two canonical, cis-spliced introns have been retained (Mair et al. 2000), demonstrating that the T. brucei splicing machinery is completely capable of cis splicing despite the loss of all other cis-spliced introns.

One mystery of trypanosome splicing is the relatively high expression level of the U1 snRNP. Although in nematodes the U1 snRNP is only required for cis splicing, in trypanosomes it is highly abundant and apparently associated with the spliceosome, even though there are only two introns on which it would function (Günzl 2010). U1's abundance suggests that trypanosomal U1 may have other roles outside of splicing (West 2012), such as human U1 snRNP's role in transcription regulation (Spiluttini et al. 2010; O'Reilly et al. 2013; Vazquez-Arango et al. 2016), or the role of the U2 snRNP in histone mRNA maturation (Friend et al. 2007; So et al. 2019; Zhang et al. 2021).

A variety of other insights into splicing evolution have come from studying organisms beyond humans and S. cerevisiae. Phylogenetic analysis of splicing proteins revealed that early spliceosomes were similarly complex to current plant and animal spliceosomes (Roy and Irimia 2014), and that many spliceosomal proteins are derived from ribosomal proteins and evolved after intron proliferation (Vosseberg et al. 2023). Phylogenetic analysis of introns showed that there had already been substantial intron loss by the time of the Last Eukaryotic Common Ancestor (LECA) (Vosseberg et al. 2022). This points to early proliferation of introns, and hints at nucleogenesis substantially predating LECA (Vosseberg et al. 2022). Comparison of U1-70k proteins in plants showed a proliferation of protein architectures that may be important for diversity of stress responses, and also revealed possible nonsplicing roles of this protein family, for example, in osmotic stress response (Chen et al. 2020). Analysis in five Entamoeba species demonstrated coevolution of introns and spliceosomes, and revealed intron proliferation in an intron-poor lineage (Roy and Bowser 2022). Deletion of splicing factors in the intron-rich yeast Cryptococcus suggested that many splicing proteins conserved in humans have a role in the fidelity of splice site detection (Sales-Lee et al. 2021). Finally, studies in diplomonads and parabasalids provide examples of divergent snRNAs (Hudson et al. 2019). These examples highlight the diversity of splicing systems outside the most commonly studied model organisms.

One potential driver of spliceosomal diversity is genome reduction. Reductions in genome size are accompanied by the loss of noncoding regions, including spliceosomal introns. As has been observed in multiple, distantly related lineages including trypanosomes (Günzl 2010) and giardia (Hudson et al. 2012), loss of introns is mirrored by a reduction in the RNA and protein components of the spliceosome (Jeffares et al. 2006; Grisdale et al. 2013; Hudson et al. 2015). These events—genome reduction and spliceosome simplification—provide us with both mechanistic and evolutionary insights into the spliceosome. In this review, we first examine diverse eukaryotic lineages with a focus on taxa with reduced genomes to identify common patterns of spliceosomal component loss, before turning to more closely examine the red alga Cyanidioschyzon merolae (C. merolae or Cm), which has extreme levels of spliceosome reduction.

Comparing spliceosomes across diverse eukaryotes

Using methods similar to Wong et al. (2021), we searched the proteomes of 40 distantly related eukaryotic taxa (Supplemental Table 1) for 71 spliceosomal proteins found in yeast and humans to assess the completeness of the spliceosome across eukaryotes, and to determine if there are any patterns to spliceosomal reduction. We increased sampling of taxa possessing fewer introns, because we predict they have reduced spliceosomes. In line with the current understanding of genome reduction in eukaryotes, we observe multiple instances of independent spliceosomal reduction, the most extreme examples of which can be seen in the microsporidia (24/71 proteins found for E. cuniculi), Giardia (17/71), the kinetoplastids (38/71 on average), and C. merolae (26/71; Fig. 1). Other lineages with notably reduced spliceosomes include P. cylindrospora (fungi; 41/71) and E. histolytica (amoebozoa; 49/71). Spliceosome reduction has therefore occurred repeatedly across the tree of life, although we have seen no evidence of it in certain groups such as plants and green algae, animals, and stramenopiles.

FIGURE 1.
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FIGURE 1.

Comparison of spliceosomes across diverse eukaryotes. Eukaryotic relationships follow (Burki et al. 2020) with common taxonomic groupings indicated (left). Parasitic taxa (P) and those that possess trans-splicing (TS) are indicated by dots in columns. Annotated introns/gene shown for each taxon with darker boxes indicating higher intron density. Phytomonas and Perkinsela have no annotated introns. Spliceosome protein presence is indicated with a filled box. Faint colors indicate proteins curated as present. Predicted absence of a protein is indicated by an unfilled box. Cm Nam8 was bioinformatically annotated but could not be identified in mass spectrometry experiments (Reimer et al. 2017).

Considering the life histories of the groups with highly reduced spliceosomes, it is clear that genome reduction correlated with parasitism plays a factor in spliceosomal composition. Microsporidia, kinetoplastids and Giardia are all parasites that have very few introns and have lost substantial numbers of spliceosomal proteins (Fig. 1). However, this pattern is not observed in all parasites. For example, the intracellular apicomplexan parasite that causes malaria, Plasmodium falciparum, does not show the same levels of spliceosome reduction, despite a significantly reduced genome. While Phytomonas sp. and Perkinsela sp. have no annotated spliceosomal introns, the kinetoplastids retain an extremely reduced spliceosome involved in trans-splicing mRNA (Fig. 1).

Some splicing factors are more frequently lost than others, suggesting they are less essential for splicing (Fig. 1). For example, components of the U1 snRNP, the Bact complex, and the Prp19/Nineteen Complex (NTC) are frequently lost. Mud1 is missing in 27 of 40 lineages, and Prp39 is missing from 22. Similarly, the U5 snRNP protein Snu40 is missing in 26 lineages, Snu71 of the A complex is missing in 20, and Spp2 and Cwc25 of the Bact complex are missing in 22 and 21, respectively. In contrast, even in the reduced taxa, the U2 snRNP remains relatively complete, as does the U5 snRNP and the set of second step proteins (Fig. 1).

The four proteins present in all 40 lineages are by inference the most indispensable. These are Hsh155 and Prp9 from the U2 snRNP, and Dib1 and Prp8 from the U5 snRNP. Other highly retained proteins include Cus1, Hsh49, and Rds3 from the U2 snRNP; Brr2 and Snu114 of the U5 snRNP; Snu13 of the U4/U6 snRNP; Msl5 of the A complex; Prp38 and Sub2 of the B complex; Cwc22 of Bact; Cef1, Clf1, Prp46, and Syf1 from the Nineteen Complex; Prp17 of the second step complex; and Ist3 of the RES complex. All of these are present in at least 90% (36) of the lineages surveyed. The absence in some lineages of proteins thought to be essential for splicing, such as Brr2 and Snu114, raises questions about the quality of the sequencing and annotation in some of these cases, but the overall trends are clear.

While we have not surveyed snRNAs here, due to the challenges of unambiguously identifying them in divergent lineages, we are not aware of any organisms that lack the U2, U4, U5, or U6 snRNAs. In contrast, U1 snRNP has been shown to be dispensable in a number of situations (e.g., for trans splicing; Günzl 2010; Stark et al. 2015) and is known to be absent in Cm (Stark et al. 2015). When considering those taxa with reduced spliceosomes, Cm stands out as being the only taxon that is extensively reduced without having a parasitic life history and while still retaining canonical splicing.

An experimentally validated reduced spliceosome

C. merolae is a rhodophyte, a deep-branching unicellular red alga that is thought to have diverged from fungi and humans approximately 1.5 billion years ago (Yoon et al. 2004). Phylogenetic evidence suggests that the ancestral state of the Cm spliceosome and intron complement was much closer to that of fungi such as S. pombe, that is, the genome contained several thousand introns and a larger spliceosome that included the U1 snRNP (Hudson et al. 2015). We have studied pre-mRNA splicing in Cm (Stark et al. 2015; Black et al. 2016; Reimer et al. 2017; Garside et al. 2019), and previously reported that it possesses a unique algal spliceosome lacking the entire U1 snRNP and several other splicing and spliceosome assembly proteins (Table 1; Fig. 2; Stark et al. 2015; Reimer et al. 2017). The Cm spliceosome therefore reflects divergence from other canonical spliceosomes, primarily in the loss of many splicing factors. It is surely not a coincidence that in the Cm lineage all but 38 introns have been eliminated (Wong et al. 2022).

FIGURE 2.
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FIGURE 2.

Overview of the Cm splicing proteins present throughout one pre-mRNA splicing cycle. Proteins are colored according to incorporation into the spliceosome, whereas proteins absent in Cm are shown in transparent gray. The color scheme is as follows: Tri-snRNP (green, PDB 3JCR); Pre-B complex (orange, PDB 6AH0); B-complex (red, PDB 5O9Z); Bact complex (purple, PDB 5GM6); C* complex (blue, PDB 5MQF); P complex (no additional proteins, PDB 5YLZ).

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TABLE 1.

Proteins present in cryo-EM spliceosomal structures missing in C. merolae

Despite their limited numbers, Cm introns appear unremarkable. Intron lengths range from 70 to 1309 nts, branchpoint to 3′ splice site distances range from 10 to 63 nt, and the 5′ and 3′ splice sites have canonical consensus sequences (GUAAGU and AG, respectively; Matsuzaki et al. 2004; Ogasawara et al. 2004). The branchpoint sequence is slightly divergent, ACUAACC, with a compensatory change in the U6 snRNA in which the canonical ACAGAGA sequence is changed to ACUGAGA (Stark et al. 2015). In contrast to S. cerevisiae, there is no obvious commonality among the intronic host genes. While two are ribosomal protein genes, and two others are also related to translation, the remainder have a wide variety of functions including protein folding, primary metabolism, and transport. No intron property yet tested correlates with the level of splicing across intron-containing genes, which is strikingly low (Schärfen et al. 2022).

Based on the presently known inventory of splicing-related factors, we propose the assembly of the Cm spliceosome to be novel. For instance, while the branchpoint sequence (BPS) is predicted to be recognized by the branchpoint bridging protein CmMsl5 (BBP, SF1) and CmMud2 (U2 auxiliary factor U2AF65), 5′ splice site (5′SS) recognition remains a mystery due to the absence of the U1 snRNP. Notably, CmMsl5 is missing the SPSP and PPxY motifs, which have been shown to interact with Prp40 and other U1 snRNP proteins absent in Cm (Chang et al. 2012). The SPSP and PPxY motifs are within a coiled-coil domain and regulate the splicing of a subset of human pre-mRNA transcripts (Lipp et al. 2015). Like yeast, Cm is also missing U2AF35, which forms a dimer with U2AF65 in S. pombe (Huang et al. 2002) and humans (Zuo and Maniatis 1996). The absence of U2AF35 is interesting because it might have a cotranscriptional function and hints at splicing and transcription being uncoupled in Cm (Wu and Fu 2015). The absence of Lea1 and Msl1, discussed later, is further possible evidence of uncoupled splicing (Gunderson and Johnson 2009). The absence of the U1 snRNP would also preclude the existence of early spliceosomal states such as the E and A complexes, at least as they are known in other organisms. If spliceosome assembly in Cm proceeds analogously to assembly in yeast, recruitment of the U2 snRNP to the pre-mRNA followed by incorporation of the tri-snRNP would presumably result in the precatalytic B complex.

To complement our experimental data, and to compare the Cm spliceosome with that from yeast and humans, we reviewed structures of spliceosomal complexes from throughout the splicing cycle. We gathered all available sequences from known Cm splicing proteins, all of which have been validated by affinity purification followed by mass spectrometry (AP-MS; Reimer et al. 2017; MR Stark, SD Rader, M Oeffinger, et al., unpubl.), and performed pairwise sequence alignments against human and yeast orthologs. Interestingly, many splicing proteins that are highly conserved between yeast (Hossain and Johnson 2014), humans, and even Galdieria sulphuraria (Qiu et al. 2018), a red alga found to cohabit extreme environments with Cm, show substantial divergence in Cm (Matsuzaki et al. 2004; Stark et al. 2015). Table 2 lists those Cm proteins that lack domains conserved in their orthologs, while Table 3 lists Cm proteins with domains that are not found in their yeast or human homologs. Supplemental Figure 1 shows multiple sequence alignments of key proteins mentioned below. Using the pairwise scores generated from the Needleman–Wunsch algorithm, we annotated the respective proteins in structures from human and yeast spliceosomes (see Table 4 for PDB accession numbers of structures used; Madeira et al. 2019) to highlight presence, absence, and degree of conservation. These data about absent proteins allowed us to model each step of the splicing pathway using cryo-EM structures, and compare Cm to both human and yeast models. Based on the resulting models, we assessed the literature to predict what functions might be conserved, lost, or unique to Cm splicing proteins. These comparisons have allowed us to propose hypotheses about spliceosome structures and mechanisms under the assumption that there are no Cm-specific proteins or snRNAs that have taken on the roles of absent splicing factors. Extensive AP-MS analysis has not yet identified novel factors (Reimer et al. 2017; MR Stark, SD Rader, M Oeffinger, et al., unpubl.).

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TABLE 2.

Conserved domains in complex spliceosomes absent in C. merolaea

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TABLE 3.

Predicted novel domains in C. merolae splicing factorsa

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TABLE 4.

Representative spliceosomal structures

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ANALYSIS OF HOMOLOGY MODELS

5′ splice site recognition: a novel role for U5 snRNA?

It is possible that spliceosome assembly in Cm begins with the B complex (Zhan et al. 2018a,b; Fig. 3). There are currently no data to support the presence in Cm of any component of the U1 snRNP or proteins whose functions are related to U1 snRNP such as the DExD/H-box ATPase Prp28 (Stark et al. 2015). In other organisms, Prp28 displaces the U1 snRNP from the 5′SS (Mathew et al. 2008), which allows U6 snRNA in the tri-snRNP to base pair at the 5′SS (Lesser and Guthrie 1993; MacRae et al. 2018). It is unknown how the Cm tri-snRNP is recruited to the spliceosome prior to B complex formation. We previously proposed that 5′SS recognition occurs during tri-snRNP integration, whereby either U6 snRNA or U5 snRNA interacts with the 5′SS (Stark et al. 2015). Evidence for the Cm U6 snRNA:5′SS direct interaction is found in U6 snRNA's extended complementarity to 5′ splice sites: six of seven positions are able to form standard Watson–Crick base pairs (Fig. 4A), which is comparable to the canonical U1 snRNA:5′SS interaction (Crispino and Sharp 1995; Maroney et al. 2000) and longer than the canonical three-base-pair U6 snRNA interaction (Sawa and Abelson 1992; Wassarman and Steitz 1992). Interestingly, the 5′ end of Cm U5 snRNA (5′-GUCUGC) is complementary to all annotated Cm 5′ splice sites (Fig. 4B; Stark et al. 2015). Furthermore, U5 snRNA has previously been found to interact with the 5′SS during B complex formation in humans (Sontheimer and Steitz 1993; Boesler et al. 2015). We previously noted that the 450-nt Cm U5 snRNA is predicted to contain two more stem–loops than yeast or human U5 snRNA (Fig. 4B; Stark et al. 2015). If U5 snRNA is indeed responsible for initial 5′SS recognition, the additional domains of U5 snRNA may play a role in this step.

FIGURE 3.
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FIGURE 3.

The human pre-B complex (PDB 6AH0). The U1 snRNP and all other proteins absent in Cm are shown in transparent gray.

FIGURE 4.
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FIGURE 4.

(A) Proposed secondary structure of the Cm U5 snRNA. Cm U5 (dark red) is overlaid on yeast U5 snRNA (black). Model was generated from the MFold RNA secondary structure prediction web server (Zuker 2003). (B) Proposed model for interaction between snRNAs and all sequence variants of 5′ splice sites in Cm. The 5′ splice site of the pre-mRNA is indicated (middle); complementary region of U5 snRNA is shown above, and that of U6 snRNA is shown below. Watson–Crick base pairs, G–U pairs, and G–A base pairs are indicated with symbols accordingly. Interactions with U5 and U6 snRNAs are proposed to occur sequentially.

There is ample precedent for redundancy in intron-recognition pathways from studies of splicing in HeLa cell extracts. High concentrations of SR proteins, and specifically SC35, can compensate for inactivation of the U1 snRNP after its depletion (Crispino et al. 1994) or blocking with an antisense oligonucleotide (Tarn and Steitz 1994), albeit only for some pre-mRNA transcripts. The transcript dependence may indicate that unknown properties of the pre-mRNA facilitate U1-independent splicing. Notably, increasing base-pairing between U6 and the 5′SS, as we see in Cm, increases the amount of U1-independent splicing in extract supplemented with SC35 (Crispino and Sharp 1995). The prior demonstration of U6-mediated suppression of U1 loss is consistent with the possibility that, in the absence of U1, intron recognition proceeds directly via U6 snRNA binding in Cm. There is a candidate SC35 homolog in Cm, but its expression level is not particularly high. Interestingly, U2AF65 can also be rendered unnecessary in HeLa extract by addition of SC35, but only in the presence of the U1 snRNP (MacMillan et al. 1997). These results point to inherent flexibility in intron recognition mediated by independent pathways, and suggest that U2, U5, or U6 snRNAs are all candidates for early roles in intron recognition.

CmBrr2 and predicted stability/regulation of the Cm tri-snRNP

As mentioned, the U4, U5, and U6 snRNPs join the assembling spliceosome as the preassembled tri-snRNP. The presence or absence and degree of similarity of Cm proteins compared to their human orthologs is revealed in the cryo-EM structure of the human tri-snRNP (Agafonov et al. 2016). Proteins that are absent in Cm are observed to be mainly peripheral in this structure, while the center of the tri-snRNP, mostly comprising Prp8 and three snRNAs, is conserved (Fig. 5). Cm lacks U5-40K, Prp6, Sad1, and the U1-associated DEAD-box helicase Prp28, which was previously discussed (Fig. 5). The absence of Prp6 is surprising, because it is required for tri-snRNP accumulation (Galisson and Legrain 1993) and bridges the U4/U6 di-snRNP and U5 snRNP in yeast and human spliceosomes (Fig. 5). U5-40K forms a trimer with Prp8 and Snu114 during assembly of the human U5 snRNP (Achsel et al. 1998), and later interacts with Prp17 in the precatalytic spliceosome (Sapra et al. 2008). It is conserved between animals and S. pombe (Cwf17), but is not present in yeast (Fabrizio et al. 2009) or Cm spliceosomes (Stark et al. 2015). The absence of Sad1 is correlated with the loss of its interacting proteins as well as other Brr2 helicase regulatory elements (Fig. 5, inset and Supplemental Figs. 1A, 2B).

FIGURE 5.
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FIGURE 5.

Surface representation of the Cm U4/U6.U5 tri-snRNP homology modeled against the human structure (PDB 3JCR). All protein sequences were annotated by conservation with their Cm homologs. Identical and similar amino acids are colored purple while dissimilar amino acids are colored wheat. Proteins absent in Cm are shown in transparent gray. The RNA is colored as follows: U6 (dark red); U4 (yellow); and U5 (green). (Inset) Surface representation of the amino- and carboxy-terminal cassettes of Cm Brr2 homology modeled against the human structure (PDB 3JCR). The amino-terminal plug domain of Brr2 that is absent in Cm is shown in magenta.

The Cm tri-snRNP could be destabilized by lack of Brr2 regulation

The Ski2-like RNA helicase Brr2 catalyzes unwinding of the U4/U6 snRNA duplex, causing the release of U4 snRNP and progression from the B complex to Bact (Mozaffari-Jovin et al. 2014). Brr2 activity is regulated by a network of proteins (Nguyen et al. 2013) including Prp8, Sad1, Snu114, Snu13, and its own amino-terminal region (Mozaffari-Jovin et al. 2013; Absmeier et al. 2015). Precise control of Brr2 activity is necessary for properly regulated splicing; unregulated helicase activity can result in disassembly of the tri-snRNP or ectopic activation of the spliceosome (Absmeier et al. 2015).

The Cm spliceosome lacks Sad1, which normally prevents premature dissociation of the tri-snRNP by regulating Brr2 (Huang et al. 2014). Specifically, Sad1 tethers Brr2 in an inactive conformation, preventing the central domain of U4 snRNA from loading into Brr2 (Agafonov et al. 2016). This inhibitory activity is necessary as Sad1 depletion results in premature tri-snRNP disassembly by Brr2 (Huang et al. 2014). Sad1 interacts with Prp31, Prp8, Snu114, and the PWI domain of Brr2 in the human tri-snRNP (Nguyen et al. 2016). Cm Brr2 lacks amino-terminal residues for the PWI domain, and Cm Snu114 has poor homology with its human ortholog at its interface with Sad1 (Supplemental Figs. 1A, 2B).

The amino-terminal region of Cm Brr2 is 300 residues shorter than many of its homologs (Supplemental Figs. 1A, 2B). It lacks the “plug” domain (Fig. 5, inset), which is necessary for preventing premature tri-snRNP dissociation as well as for efficient splicing (Absmeier et al. 2015). The amino-terminal cassette of Brr2 features a central RNA binding tunnel formed by the RecA-like, WH (winged-helix), and HB (helical bundle) domains (Absmeier et al. 2015) that binds a single-stranded region of U4 snRNA. The plug domain of the amino-terminal region interacts with the central tunnel's RecA-2 and HB domains to obstruct binding of the U4/U6 snRNA duplex and thereby prevents unwinding (Wollenhaupt et al. 2018).

In addition to the Brr2 auto-inhibition that prevents premature U4/U6 snRNA unwinding, the carboxy-terminal tail of the Prp8 Jab1 domain interacts with the Brr2 RecA-2 and HB domains and prevents their opening to accommodate U4 snRNA in the central domain; it also competes for tunnel occupation with single-stranded RNA regions (Absmeier et al. 2017). Together these two mechanisms help to ensure Brr2 activation is strictly controlled. With its absent plug domain, CmBrr2 appears to have lost a regulatory element of Brr2 activity (Fig. 5, inset) and the CmJab1 carboxy-terminal tail is also absent (Supplemental Fig. 2A). The Prp8 RNase H-like (RH) domain, however, also inhibits Brr2 activity by competing for U4 and U6 snRNA binding with Brr2 and thereby blocking Brr2 from loading its substrate (Mozaffari-Jovin et al. 2013). This domain is conserved in Cm and presumably inhibits Brr2 activity prior to activation. In the absence of the Brr2 auto-inhibitory plug, the Prp8 Jab1 tail, and Sad1, we suggest that U4 snRNA is preloaded in the Brr2 RNA binding tunnel as seen in the yeast tri-snRNP.

Cm lacks the U6 snRNP-associated protein Prp24, which promotes reannealing of the U4 and U6 snRNAs during tri-snRNP recycling in yeast (Raghunathan and Guthrie 1998a; Didychuk et al. 2016) and humans (Medenbach et al. 2004). It has been suggested that the U4 and U6 snRNAs can anneal via a Prp24-independent pathway if they are not completely dissociated by Brr2 (Huang et al. 2014). It is possible that Cm U4 and U6 snRNPs have evolved such a Prp24-independent annealing pathway related to the extra domain in U4 snRNA or the higher temperature at which Cm grows. For example, U4's extra domain could mediate interactions with the U6 snRNP that stabilize U4–U6 interaction (but not necessarily base-pairing) that prevents complete dissociation.

C. merolae B complex

The precatalytic spliceosome is formed upon displacement of the U1 snRNP from the 5′SS by U6 snRNA resulting in the departure of U1 and U4 snRNPs and the formation of the precatalytic B complex. Cm homology modeling reveals many peripheral proteins missing between Cm and other spliceosomes (Fig. 6). Besides the absent proteins already discussed, the Cm B complex lacks U2 snRNP-associated proteins Lea1, Msl1, SF3B6, Ysf3, and the B complex-specific proteins FBP21, PPIH, Snu23, Snu66, Smu1, and Spp381 (Fig. 6). Similar to U5-40K, the peptidyl-prolyl isomerase PPIH, Brr2-regulator FBP21 and alternative splicing factor Smu1 are conserved among metazoans, but are not present in yeast (Agafonov et al. 2011; Henning et al. 2017; Keiper et al. 2019). In humans, Smu1 and RED are required for spliceosome activation when the distance between the 5′ splice site and branchpoint sequence is short (50 nt), and for splicing efficiency in general (Keiper et al. 2019). In Cm, almost all introns have more than 50 nt between the 5′SS and the BPS.

FIGURE 6.
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FIGURE 6.

Surface representation of the Cm B complex homology modeled against the human structure (PDB 5O9Z). All protein sequences were annotated by conservation with their Cm homologs. Identical and similar amino acids are colored purple while dissimilar amino acids are colored wheat. Proteins absent in Cm are shown in transparent gray. The RNA is colored as follows: U6 (dark red); U4 (yellow); U5 (green); U2 (blue); and pre-mRNA (cyan).

The heterodimer Lea1/Msl1 has been implicated in cotranscriptional splicing of a subset of yeast genes (Gunderson and Johnson 2009). Specifically, histone acetylation of actively transcribed genes by the histone acetyltransferase (HAT) Gcn5 promotes the recruitment of Lea1/Msl1, and in turn the U2 snRNP (Gunderson and Johnson 2009). This raises the question of whether or not pre-mRNA splicing occurs cotranscriptionally in Cm. While there is no evidence for Lea1/Msl1 in Cm (Stark et al. 2015), the HAT Gcn5 may exist, as it was the Reciprocal Best Hit for Hypothetical Protein CMA025C. With both U2AF35 and the Lea1/Msl1 heterodimer missing, we predict that splicing and transcription are uncoupled in C. merolae.

The tri-snRNP protein Snu66 has a conserved “HIND” element, and its interaction with the ubiquitin-like protein Hub1 is necessary for alternative splicing and for recognition of noncanonical 5′SS in humans and yeast (Wilkinson et al. 2004). With preliminary RNA sequencing data, we have not found evidence of alternative splicing of the single Cm gene that contains more than one intron, nor have we found evidence for noncanonical splice sites (Stark et al. 2015). These observations are consistent with the absence of Snu66.

When looking at features associated with the RNA core, the Cm B complex is relatively conserved (Fig. 7). Cm Hsh155 and the LSm ring that are proximal to the proposed branch duplex show strong sequence conservation (Fig. 7). In addition, the Prp8 amino-terminal domain is conserved, where the U6 snRNA ACAGAGA helix tethers the 5′SS prior to the first transesterification (Fig. 7, bottom right).

FIGURE 7.
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FIGURE 7.

Overview of the Cm B complex homology model (PDB 5O9Z). All protein sequences were annotated by conservation with their Cm homologs. Identical and similar amino acids are colored purple while dissimilar amino acids are colored wheat. Right insets show regions of high conservation, which are also positions of helices formed by U2 snRNA and the BPS (upper right), as well as U6 snRNA and the 5′ splice site (lower right). (Left inset) Surface representation of Prp38 and associated proteins in the Cm B complex (PDB 5O9Z). Cm Prp38 lacks Prp38 complex-associated proteins FBP21, Spp381 and Snu23. Proteins absent in Cm are shown in transparent gray. The RNA is colored as follows: U6 (dark red); U4 (yellow); U5 (green); U2 (blue); and pre-mRNA (cyan).

Snu23 and Spp381, absent in Cm, are part of the Prp38 complex that stabilizes the catalytic RNA prior to the branching reaction (Fig. 7, left inset). Prp38 is an essential splicing protein necessary for formation of the activated Bact complex in humans (Schütze et al. 2016), yeast (Xie et al. 1998), and fruit flies (Andersen and Tapon 2008). Snu23, Spp381, and Prp38 form a trimer that is tri-snRNP-specific in yeast and B complex-specific in animals, which suggests these proteins coevolved (Ulrich and Wahl 2017). The interface between Prp38 and the Prp8 amino-terminal domain reveals those interacting surfaces are well conserved, the Spp381 interacting surface is partially conserved, and the Snu23 surface is not conserved (Fig. 7, left inset). The Prp38-interacting interface of the Prp8 amino-terminal domain is conserved in Cm highlighting the importance of Prp38 in stabilizing the U6 snRNA ACAGAGA helix formed with the 5′SS during the activation step in Cm (Fig. 7, left inset; Supplemental Fig. 1O; Plaschka et al. 2017).

C. merolae Bact complex

In yeast and humans, transition from the B complex to the activated Bact complex requires departure of the U1 and U4 snRNPs, recruitment of the NTC and related proteins, and formation of the U6 snRNA intramolecular stem–loop (ISL) and U2/U6 Helix I. These structures, together with loop I of U5 snRNA, form the RNA center of the spliceosome that coordinates catalytic Mg2+ ions responsible for the actual chemistry of splicing. The formation of the active site occurs in the activated spliceosome or Bact complex. This complex is not yet chemically competent, because the BPS is too distant from the 5′SS for splicing chemistry to occur (Rauhut et al. 2016; Yan et al. 2016).

Cm lacks 16 of the 38 proteins of the yeast Bact complex (Table 1; Fig. 8). The majority of the proteins missing in the Cm Bact complex are non-snRNP or otherwise associated with the NTC-, RES-, or Bact complexes (Fig. 8; Bao et al. 2017a). Notably, the Cm NTC is missing Prp17 and the Prp17 yeast synthetic lethal proteins Syf1 and Syf2, Ecm2, Snt309 (SPF27), Prp45, Cwc2, Cwc25, and peptidyl-prolyl isomerases PPIL1 and PPIE (Fig. 8). Syf1, but not Syf2, is required for cell cycle progression and splicing activity in yeast (Russell et al. 2006). In Cm, Syf1, Syf2, and their interacting partner Prp17 are all absent, which suggests these proteins function cohesively and perhaps coevolved (Russell et al. 2006). In yeast, Syf1, Syf2, Ecm2, Cwc2, and Prp17 are connected to each other in a chain-like fashion, as well as to the core of the Bact complex (Fig. 8). These missing proteins surround helix I of the yeast U2/U6 snRNA duplex, which is therefore presumably exposed in Cm (Fig. 8, right). In contrast, the Prp8 N-domain that coordinates the branch helix formed by the 3′ end of U2 snRNA and the intron branchpoint sequence is highly conserved (Fig. 8, right). Cm lacks Cwc2, which contacts the U6 snRNA internal stem–loop (ISL) and is involved in making the active site catalytically competent (Rasche et al. 2012). The absence of the cotranscriptional splicing factor Prp45 (Hálová et al. 2017) is reminiscent of the loss of the Lea1/Msl1 heterodimer, because they are both associated with histone modification-dependent recruitment of splicing machinery to actively transcribed genes (Gunderson and Johnson 2009; Leung et al. 2019). Prp45, and subsequently the NTC, is cotranscriptionally recruited to the precatalytic B complex by the chromodomain protein Eaf3 (Leung et al. 2019). Eaf3 interacts with both the Set2-trimethylated histone H3K36 as well as Prp45 (Leung et al. 2019).

FIGURE 8.
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FIGURE 8.

Surface representation of the Cm Bact complex homology modeled against the Sc structure (PDB 5GM6). All protein sequences were annotated by conservation with their Cm homologs. Identical and similar amino acids are colored purple while dissimilar amino acids are colored wheat. Proteins absent in Cm are shown in transparent gray. The RNA is colored as follows: U6 (dark red); U5 (green); U2 (blue); and pre-mRNA (cyan).

Several proteins that interact with Prp45 in the Bact complex and subsequent structures are also absent in Cm including Ecm2 and Syf2 of the NTC and the RES complex (Fig. 8, right; Yan et al. 2016). The RES complex (Bud13, Pml1, and Snu17) is dispensable for splicing, but RES-depleted yeast have inefficient Bact complex assembly and splicing activity (Bao et al. 2017b). The activated Cm spliceosome also lacks the NTC-associated heat resistant protein Cwc25, which, together with Yju2, stabilizes the U2 snRNA/branchpoint helix during the branching reaction (Chiang and Cheng 2013; Zhan et al. 2018a). While Cwc25 and Yju2 are both essential in yeast, only Yju2 has been identified in Cm (Supplemental Fig. 1R).

The simplified Cm spliceosome offers a glimpse into essential spliceosomal components (Stark et al. 2015). Cm lacks the cotranscriptional pre-mRNA splicing regulation offered by either Lea1/Msl1 (Gunderson and Johnson 2009) or Prp45 (Hálová et al. 2017; Leung et al. 2019), consistent with its inefficient splicing (Stark et al. 2015; Schärfen et al. 2022). We assume the first transesterification is inefficient in Cm based on the splicing factors that are absent in the activated Cm spliceosome, particularly Prp17 and NTC proteins.

Absent Ysf3 protein and destabilization of SF3B in C. merolae

The SF3B complex in the U2 snRNP is responsible for catalytic latency, i.e., for preventing premature nucleophilic attack of the chemically reactive hydroxyl of the branchpoint adenosine (McPheeters and Muhlenkamp 2003). Comparison of the homology with the electrostatic surface potential of the SF3B complex suggests that the important RNA-binding surfaces (electropositive) are conserved in Cm (Fig. 9A,B). Six proteins fold into the compact SF3B structure (Rse1, Hsh155, Cus1, Rds3, Hsh49, and Ysf3). Ysf3 (SF3B5) is a wedge-like protein that is missing in the Cm spliceosome (Fig. 9A). In yeast, Ysf3 is essential for U2 snRNP entry during spliceosomal assembly and splicing (Wang et al. 2005). The SF3B complex can assemble without Ysf3, but loss of Ysf3 causes reduction in Cus1 and a decrease in stability of the SF3B complex (Wang et al. 2005). Ysf3 absence may result in slower splicing, but may also obviate the need for proteins that would otherwise be required to help remove the SF3B complex such as the Prp2 cofactors Cwc22 and Spp2 (Silverman et al. 2004; Yeh et al. 2011), which are indeed absent in Cm.

FIGURE 9.
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FIGURE 9.

The SF3B complex from the Sc Bact complex (PDB 5GM6), showing both (A) Cm homology model and (B) electrostatic surface representations. Ysf3, shown in black, is absent in the Cm SF3B complex. In A, all protein sequences were annotated by conservation with their Cm homologs. Identical and similar amino acids are colored purple while dissimilar amino acids are colored wheat. mRNA is colored cyan. In B, the protein surface is colored from blue (electropositive) to red (electronegative) with mRNA as in A.

Spp2/GPKOW is a G-patch protein that activates the DEAH-box helicase Prp2/DHX16 (Robert-Paganin et al. 2015). There are five known G-patch proteins that activate DEAH-box helicases in both yeast and humans: Cgm1/GPATCH11, Gno1/PINX1, Pfa1/NKRF, Spp382/TFIP11, and Spp2/GPKOW (Robert-Paganin et al. 2015; Studer et al. 2020). Of those five, Spp2/GPKOW activates Prp2/DHX16 (Warkocki et al. 2015), while the other four all activate Prp43/DHX15 (Hamann et al. 2019; Bohnsack et al. 2021). Mass spectrometry and bioinformatics suggest there do not appear to be any DEAH-box helicase-activating G-patch proteins present in Cm. The absence of G-patch activators of DEAH-box helicases suggests that Cm lacks regulation and efficiency during ATP-dependent conformational changes.

In humans, the U2 protein p14/SF3B6, a component of the SF3B complex, associates with the BPS at early stages of spliceosome assembly and directly contacts the BPS adenosine (MacMillan et al. 1994; Schellenberg et al. 2006; Tholen et al. 2022); this protein is also missing in Cm. It is also notably absent in yeast (Dziembowski et al. 2004) despite the presence of the six other protein components of SF3B, but is conserved in other fungi including the fission yeast S. pombe. Overall, the presence of SF3B6 appears to correlate with degeneracy of the intron BPS (the BPS in both Cm and yeast is highly conserved while that in S. pombe is of intermediate complexity compared to the highly degenerate human BPS). Thus, SF3B6 may play a role in the selection of the BPS as a splicing regulatory mechanism.

C. merolae Prp8 lacks structural motifs necessary for splicing efficiency

The human and yeast Prp8 RNase H domain appears to act as a spliceosomal throttle. It is hypothesized to toggle assembly between the “closed” and “open” catalytic states associated with hyper-accurate/slow and inaccurate/fast splicing, respectively (Schellenberg et al. 2013; Mayerle et al. 2017). It contains a 17 amino acid insertion between two adjacent β-strands termed the “Toggle loop” motif (Schellenberg et al. 2013; Mayerle et al. 2017). This loop motif is not conserved in Cm; absence of the 17 amino acid insertion was suggested bioinformatically and confirmed crystallographically (Garside et al. 2019). In place of the Toggle loop, the Cm Prp8 RH domain contains a well-ordered type II β-turn that locks the RH domain in the “Closed loop” conformation (Garside et al. 2019). The Closed loop conformation of the Cm Prp8 RH domain, combined with the absence of the Toggle loop and other regulatory proteins previously discussed, leads us to predict the Cm spliceosome is slow during assembly, transitional, and catalytic stages.

Similar to the observation with Sad1, the loss of the RH Toggle loop motif is associated with the loss of proteins that interact with it such as Prp6 in the tri-snRNP. Along with the RNase H domain Toggle loop, Cm Prp8 is also predicted to be missing the “Switch loop” of its linker domain (Supplemental Fig. 1J). In the structure of the yeast Bact complex, the Switch loop interacts with Cwc21, which is also missing in Cm (Jia and Sun 2018; Supplemental Fig. 1J). The absence of these two features from the Cm Bact complex active site is predicted to translate to fewer and weaker interactions with the 5′SS prior to catalysis. Thus, splicing accuracy in Cm is also predicted to be lower than in highly regulated spliceosomes. The absence of these two features would also mean that a canonical exon binding channel would not exist in Cm (Figs. 10, 11).

FIGURE 10.
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FIGURE 10.

Surface representation of the Cm C* complex homology modeled against the human structure (PDB 5MQF). All protein sequences were annotated by conservation with their Cm homologs. Identical and similar amino acids are colored purple while dissimilar amino acids are colored wheat. (Inset) Several proteins are missing or are poorly conserved within the Cm 3′SS/BPS region, highlighting the importance of CmPrp8 in the second transesterification. Proteins that are absent in Cm are shown in transparent gray. The RNA is colored as follows: U6 (dark red); U5 (green); U2 (blue); and pre-mRNA (cyan).

FIGURE 11.
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FIGURE 11.

Surface representation of the Cm P complex homology modeled against the Sc structure (PDB 5YLZ). All protein sequences were annotated by conservation with their Cm homologs. Identical and similar amino acids are colored purple while dissimilar amino acids are colored wheat. (Inset) the intron lariat junction from the Sc P complex active site (PDB 5YLZ). The 1585 loop (present in CmPrp8) and Toggle loop (missing in CmPrp8) are represented in cartoon, while the respective linker and RNase H domains of CmPrp8 are represented as surfaces. Proteins absent in Cm are shown in transparent gray. The RNA is colored as follows: U6 (dark red); U5 (green); U2 (blue); and pre-mRNA (cyan).

Despite the lack of protein coordination, we predict there are novel, compensatory RNA–RNA interactions that contribute to splicing in Cm. Cm U5 snRNA is 450 nt in length, which is much larger than both the 214 nt yeast U5 snRNA and the 116 nt human equivalent. Secondary structure prediction of Cm U5 snRNA suggests several additional stems and loops are present (Fig. 4). It is possible that Cm U5 snRNA stabilizes the 5′ exon during the first transesterification in a role otherwise filled by the Prp8 Switch loop (Stark et al. 2015).

C. merolae C* complex

The catalytically activated C complex (C*) is formed after the ATP-driven DEAH-box helicase activity of Prp16 results in dissociation of first step factors and rearrangement of the branched intron for the second transesterification (Chiang and Cheng 2013). Cm lacks a third of the proteins (15) present in the cryo-EM structure of the human C* complex (Fig. 10).

The majority of proteins missing from the C* complex—such as Cwc2, Cwc21, Cwc22, Lea1, Msl1, U5-40K, Prp45, Snt309, Syf1, and Syf2—are not associated with a specific snRNP and have been discussed previously (Fig. 10). Some of these proteins are functionally relevant earlier in the Bact complex and the first transesterification reaction, including the post-first step branched complex. Each of these missing proteins plays an important role in structures of human or yeast C*. Msl1 and Lea1 are part of a bridge between stem IV of U2 snRNA and proteins interacting with the active site (Price et al. 1998; Galej et al 2016). The splicing factor Cwc21 binds and stabilizes the 5′ exon along with portions of Prp8 (Bertram et al. 2017a; Fica et al. 2017). The NTC protein Syf2 binds to Helix II of the annealed U2/U6 snRNA as well as to Prp45 and Clf1 (Bertram et al. 2017b). Thus, the catalytic site of the spliceosome is enveloped by PRP45/NTC components as well as elements of Prp8 (Galej et al. 2016). The Prp18–Slu7 complex that joins the spliceosome for the second transesterification is also absent in Cm, as is Cwc22 that would contact Slu7 at this stage (Fica et al. 2017; Zhang et al. 2017). Prp18, Slu7, and Prp22 are required for efficient positioning of the 3′SS at the catalytic center, and displace first step factors Cwc25 and Yju2 in yeast (Schwer 2008). The absence of Msl1, Lea1, and Prp45 in Cm suggests that stabilization of spliceosomal assembly intermediates may involve the participation of more complex RNA structures or elements of proximal, conserved core proteins.

Based on the above analysis, the transition from the C complex to C* would presumably be less complex in Cm; Yju2 would either depart or undergo a conformational rearrangement upon Prp22 entry, but there would be no exchange between Cwc25, Prp18, or Slu7. This last factor is unnecessary in exon ligation events where the distance between the BPS and 3′SS is 9 nucleotides or fewer (Frank and Guthrie 1992; Brys and Schwer 1996; James et al. 2002); 18 of the 38 Cm introns contain 20 nucleotides or more between the BPS and 3′SS, so the absence of Slu7 is surprising. It has been reported that splicing occurs at a low efficiency in yeast extracts depleted of Cwc25, Prp18, and Slu7 (Tseng et al. 2017). Prp17 is also absent from the Cm C* complex and might cause the U2 snRNA/branchpoint helix to be relatively less stable (Fig. 10). The majority of proteins at the catalytic center that contact snRNAs, such as Cdc5 and Crn, are present in the Cm C* spliceosome (Fig. 10). Interestingly, the amino acid conservation between these proteins is not high; perhaps their structures remain conserved while their sequence-specific RNA recognition does not (Fig. 10).

C. merolae P complex

Recent structures of yeast P complexes have provided several snapshots immediately following the transesterification reaction that results in exon ligation (Bai et al. 2017; Liu et al. 2017; Wilkinson et al. 2017; Fica et al. 2019). The yeast P complex contains approximately 40 proteins (Fica et al. 2019) of which only 15 have orthologs that have been identified in Cm (Fig. 11). Of the 16 proteins that stabilize RNA elements at the P complex active site, Cm is missing 10 (Table 1).

A recurring theme in the analysis of the Cm spliceosome is that most of the proteins absent in the Cm P complex are peripheral to the RNA helices or stabilize the active site (Fig. 11). We hypothesize that the proteins present in the Cm P complex represent an essential catalytic core of proteins and RNA necessary for exon ligation. These include Prp8, Snu114, Cef1, Clf1, Bud31, and Prp46. With the exception of Bud31, these are all among the phylogenetically most highly retained splicing factors. Many of the missing proteins in the C* complex are of course absent in the Cm P complex, such as the intron-binding NTR protein Cwc2 or the NTC component Syf2, which stabilizes the U2/U6 snRNA Helix II (Rasche et al. 2012; Bertram et al. 2017b). Cwc21 and Cwc22, also absent, stabilize the 5′ exon during and after ligation (Yeh et al. 2011; Chen et al. 2013; Bai et al. 2017; Bertram et al. 2017b). The absence of Prp17 and the Prp8 Toggle loop motif could have substantial effects on the stability of the spliceosome active site in the C* and P complexes (Fig. 11 inset; Sapra et al. 2008; Mayerle et al. 2017). Some of the missing proteins modulate the efficiency of splicing as described for Prp18 and Slu7.

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DISCUSSION

Our analysis of yeast and human spliceosome structures through a C. merolae lens reveals an interesting trend, namely the increased number of missing proteins as a function of step in the splicing cycle (Fig. 2). Most of the proteins present in the catalytic core of the spliceosome are present in Cm with the exception of Prp17 (Figs. 10, 11). An in-depth look at the catalytic centers of these spliceosomes shows that as assembly and catalysis progress in Cm, more proteins and structural motifs are missing at each step. Interestingly, some of these absent proteins and domains, such as the Prp18–Slu7 heterodimer, or the Toggle loop of the Prp8 RNase H domain, are proposed to act as throttles that normally allow the spliceosome to shift between two states: slow and accurate versus fast and inaccurate. The absence of such domains is consistent with the low fraction of spliced transcripts observed in Cm (Stark et al. 2015; Schärfen et al. 2022), although we have not observed evidence of inaccuracy: while some cryptic splice site use is detected by Illumina and PacBio sequencing, there do not appear to be more of these in Cm than in other organisms (Schärfen et al. 2022).

We have observed an absence of proteins or motifs that are coupled: Sad1 and the Brr2 plug domain during tri-snRNP incorporation (Fig. 5, inset); Cwc21 and the Prp8 RH Toggle loop during spliceosome activation; and Cwc22 and Slu7 in the C* complex. In the ligation reaction, Prp17 and the Prp8 RH Toggle loop interact with each other to stabilize the 3′SS and are both missing in Cm (Fig. 11). The Prp8 RH Toggle loop motif, together with the 1585 loop/α-finger motif of the Prp8 linker domain, sandwiches the intron lariat/active site in the yeast P complex (Fig. 11). The most surprising protein absences from the Cm spliceosome are those from the active site, such as Cwc21 and Prp17, as well as cotranscriptional regulatory factors Lea1/Msl1 and Prp45.

Pairwise and multiple sequence alignments of the Cm proteins suggest many proteins are either missing canonical domains or contain novel ones. Prp19 is dramatically truncated, lacking the amino-terminal RING domains and the carboxy-terminal WD40 motifs (Supplemental Fig. 2D). The Toggle loop motif of the Prp8 RNase H domain is another example (Fig. 11; Supplemental Figs. 1J, 2A; Mayerle et al. 2017). Conversely, CmMud2 has a large amino-terminal extension compared to its human and yeast orthologs and is ∼800 aa longer (Supplemental Fig. 1F), and the U5 snRNA is nearly twice as large as its homologs in other organisms (Fig. 4).

Homology-based annotation of human and yeast structures revealed that the reduced C. merolae spliceosome lacks protein–RNA and protein–protein interactions that together suggest less coordination of RNA elements and regulation of protein activity during the splicing cycle. We suggest Cm splicing—and by analogy that of other organisms with reduced spliceosomes—to be inefficient, inaccurate, and post-transcriptional. The inefficiency of Cm splicing contrasts strongly with splicing efficiency in other organisms including yeast (Stark et al. 2015; Schärfen et al. 2022). Furthermore, Cm lacks the DEAD-box RNA helicase Prp28, which is not only associated with release of U1 snRNP, but also with proofreading the 5′SS and splicing fidelity (Price et al. 2013). These properties of Cm splicing may be more similar to an ancestral (pre-LECA) spliceosomal ribozyme—with few associated proteins—than to the highly elaborated, protein-rich spliceosomes present in animals and plants.

The discovery of a spliceosome lacking the U1 snRNP was surprising. The comparative analysis performed here will be useful to the extent that it stimulates questions about the precise role of each splicing factor and the evolutionary pressures that have led to the great divergence in splicing systems seen across organisms. Much mechanistic work is still required before we understand the many layers of splicing regulation and splice site selection.

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SUPPLEMENTAL MATERIAL

Supplemental material is available for this article.

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ACKNOWLEDGMENTS

We acknowledge Kevin Huolt for initial development of algorithms used to color structures by sequence conservation. We thank Dr. Martha Stark for helpful feedback on the manuscript. This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grants to S.D.R., N.M.F., and A.M.M.

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Footnotes

This article, published in RNA, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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REFERENCES

  1. ↵
    Abelson J. 2017. A close-up look at the spliceosome, at last. Proc Natl Acad Sci 114: 4288–4293. doi:10.1073/pnas.1700390114
    Abstract/FREE Full Text
  2. ↵
    Absmeier E, Wollenhaupt J, Mozaffari-Jovin S, Becke C, Lee C-T, Preussner M, Heyd F, Urlaub H, Lührmann R, Santos KF, et al. 2015. The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation. Genes Dev 29: 2576–2587. doi:10.1101/gad.271528.115
    Abstract/FREE Full Text
  3. ↵
    Absmeier E, Becke C, Wollenhaupt J, Santos KF, Wahl MC. 2017. Interplay of cis- and trans-regulatory mechanisms in the spliceosomal RNA helicase Brr2. Cell Cycle 16: 100–112. doi:10.1080/15384101.2016.1255384
    CrossRefMedlineGoogle Scholar
  4. ↵
    Achsel T, Ahrens K, Brahms H, Teigelkamp S, Lührmann R. 1998. The human U5-220kD protein (hPrp8) forms a stable RNA-free complex with several U5-specific proteins, including an RNA unwindase, a homologue of ribosomal elongation factor EF-2, and a novel WD-40 protein. Mol Cell Biol 18: 6756–6766. doi:10.1128/MCB.18.11.6756
    Abstract/FREE Full Text
  5. ↵
    Agafonov DE, Deckert J, Wolf E, Odenwälder P, Bessonov S, Will CL, Urlaub H, Lührmann R. 2011. Semiquantitative proteomic analysis of the human spliceosome via a novel two-dimensional gel electrophoresis method. Mol Cell Biol 31: 2667–2682. doi:10.1128/MCB.05266-11
    Abstract/FREE Full Text
  6. ↵
    Agafonov DE, Kastner B, Dybkov O, Hofele RV, Liu W-T, Urlaub H, Lührmann R, Stark H. 2016. Molecular architecture of the human U4/U6.U5 tri-snRNP. Science 351: 1416–1420. doi:10.1126/science.aad2085
    Abstract/FREE Full Text
  7. ↵
    Andersen DS, Tapon N. 2008. Drosophila MFAP1 is required for pre-mRNA processing and G2/M progression. J Biol Chem 283: 31256–31267. doi:10.1074/jbc.M803512200
    Abstract/FREE Full Text
  8. ↵
    Bai R, Yan C, Wan R, Lei J, Shi Y. 2017. Structure of the post-catalytic spliceosome from Saccharomyces cerevisiae. Cell 171: 1–10. doi:10.1016/j.cell.2017.09.011
    CrossRefGoogle Scholar
  9. ↵
    Bai R, Wan R, Yan C, Jia Q, Lei J, Shi Y. 2021. Mechanism of spliceosome remodeling by the ATPase/helicase Prp2 and its coactivator Spp2. Science 371: eabe8863. doi:10.1126/science.abe8863
    Abstract/FREE Full Text
  10. ↵
    Bao P, Höbartner C, Hartmuth K, Lührmann R. 2017a. Yeast Prp2 liberates the 5′ splice site and the branch site adenosine for catalysis for pre-mRNA splicing. RNA 23: 1770–1779. doi:10.1261/rna.063115.117
    Abstract/FREE Full Text
  11. ↵
    Bao P, Will CL, Urlaub H, Boon K-L, Lührmann R. 2017b. The RES complex is required for efficient transformation of the precatalytic B spliceosome into an activated Bact complex. Genes Dev 31: 2416–2429. doi:10.1101/gad.308163.117
    Abstract/FREE Full Text
  12. ↵
    Berget SM, Moore C, Sharp PA. 1977. Spliced segments at the 5′ terminus of adenovirus 2 late mRNA. Proc Natl Acad Sci 74: 3171–3175. doi:10.1073/pnas.74.8.3171
    Abstract/FREE Full Text
  13. ↵
    Berglund JA, Chua K, Abovich N, Reed R, Rosbash N. 1997. The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC. Cell 89: 781–787. doi:10.1016/S0092-8674(00)80261-5
    CrossRefMedlineWeb of ScienceGoogle Scholar
  14. ↵
    Bertram K, Agafonov DE, Dybkov O, Haselbach D, Leelaram MN, Will CL, Urlaub H, Kastner B, Lührmann R, Stark H. 2017a. Cryo-EM structure of a pre-catalytic human spliceosome primed for activation. Cell 170: 701–713. doi:10.1016/j.cell.2017.07.011
    CrossRefMedlineGoogle Scholar
  15. ↵
    Bertram K, Agafonov DE, Liu W-T, Dybkov O, Will CL, Hartmuth K, Urlaub H, Kastner B, Stark H, Lührmann R. 2017b. Cryo-EM structure of a human spliceosome activated for step 2 of splicing. Nature 542: 318–323. doi:10.1038/nature21079
    CrossRefMedlineGoogle Scholar
  16. ↵
    Black CS, Garside EL, MacMillan AM, Rader SD. 2016. Conserved structure of Snu13 from the highly reduced spliceosome of Cyanidioschyzon merolae. Protein Sci 25: 911–916. doi:10.1002/pro.2894
    CrossRefGoogle Scholar
  17. ↵
    Boesler C, Rigo N, Agafonov DE, Kastner B, Urlaub H, Will CL, Lührmann R. 2015. Stable tri-snRNP integration is accompanied by a major structural rearrangement of the spliceosome that is dependent on Prp8 interaction with the 5′ splice site. RNA 21: 1993–2005. doi:10.1261/rna.053991.115
    Abstract/FREE Full Text
  18. ↵
    Bohnsack KE, Ficner R, Bohnsack MT, Jonas S. 2021. Regulation of DEAH-box RNA helicases by G-patch proteins. Biol Chem 402: 561–579. doi:10.1515/hsz-2020-0338
    CrossRefGoogle Scholar
  19. ↵
    Boothroyd JC, Cross GA. 1982. Transcripts coding for variant surface glycoproteins of Trypanosoma brucei have a short, identical exon at their 5′ end. Gene 20: 281–289. doi:10.1016/0378-1119(82)90046-4
    CrossRefMedlineWeb of ScienceGoogle Scholar
  20. ↵
    Brys A, Schwer B. 1996. Requirement for SLU7 in yeast pre-mRNA splicing is dictated by the distance between the branchpoint and the 3′ splice site. RNA 2: 707–717.
    Abstract
  21. ↵
    Burki F, Roger AJ, Brown MW, Simpson AGB. 2020. The new tree of eukaryotes. Trends Ecol Evol 35: 43–55. doi:10.1016/j.tree.2019.08.008
    CrossRefMedlineGoogle Scholar
  22. ↵
    Chang J, Schwer B, Shuman S. 2012. Structure-function analysis and genetic interactions of the yeast branchpoint binding protein Msl5. Nucleic Acids Res 40: 4539–4552. doi:10.1093/nar/gks049
    CrossRefMedlineWeb of ScienceGoogle Scholar
  23. ↵
    Charenton C, Wilkinson ME, Nagai K. 2019. Mechanism of 5′ splice site transfer for human spliceosome activation. Science 364: 362–367. doi:10.1126/science.aax3289
    Abstract/FREE Full Text
  24. ↵
    Chen HC, Cheng SC. 2012. Functional roles of protein splicing factors. Biosci Rep 32: 345–359. doi:10.1042/BSR20120007
    Abstract/FREE Full Text
  25. ↵
    Chen HC, Tseng CK, Tsai RT, Chung CS, Cheng SC. 2013. Link of NTR-mediated spliceosome disassembly with DEAH-box ATPases Prp2, Prp16, and Prp22. Mol Cell Biol 33: 514–525. doi:10.1128/MCB.01093-12
    Abstract/FREE Full Text
  26. ↵
    Chen M, Zhang K, Gao B, Yang J, Tian Y, Das D, Fan T, Dai L, Hao G, Yang G, et al. 2020. Phylogenetic comparison of 5′ splice site determination in central spliceosomal proteins of the U1-70K gene family, in response to developmental cues and stress conditions. Plant J 103: 357–378. doi:10.1111/tpj.14735
    CrossRefGoogle Scholar
  27. ↵
    Cheng SC, Abelson J. 1987. Spliceosome assembly in yeast. Genes Dev 1: 1014–1027. doi:10.1101/gad.1.9.1014
    Abstract/FREE Full Text
  28. ↵
    Chiang T-W, Cheng S-C. 2013. A weak splicesome-binding domain of Yju2 functions in the first step and bypasses Prp16 in the second step of splicing. Mol Cell Biol 33: 1746–1755. doi:10.1128/MCB.00035-13
    Abstract/FREE Full Text
  29. ↵
    Chow LT, Roberts JM, Lewis JB, Broker TR. 1977. A map of cytoplasmic RNA transcripts from lytic adenovirus type 2, determined by electron microscopy of RNA:DNA hybrids. Cell 11: 819–836. doi:10.1016/0092-8674(77)90294-X
    CrossRefMedlineWeb of ScienceGoogle Scholar
  30. ↵
    Cordin O, Beggs JD. 2013. RNA helicases in splicing. RNA Biol 10: 83–95. doi:10.4161/rna.22547
    CrossRefMedlineWeb of ScienceGoogle Scholar
  31. ↵
    Crispino JD, Sharp PA. 1995. A U6 snRNA:pre-mRNA interaction can be rate-limiting for U1-independent splicing. Genes Dev 9: 2314–2323. doi:10.1101/gad.9.18.2314
    Abstract/FREE Full Text
  32. ↵
    Crispino JD, Blencowe BJ, Sharp PA. 1994. Complementation by SR proteins of pre-mRNA splicing reactions depleted of U1 snRNP. Science 265: 1866–1869. doi:10.1126/science.8091213
    Abstract/FREE Full Text
  33. ↵
    Das R, Reed R. 1999. Resolution of the mammalian E complex and the ATP-dependent spliceosomal complexes on native agarose mini-gels. RNA 5: 1504–1508. doi:10.1017/s1355838299991501
    Abstract
  34. ↵
    Didychuk AL, Montemayor EJ, Brow DA, Butcher SE. 2016. Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly. Nucleic Acids Res 44: 1398–1410. doi:10.1093/nar/gkv1374
    CrossRefMedlineGoogle Scholar
  35. ↵
    Dziembowski A, Ventura AP, Rutz B, Caspary F, Faux C, Halgand F, Laprévote O, Séraphin B. 2004. Proteomic analysis identifies a new complex required for nuclear pre-mRNA retention and splicing. EMBO J 23: 4847–4856. doi:10.1038/sj.emboj.7600482
    Abstract/FREE Full Text
  36. ↵
    Fabrizio P, Dannenberg J, Dube P, Kastner B, Stark H, Urlaub H, Lührmann R. 2009. The evolutionarily conserved core design of the catalytic activation step of the yeast spliceosome. Mol Cell 36: 593–608. doi:10.1016/j.molcel.2009.09.040
    CrossRefMedlineWeb of ScienceGoogle Scholar
  37. ↵
    Fica S, Oubridge C, Galej W, Wilkinson ME, Bai XC, Newman AJ, Nagai K. 2017. Structure of a spliceosome remodelled for exon ligation. Nature 542: 377–380. doi:10.1038/nature21078
    CrossRefMedlineGoogle Scholar
  38. ↵
    Fica SM, Oubridge C, Wilkinson ME, Newman AJ, Nagai K. 2019. A human postcatalytic spliceosome structure reveals essential roles of metazoan factors for exon ligation. Science 363: 710–714. doi:10.1126/science.aaw5569
    Abstract/FREE Full Text
  39. ↵
    Frank D, Guthrie C. 1992. An essential splicing factor, SLU7, mediates 3′ splice site choice in yeast. Genes Dev 6: 2112–2124. doi:10.1101/gad.6.11.2112
    Abstract/FREE Full Text
  40. ↵
    Friend K, Lovejoy AF, Steitz JA. 2007. U2 snRNP binds intronless histone pre-mRNAs to facilitate U7-snRNP-dependent 3′ end formation. Mol Cell 28: 240–252. doi:10.1016/j.molcel.2007.09.026
    CrossRefMedlineWeb of ScienceGoogle Scholar
  41. ↵
    Galej WP, Wilkinson ME, Fica SM, Oubridge C, Newman AJ, Nagai K. 2016. CryoEM structure of the spliceosome immediately after branching. Nature 537: 197–201. doi:10.1038/nature19316
    CrossRefMedlineGoogle Scholar
  42. ↵
    Galisson F, Legrain P. 1993. The biochemical defects of prp4-1 and prp6-1 yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP. Nucleic Acids Res 21: 1555–1562. doi:10.1093/nar/21.7.1555
    CrossRefMedlineWeb of ScienceGoogle Scholar
  43. ↵
    Garside EL, Whelan T, Stark MR, Rader SD, Fast NM, MacMillan AM. 2019. Prp8 in a reduced spliceosome lacks a conserved toggle that correlates with splicing complexity across diverse taxa. J Mol Biol 431: 2543–2553. doi:10.1016/j.jmb.2019.04.047
    CrossRefGoogle Scholar
  44. ↵
    Grisdale CJ, Bowers LC, Dider ES, Fast NM. 2013. Transcriptome analysis of the parasite Encephalitozoon cuniculi: an in-depth examination of pre-mRNA splicing in a reduced eukaryote. BMC Genomics 14: 207. doi:10.1186/1471-2164-14-207
    CrossRefMedlineGoogle Scholar
  45. ↵
    Gunderson FQ, Johnson TL. 2009. Acetylation by the transcriptional coactivator Gcn5 plays a novel role in co-transcriptional spliceosome assembly. PLoS Genet 5: 1000682. doi:10.1371/journal.pgen.1000682
    CrossRefGoogle Scholar
  46. ↵
    Günzl A. 2010. The pre-mRNA splicing machinery of trypanosomes: complex or simplified? Eukaryot Cell 9: 1159–1170. doi:10.1128/EC.00113-10
    Abstract/FREE Full Text
  47. ↵
    Hálová M, Gahura O, Prevorovsky M, Cit Z, Novotny M, Valentova A, Abrhámová K, Puta F, Folk P. 2017. Ninteteen complex-related factor Prp45 is required for the early stages of cotranscriptional spliceosome assembly. RNA 23: 1512–1524. doi:10.1261/rna.061986.117
    Abstract/FREE Full Text
  48. ↵
    Hamann F, Enders M, Ficner R. 2019. Structural basis for RNA translocation by DEAH-box ATPases. Nucleic Acids Res 47: 4349–4362. doi:10.1093/nar/gkz150
    CrossRefMedlineGoogle Scholar
  49. ↵
    Henning LM, Santos KF, Sticht J, Jehle S, Lee C-T, Wittwer M, Urlaub H, Stezl U, Wahl MC, Freund C. 2017. A new role for FBP21 as a regulator of Brr2 helicase activity. Nucleic Acids Res 45: 7922–7937. doi:10.1093/nar/gkx535
    CrossRefMedlineGoogle Scholar
  50. ↵
    Hoskins AA, Friedman LJ, Gallagher SS, Crawford DJ, Anderson EG, Wombacher R, Ramirez N, Cornish VW, Gelles J, Moore MJ. 2011. Ordered and dynamic assembly of single spliceosomes. Science 331: 1289–1295. doi:10.1126/science.1198830
    Abstract/FREE Full Text
  51. ↵
    Hossain MA, Johnson TL. 2014. Using yeast genetics to study splicing mechanisms. Methods Mol Biol 1126: 285–298. doi:10.1007/978-1-62703-980-2_21
    CrossRefMedlineWeb of ScienceGoogle Scholar
  52. ↵
    Huang T, Vilardell J, Query CC. 2002. Pre-spliceosome formation in S. pombe requires a stable complex of SF1-U2AF59-U2AF23. EMBO J 21: 5516–5526. doi:10.1093/emboj/cdf555
    Abstract
  53. ↵
    Huang YH, Chung CS, Kao DI, Kao TC, Cheng SC. 2014. Sad1 counteracts Brr2-mediated dissociation of U4/U6.U5 in tri-snRNP homeostasis. Mol Cell Biol 34: 210–220. doi:10.1128/MCB.00837-13
    Abstract/FREE Full Text
  54. ↵
    Hudson AJ, Moore AN, Elniski D, Joseph J, Yee J, Russell AG. 2012. Evolutionaryily divergent spliceosomal snRNAs and a conserved non-coding RNA processing motif in Giardia lamblia. Nucleic Acids Res 40: 10995–11008. doi:10.1093/nar/gks887
    CrossRefMedlineWeb of ScienceGoogle Scholar
  55. ↵
    Hudson AJ, Stark MR, Fast NM, Russell AG, Rader SD. 2015. Splicing diversity revealed by reduced spliceosomes in C. merolae and other organisms. RNA Biol 12: 1–8. doi:10.1080/15476286.2015.1094602
    CrossRefGoogle Scholar
  56. ↵
    Hudson AJ, Mcwatters DC, Bowser BA, Moore AN, Larue GE, Roy SW, Russell AG. 2019. Patterns of conservation of spliceosomal intron structures and spliceosome divergence in representatives of the diplomonad and parabasalid lineages. BMC Evol Biol 19: 162. doi:10.1186/s12862-019-1488-y
    CrossRefGoogle Scholar
  57. ↵
    James SA, Turner W, Schwer B. 2002. How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing. RNA 8: 1068–1077. doi:10.1017/S1355838202022033
    Abstract
  58. ↵
    Jeffares DC, Mourier T, Penny D. 2006. The biology of intron gain and loss. Trends Genet 22: 16–22. doi:10.1016/j.tig.2005.10.006
    CrossRefMedlineWeb of ScienceGoogle Scholar
  59. ↵
    Jia X, Sun C. 2018. Structural dynamics of the N-terminal domain and the Switch loop of Prp8 during spliceosome assembly and activation. Nucleic Acids Res 46: 3833–3840. doi:10.1093/nar/gky242
    CrossRefGoogle Scholar
  60. ↵
    Jurica MS, Moore MJ. 2003. Pre-mRNA splicing: awash in a sea of proteins. Mol Cell 12: 5–14. doi:10.1016/S1097-2765(03)00270-3
    CrossRefMedlineWeb of ScienceGoogle Scholar
  61. ↵
    Keiper S, Papasaikas P, Will CL, Valcárcel J, Girard C, Lührmann R. 2019. Smu1 and RED are required for activation of spliceosomal B complexes assembled on short introns. Nat Commun 10: 3639. doi:10.1038/s41467-019-11293-8
    CrossRefMedlineGoogle Scholar
  62. ↵
    Konarska MM, Sharp PA. 1986. Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs. Cell 46: 845–855. doi:10.1016/0092-8674(86)90066-8
    CrossRefMedlineWeb of ScienceGoogle Scholar
  63. ↵
    Konarska MM, Sharp PA. 1987. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell 49: 763–774. doi:10.1016/0092-8674(87)90614-3
    CrossRefMedlineWeb of ScienceGoogle Scholar
  64. ↵
    Koonin EV. 2006. The origin of introns and their role in eukaryogensis: a compromise solution to the introns-early versus introns-late debate? Biol Direct 1: 22. doi:10.1186/1745-6150-1-22
    CrossRefMedlineGoogle Scholar
  65. ↵
    Lesser CF, Guthrie C. 1993. Mutations in U6 snRNA that alter splice site specificity: implications for the active site. Science 262: 1982–1988. doi:10.1126/science.8266093
    Abstract/FREE Full Text
  66. ↵
    Leung CS, Douglass SM, Morselli M, Obusan MB, Pavlyukov MS, Pellegrini M, Johnson TL. 2019. H3K36 methylation and the chromodomain protein Eaf3 are required for proper cotranscriptional spliceosome assembly. Cell Rep 27: 3760–3769. doi:10.1016/j.celrep.2019.05.100
    CrossRefGoogle Scholar
  67. ↵
    Lipp JL, Marvin MC, Shokat KM, Guthrie C. 2015. SR protein kinases promote splicing of nonconsensus introns. Nat Struct Mol Biol 22: 611–617. doi:10.1038/nsmb.3057
    CrossRefMedlineGoogle Scholar
  68. ↵
    Liu HL, Cheng SC. 2012. The interaction of Prp2 with a defined region of the intron is required for the first splicing reaction. Mol Cell Biol 32: 5056–5066. doi:10.1128/MCB.01109-12
    Abstract/FREE Full Text
  69. ↵
    Liu S, Li X, Zhang L, Jiang J, Hill RC, Cui Y, Hansen KC, Zhou ZH, Zhao R. 2017. Structure of the yeast spliceosomal postcatalytic P complex. Science 358: 1278–1283. doi:10.1126/science.aar3462
    Abstract/FREE Full Text
  70. ↵
    MacMillan AM, Query CC, Allerson CR, Chen S, Verdine GL, Sharp PA. 1994. Dynamic association of proteins with the pre-mRNA branch region. Genes Dev 8: 3008–3020. doi:10.1101/gad.8.24.3008
    Abstract/FREE Full Text
  71. ↵
    MacMillan A, McCaw PS, Crispino JD, Sharp PA. 1997. SC35-mediated reconstitution of splicing in UFAF-depleted nuclear extract. Proc Natl Acad Sci 94: 133–136. doi:10.1073/pnas.94.1.133
    Abstract/FREE Full Text
  72. ↵
    MacRae AJ, Mayerle M, Hrabeta-Robinson E, Chalkley RJ, Guthrie C, Burlingame AL, Jurica MS. 2018. Prp8 positioning of U5 snRNA is linked to 5′ splice site recognition. RNA 24: 769–777. doi:10.1261/rna.065458.117
    Abstract/FREE Full Text
  73. ↵
    Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, Basutkar P, Tivey ARN, Potter SC, Finn RD, et al. 2019. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res 47: 636–641. doi:10.1093/nar/gkz268
    CrossRefMedlineGoogle Scholar
  74. ↵
    Mair G, Shi H, Li H, Djikeng A, Aviles HO, Bishop JR, Falcone FH, Gavrilescu C, Montgomery JL, Santori MI, et al. 2000. A new twist in trypanosome RNA metabolism: cis-splicing of pre-mRNA. RNA 6: 163–169. doi:10.1017/S135583820099229X
    Abstract
  75. ↵
    Maroney PA, Romfo CM, Nilsen TW. 2000. Functional recognition of 5′ splice site by U4/U6.U5 tri-snRNP defines a novel ATP-dependent step in early spliceosome assembly. Mol Cell 6: 317–328. doi:10.1016/S1097-2765(00)00032-0
    CrossRefMedlineWeb of ScienceGoogle Scholar
  76. ↵
    Mathew R, Hartmuth K, Möhlmann S, Urlaub H, Ficner R, Lührmann R. 2008. Phosphorylation of human PRP28 by SRPK2 is required for integration of the U4/U6-U5 tri-snRNP into the spliceosome. Nat Struct Mol Biol 15: 435–443. doi:10.1038/nsmb.1415
    CrossRefMedlineGoogle Scholar
  77. ↵
    Matsuzaki M, Misumi O, Shin-I T, Maruyama S, Takahara M, Miyagishima SY, Mori T, Nishida K, Yagisawa F, Nishida K, et al. 2004. Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature 428: 653–657. doi:10.1038/nature02398
    CrossRefMedlineWeb of ScienceGoogle Scholar
  78. ↵
    Mayerle M, Raghavan M, Ledoux S, Price A, Stepankiw N, Hadjivassiliou H, Moehle EA, Mendoza SD, Pleiss JA, Guthrie C, et al. 2017. Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency. Proc Natl Acad Sci 114: 4739–4744. doi:10.1073/pnas.1701462114
    Abstract/FREE Full Text
  79. ↵
    McPheeters DS, Muhlenkamp P. 2003. Spatial organization of protein-RNA interactions in the branch site-3'splice site region during pre-mRNA splicing in yeast. Mol Cell Biol 23: 4174–4186. doi:10.1128/MCB.23.12.4174-4186.2003
    Abstract/FREE Full Text
  80. ↵
    Medenbach J, Schreiner S, Liu S, Lührmann R, Bindereif A. 2004. Human U4/U6 snRNP recycling factor p110: mutational analysis reveals the function of the tetratricopeptide repeat domain in recycling. Mol Cell Biol 24: 7392–7401. doi:10.1128/MCB.24.17.7392-7401.2004
    Abstract/FREE Full Text
  81. ↵
    Mozaffari-Jovin S, Wandersleben T, Santos KF, Will CL, Lührmann R, Wahl MC. 2013. Inhibition of RNA helicase Brr2 by the C-terminal tail of the spliceosomal protein Prp8. Science 341: 80–84. doi:10.1126/science.1237515
    Abstract/FREE Full Text
  82. ↵
    Mozaffari-Jovin S, Wandersleben T, Santos KF, Will CL, Lührmann R, Wahl MC. 2014. Novel regulatory principles of the spliceosomal Brr2 RNA helicase and links to retinal disease in humans. RNA Biol 11: 298–312. doi:10.4161/rna.28353
    CrossRefMedlineWeb of ScienceGoogle Scholar
  83. ↵
    Nguyen THD, Li J, Galej WP, Oshikane H, Newman AJ, Nagai K. 2013. Structural basis of Brr2-Prp8 interactions and implications for U5 snRNP biogenesis and the spliceosome active site. Structure 21: 910–919. doi:10.1016/j.str.2013.04.017
    CrossRefMedlineGoogle Scholar
  84. ↵
    Nguyen TH, Galej WP, Bai XC, Savva CG, Newman AJ, Scheres SH, Nagai K. 2015. The architecture of the spliceosomal U4/U6.U5 tri-snRNP. Nature 523: 47–52. doi:10.1038/nature14548
    CrossRefMedlineGoogle Scholar
  85. ↵
    Nguyen THD, Galej WP, Bai XC, Oubridge C, Newman AJ, Scheres SHW, Nagai K. 2016. Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution. Nature 530: 298–302. doi:10.1038/nature16940
    CrossRefMedlineGoogle Scholar
  86. ↵
    Ogasawara N, Kohara Y, Kuroiwa T. 2004. Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature 428: 653–657. doi:10.1038/nature02398
    CrossRefMedlineWeb of ScienceGoogle Scholar
  87. ↵
    Ohrt T, Prior M, Dannenberg J, Odenwälder P, Dybov O, Rasche N, Schmitzová J, Gregor I, Fabrizio P, Enderlein J, et al. 2012. Prp2-mediated protein rearrangements at the catalytic core of the spliceosome as revealed by dcFCCS. RNA 18: 1244–1256. doi:10.1261/rna.033316.112
    Abstract/FREE Full Text
  88. ↵
    O'Reilly D, Dienstbier M, Cowley SA, Vazquez P, Drozdz M, Taylor S, James WS, Murphy S. 2013. Differentially expressed, variant U1 snRNAs regulate gene expression in human cells. Genome Res 23: 281–291. doi:10.1101/gr.142968.112
    Abstract/FREE Full Text
  89. ↵
    Plaschka C, Lin PC, Nagai K. 2017. Structure of a pre-catalytic spliceosome. Nature 546: 617–621. doi:10.1038/nature22799
    CrossRefMedlineGoogle Scholar
  90. ↵
    Price SR, Evans PR, Nagai K. 1998. Crystal structure of the spliceosomal U2B”-U2A' protein complex bound to a fragment of U2 small nuclear RNA. Nature 394: 645–650. doi:10.1038/29234
    CrossRefMedlineGoogle Scholar
  91. ↵
    Price AM, Görnemann J, Guthrie C, Brow DA. 2013. An unanticipated early function of DEAD-box ATPase Prp28 during commitment to splicing is modulated by U5 snRNP protein Prp8. RNA 20: 46–60. doi:10.1261/rna.041970.113
    Abstract/FREE Full Text
  92. ↵
    Qiu H, Rossoni AW, Weber APM, Yoon HS, Bhattacharya D. 2018. Unexpected conservation of the RNA splicing apparatus in the highly streamlined genome of Galdieria sulphuraria. BMC Evol Biol 18: 1–11. doi:10.1186/s12862-017-1072-2
    CrossRefGoogle Scholar
  93. ↵
    Raghunathan PL, Guthrie C. 1998a. A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles. Science 279: 857–860. doi:10.1126/science.279.5352.857
    Abstract/FREE Full Text
  94. ↵
    Raghunathan PL, Guthrie C. 1998b. RNA unwinding in U4/U6 snRNPs require ATP hydrolysis and the DEIH-box splicing factor Brr2. Curr Biol 8: 847–855. doi:10.1016/S0960-9822(07)00345-4
    CrossRefMedlineWeb of ScienceGoogle Scholar
  95. ↵
    Rasche N, Dybkov O, Schmitzová J, Akyildiz B, Fabrizio P, Lührmann R. 2012. Cwc2 and its human homologue RBM22 promote an active conformation of the spliceosome catalyic centre. EMBO J 31: 1591–1604. doi:10.1038/emboj.2011.502
    CrossRefMedlineWeb of ScienceGoogle Scholar
  96. ↵
    Rauhut R, Fabrizio P, Dybkov O, Hartmuth K, Pena V, Chari A, Kumar V, Lee C-T, Urlaub H, Kastner B, et al. 2016. Molecular architecture of the Saccharomyces cerevisiae activated spliceosome. Science 353: 1399–1405. doi:10.1126/science.aag1906
    Abstract/FREE Full Text
  97. ↵
    Reimer KA, Stark MR, Aguilar L-C, Stark SR, Burke RD, Moore J, Fahlman RP, Yip CK, Kuroiwa H, Oeffinger M, Rader SD, et al. 2017. The sole LSm complex in Cyanidioschyzon merolae associates with pre-mRNA splicing and mRNA degradation factors. RNA 23: 952–967. doi:10.1261/rna.058487.116
    Abstract/FREE Full Text
  98. ↵
    Robert-Paganin J, Rety S, Leulliot N. 2015. Regulation of DEAH/RHA helicases by G-patch proteins. Biomed Res Int 2015: 931857. doi:10.1155/2015/931857
    CrossRefMedlineGoogle Scholar
  99. ↵
    Roy SW, Bowser BA. 2022. Genomic analysis in Entamoeba reveals intron gain and unbiased intron loss, transformed splicing signals, and a coevolved snRNA. bioRxiv doi:10.1101/2022.06.08.495308
    Abstract/FREE Full Text
  100. ↵
    Roy SW, Gilbert W. 2006. The evolution of spliceosomal introns: patterns, puzzles and progress. Nat Rev Genet 7: 211–221.
    CrossRefMedlineWeb of ScienceGoogle Scholar
  101. ↵
    Roy SW, Irimia M. 2014. Diversity and evolution of spliceosomal systems. Methods Mol Biol 1126: 13–33. doi:10.1007/978-1-62703-980-2_2
    CrossRefMedlineGoogle Scholar
  102. ↵
    Russell CS, Ben-Yehuda S, Dix I, Kupiec M, Beggs D. 2006. Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae. RNA 6: 1565–1572. doi:10.1017/S1355838200000984
    CrossRefGoogle Scholar
  103. ↵
    Sales-Lee J, Perry DS, Bowser BA, Diedrich JK, Rao B, Beusch I, Yates JR III., Roy SW, Madhani HD. 2021. Coupling of spliceosome complexity to intron diversity. Curr Biol 31: 4898–4910.e4. doi:10.1016/j.cub.2021.09.004
    CrossRefGoogle Scholar
  104. ↵
    Sapra AK, Khandelia P, Vijayraghavan U. 2008. The splicing factor Prp17 interactions with U2, U5, and U6 snRNPs and associates with the spliceosome pre- and post-catalysis. Biochem J 416: 365–374. doi:10.1042/BJ20081195
    Abstract/FREE Full Text
  105. ↵
    Sawa H, Abelson J. 1992. Evidence for a base-pairing interaction between U6 small nuclear RNA and 5′ slice site during the splicing reaction in yeast. Proc Natl Acad Sci 89: 11269–11273. doi:10.1073/pnas.89.23.11269
    Abstract/FREE Full Text
  106. ↵
    Schärfen L, Zigackova D, Reimer K, Stark M, Slat V, Francoeur N, Wells M, Zhou L, Blackshear P, Neugebauer K, et al. 2022. Identification of alternative polyadenylation in Cyanidioschyzon merolae through long-read sequencing of mRNA. Front Genet 12: 818697. doi:10.3389/fgene.2021.818697
    CrossRefGoogle Scholar
  107. ↵
    Schellenberg MJ, Edwards RA, Ritchie DB, Kent OA, Golas MM, Stark H, Lührmann R, Glover JN, MacMillan AM. 2006. Crystal structure of a core spliceosomal protein interface. Proc Natl Acad Sci 103: 1266–1271. doi:10.1073/pnas.0508048103
    Abstract/FREE Full Text
  108. ↵
    Schellenberg MJ, Wu T, Ritchie DB, Fica S, Staley JP, Atta KA, LaPointe P, MacMillan AM. 2013. A conformational switch in PRP8 mediates metal ion coordination that promotes pre-mRNA exon ligation. Nat Struct Mol Biol 20: 728–734. doi:10.1038/nsmb.2556
    CrossRefMedlineGoogle Scholar
  109. ↵
    Schütze T, Ulrich AKC, Apelt L, Will CL, Bartlick N, Seeger M, Weber G, Lührmann R, Stelzl U, Wahl MC. 2016. Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome. RNA 22: 265–277. doi:10.1261/rna.054296.115
    Abstract/FREE Full Text
  110. ↵
    Schwer B. 2008. A conformational rearrangement in the spliceosome sets the stage for Prp22-dependent mRNA release. Mol Cell 30: 743–754. doi:10.1016/j.molcel.2008.05.003
    CrossRefMedlineWeb of ScienceGoogle Scholar
  111. ↵
    Schwer B, Gross CH. 1998. Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing. EMBO J 17: 2086–2094. doi:10.1093/emboj/17.7.2086
    Abstract
  112. ↵
    Seraphin B, Rosbash M. 1989. Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing. Cell 59: 349–358. doi:10.1016/0092-8674(89)90296-1
    CrossRefMedlineWeb of ScienceGoogle Scholar
  113. ↵
    Silverman EJ, Maeda A, Wei J, Smith P, Beggs JD, Lin RJ. 2004. Interaction between a G-patch protein and a spliceosomal DEXD/H-box ATPase that is critical for splicing. Mol Cell Biol 24: 10101–10110. doi:10.1128/MCB.24.23.10101-10110.2004
    Abstract/FREE Full Text
  114. ↵
    So BR, Di C, Cai Z, Venters CC, Guo J, Oh JM, Arai C, Dreyfuss G. 2019. A complex of U1 snRNP with cleavage and polyadenylation factors controls telescripting, regulating mRNA transcription in human cells. Mol Cell 76: 590–599.e4. doi:10.1016/j.molcel.2019.08.007
    CrossRefGoogle Scholar
  115. ↵
    Sontheimer E, Steitz J. 1993. The U5 and U5 small nuclear RNAs as active site components of the spliceosome. Science 262: 1989–1996. doi:10.1126/science.8266094
    Abstract/FREE Full Text
  116. ↵
    Spiluttini B, Gu B, Belagal P, Smirnova AS, Nguyen VT, Hébert C, Schmidt U, Bertrand E, Darzacq X, Bensaude O. 2010. Splicing-independent recruitment of U1 snRNP to a transcription unit in living cells. J Cell Sci 123: 2085–2093. doi:10.1242/jcs.061358
    Abstract/FREE Full Text
  117. ↵
    Staley JP, Guthrie C. 1998. Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell 92: 315–326. doi:10.1016/S0092-8674(00)80925-3
    CrossRefMedlineWeb of ScienceGoogle Scholar
  118. ↵
    Stark MR, Dunn EA, Dunn WSC, Grisdale CJ, Daniele AR, Halstead MRG, Fast NM, Rader SD. 2015. Dramatically reduced spliceosome in Cyandioschyzon merolae. Proc Natl Acad Sci 112: 1191–1200.
    Google Scholar
  119. ↵
    Strauss EJ, Guthrie C. 1991. A cold-sensitive mRNA splicing mutant is a member of the RNA helicase gene family. Genes Dev 5: 629–641. doi:10.1101/gad.5.4.629
    Abstract/FREE Full Text
  120. ↵
    Studer MK, Ivanovic L, Weber ME, Marti S, Jonas S. 2020. Structural basis for DEAH-helicase activation by G-patch proteins. Proc Natl Acad Sci 117: 7159–7170. doi:10.1073/pnas.1913880117
    Abstract/FREE Full Text
  121. ↵
    Tarn WY, Steitz JA. 1994. SR proteins can compensate for the loss of U1 snRNP functions in vitro. Genes Dev 8: 2704–2717. doi:10.1101/gad.8.22.2704
    Abstract/FREE Full Text
  122. ↵
    Tholen J, Razew M, Weis F, Galej WP. 2022. Structural basis of branch site recognition by the human spliceosome. Science 375: 50–57. doi:10.1126/science.abm4245
    CrossRefGoogle Scholar
  123. ↵
    Townsend C, Leelaram MN, Agafonov DE, Dybkov O, Will CL, Bertram K, Urlaub H, Kastner B, Stark H, Lührmann R. 2020. Science 370: eabc3753. doi:10.1126/science.abc3753
    Abstract/FREE Full Text
  124. ↵
    Tseng CK, Chung CS, Chen HC, Cheng SC. 2017. A central role of Cwc25 in spliceosome dynamics during catalytic phase of pre-mRNA splicing. RNA 23: 546–556. doi:10.1261/rna.059204.116
    Abstract/FREE Full Text
  125. ↵
    Ulrich AK, Wahl MC. 2017. Human MFAP1 is a cryptic ortholog of the Saccharomyces cerevisiae Spp381 splicing factor. BMC Evol Biol 17: 91. doi:10.1186/s12862-017-0923-1
    CrossRefGoogle Scholar
  126. ↵
    Vazquez-Arango P, Vowles J, Browne C, Hartfield E, Fernandes HJR, Mandefro B, Sareen D, James W, Wade-Martins R, Cowley SA, et al. 2016. Variant U1 snRNAs are implicated in human pluripotent stem cell maintenance and neuromuscular disease. Nucleic Acids Res 44: 10960–10973. doi:10.1093/nar/gkw711
    CrossRefMedlineGoogle Scholar
  127. ↵
    Vosseberg J, Schinkel M, Gremmen S, Snel B. 2022. The spread of the first introns in proto-eukaryotic paralogs. Commun Biol 5: 476. doi:10.1038/s42003-022-03426-5
    CrossRefGoogle Scholar
  128. ↵
    Vosseberg J, Stolker D, von der Dunk SHA, Snel B. 2023. Integrating phylogenetics with intron positions illuminates the origin of the complex spliceosome. Mol Biol Evol 40: msad011. doi:10.1093/molbev/msad011
    CrossRefGoogle Scholar
  129. ↵
    Wang Q, He J, Lynn B, Rymond BC. 2005. Interactions of the yeast SF3b splicing factor. Mol Cell Biol 25: 10745–10754. doi:10.1128/MCB.25.24.10745-10754.2005
    Abstract/FREE Full Text
  130. ↵
    Warkocki Z, Schneider C, Mozaffari-Jovin S, Schmitzova J, Hobartner C, Fabrizio P, Lührmann R. 2015. The G-patch protein Spp2 couples the spliceosome-stimulated ATPase activity of the DEAH-box protein Prp2 to catalytic activation of the spliceosome. Genes Dev 29: 94–107. doi:10.1101/gad.253070.114
    Abstract/FREE Full Text
  131. ↵
    Wassarman DA, Steitz JA. 1992. Interactions of small nuclear RNA's with precursor messenger RNA during in vitro splicing. Science 257: 1918–1925. doi:10.1126/science.1411506
    Abstract/FREE Full Text
  132. ↵
    West S. 2012. The increasing functional repertoire of U1 snRNA. Biochem Soc Trans 40: 846–849. doi:10.1042/BST20120058
    Abstract/FREE Full Text
  133. ↵
    Wilkinson CR, Dittmar GA, Ohi MD, Uetz P, Jones N, Finley D. 2004. Ubiquitin-like protein Hub1 is required for pre-mRNA splicing and localization of an essential splicing factor in fission yeast. Curr Biol 14: 2283–2288. doi:10.1016/j.cub.2004.11.058
    CrossRefMedlineGoogle Scholar
  134. ↵
    Wilkinson ME, Fica SM, Galej WP, Norman CM, Newman AJ, Nagai K. 2017. Postcatalytic spliceosome structure reveals mechanism of 3'-splice site selection. Science 358: 1283–1288. doi:10.1126/science.aar3729
    Abstract/FREE Full Text
  135. ↵
    Wilkinson ME, Fica SM, Galej WP, Nagai K. 2021. Structural basis for conformational equilibrium of the catalytic spliceosome. Mol Cell 81: 1439–1452. doi:10.1016/j.molcel.2021.02.021
    CrossRefGoogle Scholar
  136. ↵
    Will CL, Lührmann R. 2011. Spliceosome structure and function. Cold Spring Harb Perspect Biol 3: a003707. doi:10.1101/cshperspect.a003707
    Abstract/FREE Full Text
  137. ↵
    Wollenhaupt J, Henning LM, Sticht J, Becke C, Freund C, Santos KF, Wahl MC. 2018. Intrinsically disordered protein Ntr2 modulates the spliceosomal RNA helicase Brr2. Biophys J 114: 788–799. doi:10.1016/j.bpj.2017.12.033
    CrossRefMedlineGoogle Scholar
  138. ↵
    Wong D, Stark M, Rader S, Fast N. 2021. Characterization of pre-mRNA splicing and spliceosomal machinery in Porphyridium purpureum and evolutionary implications for red algae. J Eukaryot Microbiol e12844. doi:10.1111/jeu.12844
    CrossRefGoogle Scholar
  139. ↵
    Wong D, Grisdale C, Slat V, Rader S, Fast N. 2022. The evolution of pre-mRNA splicing and its machinery revealed by reduced extremophilic red algae. J Eukaryot Microbiol 70: e12927. doi:10.1111/jeu.12927
    CrossRefGoogle Scholar
  140. ↵
    Wu T, Fu X-D. 2015. Genomic functions of U2AF in constitutive and regulated splicing. RNA Biol 12: 479–485. doi:10.1080/15476286.2015.1020272
    CrossRefMedlineGoogle Scholar
  141. ↵
    Xie J, Beickman K, Otte E, Rymond BC. 1998. Progression through the spliceosome cycle requires Prp38 function for U4/U6 snRNA dissociation. EMBO J 17: 2938–2946. doi:10.1093/emboj/17.10.2938
    Abstract/FREE Full Text
  142. ↵
    Yan D, Ares M. 1996. Invariant U2 sequences bordering the branchpoint interaction region are essential for interaction with SF3a and SF3b subunits. Mol Cell Biol 16: 818–828. doi:10.1128/MCB.16.3.818
    Abstract/FREE Full Text
  143. ↵
    Yan C, Hang J, Wan R, Huang M, Wong CCL, Shi Y. 2015. Structure of a yeast spliceosome at 3.6-Å resolution. Science 349: 1182–1191. doi:10.1126/science.aac7629
    Abstract/FREE Full Text
  144. ↵
    Yan C, Wan R, Bai R, Huang G, Shi Y. 2016. Structure of a yeast activated spliceosome at 3.5 Å resolution. Science 353: 904–911. doi:10.1126/science.aag0291
    Abstract/FREE Full Text
  145. ↵
    Yeh TC, Liu HL, Chung CS, Wu NY, Liu YC, Cheng SC. 2011. Splicing factor Cwc22 is required for the function of Prp2 and for the spliceosome to escape from a futile pathway. Mol Cell Biol 31: 43–53. doi:10.1128/MCB.00801-10
    Abstract/FREE Full Text
  146. ↵
    Yoon HS, Hackett JD, Ciniglia C, Pinto G, Bhattacharya D. 2004. A molecular timeline for the origin of photosynthetic eukaryotes. Mol Biol Evol 21: 809–818. doi:10.1093/molbev/msh075
    CrossRefMedlineWeb of ScienceGoogle Scholar
  147. ↵
    Zhan X, Yan C, Zhang X, Lei J, Shi Y. 2018a. Structure of a human catalytic step I spliceosome. Science 359: 537–545. doi:10.1126/science.aar6401
    Abstract/FREE Full Text
  148. ↵
    Zhan X, Yan C, Zhang X, Lei J, Shi Y. 2018b. Structures of the human pre-catalytic spliceosome and its precursor splicesosome. Cell Res 28: 1129–1140. doi:10.1038/s41422-018-0094-7
    CrossRefMedlineGoogle Scholar
  149. ↵
    Zhang X, Yan C, Hang J, Finci LI, Lei J, Shi Y. 2017. An atomic structure of the human spliceosome. Cell 169: 918–929. doi:10.1016/j.cell.2017.04.033
    CrossRefMedlineGoogle Scholar
  150. ↵
    Zhang X, Zhan X, Yan C, Zhang W, Liu D, Lei J, Shi Y. 2019. Structures of the human spliceosomes before and after release of the ligated exon. Cell Res 29: 274–285. doi:10.1038/s41422-019-0143-x
    CrossRefMedlineGoogle Scholar
  151. ↵
    Zhang S, Aibara S, Vos SM, Agafonov DE, Lührmann R, Cramer P. 2021. Structure of a transcribing RNA polymerase II–U1 snRNP complex. Science 371: 305–309. doi:10.1126/science.abf1870
    Abstract/FREE Full Text
  152. ↵
    Zuker M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 3: 3406–3415. doi:10.1093/nar/gkg595
    CrossRefGoogle Scholar
  153. ↵
    Zuo P, Maniatis T. 1996. The splicing factor U2AF35 mediats critical protein-protein interactions in constitutive and enhancer-dependent splicing. Genes Dev 10: 1356–1368. doi:10.1101/gad.10.11.1356
    Abstract/FREE Full Text
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