The unfolding landscape of RNA and disease

The diversity of fundamental functions that RNA molecules carry out in living cells is truly overwhelming. Their molecular roles span from being universal conveyors of genetic information, to catalyzing key processes such as protein synthesis or RNA splicing, to serving as molecular scaffolds for macromolecular assemblies and condensates that help to organize and regulate cellular functions. Not surprisingly, alterations in RNA metabolism are at the root of a wide (and expanding) variety of pathologies. Given the intense research efforts and volume of literature on the topic of RNA and disease, our aim for this Special Issue has been to recruit inspiring leaders in this area and ask them to provide their visions of the evolution of the field and forecast the future, encouraging them to provide their own opinions and personal perspectives.

It has long been assumed that a detailed understanding of mechanisms involved in RNA transactions will pave the way to the development of novel therapies. How this assumption is becoming a reality in recent years was powerfully shown in the previous Special Issue on “RNA Therapeutics,” coordinated by Michelle Hastings and Adrian Krainer (April 2023) and is further illustrated in many of the chapters of this one. We may be indeed at a unique juncture for the development of RNA-focused therapies, armed with unprecedented technical capacities, deep mechanistic knowledge, boldness triggered by some early spectacular successes, a booming industry and even social awareness of the wonders that RNA has in store.

The articles in this Special Issue provide striking examples—many of which likely to surprise even seasoned connoisseurs—of how even subtle alterations in RNA, its modifications and its dynamic interactions with other macromolecules induce pathological states affecting very different organs and systems, often in unexpected ways. The articles cover a wide breadth of topics and are also diverse in purpose and style.

Gideon Dreyfuss provides a lively personal reflection of how his lab's thinking stream captured, often from serendipitous observations followed for curiosity's sake, some of the earliest links between RNA-binding proteins and disease, including spinal muscular atrophy (SMA) and the fragile X mental retardation syndrome. And how even low-tech approaches (e.g., color pencils), obsession with an important question regardless of feasibility or fashion, and a flexible mindset can lead to powerful insights. It was difficult to predict, at the time that the Dreyfuss lab first described the family of hnRNP proteins coating nascent RNAs, that these assemblies are in fact the real substrate for all the processing steps, cellular voyages that RNAs endure in eukaryotic cells and the determinants of their fates. And even more difficult to predict was that disruption of one specific such interaction by an antisense oligonucleotide (ASO) would provide the first efficacious therapy for children with SMA.

Unexpected discoveries continue to reshape our understanding of RNA biology, revealing new and surprising links to disease. One such breakthrough was the identification in the 1990s of the minor spliceosome, challenging the previous long-standing view that all splicing was catalyzed by a single, evolutionarily conserved spliceosome. The revelation that most eukaryotes have two distinct classes of introns, each requiring a dedicated splicing machinery, was both unanticipated and intriguing. Antto Norppa, Mariia Shcherbii, and Mikko Frilander discuss how recent studies are uncovering the mechanisms driving minor spliceosomopathies, that is, diseases caused by dysfunction of the minor spliceosome. Despite its low cellular abundance, comprising just 1% of the major spliceosome and processing fewer than 0.5% of all introns, the minor spliceosome plays an essential role in human physiology. Mutations in its core components, including both snRNAs and associated proteins, underlie several developmental disorders and may represent a novel cancer vulnerability.

Another striking example involves the splicing factor RBM20, an RNA-binding protein that regulates pre-mRNA splicing. Mutations in RBM20 are associated with dilated cardiomyopathy, an inherited heart condition. However, as discussed by Zachery Gregorich and Wei Guo, disease mechanisms extend beyond the expected disruption of mRNA splicing. While knocking out the Rbm20 gene in mice leads to mRNA missplicing in cardiac cells, patient-derived RBM20 mutations primarily impair the protein's nuclear localization signal. This mislocalization results in the formation of cytoplasmic granules, suggesting that RBM20 mutations may drive disease through toxic protein aggregation, a mechanism that parallels pathological processes seen in neurological disorders, such as amyotrophic lateral sclerosis and Alzheimer's disease. Protein aggregation through the lens of RNA in biological condensates was covered in a previous Special Issue of RNA coordinated by Tom Cech (January 2022).

Suna Jung and Joel Richter highlight an additional surprising breakthrough that emerged from testing a long-held hypothesis about fragile X syndrome (FXS). Scientists initially sought to confirm that FMRP, the protein encoded by the FMR1 gene implicated in FXS, stalls ribosomes on specific mRNAs. Unexpectedly, they discovered widespread dysregulation of RNA splicing. Subsequent studies confirmed that splicing dysregulation is a hallmark of fragile X syndrome. Remarkably, splice-switching ASOs reduced FMR1 missplicing, rescued full-length FMR1 RNA and restored FMRP to normal levels, suggesting that ASOs could represent a novel therapeutic approach for treating FXS and other related disorders.

RNA splicing is frequently dysregulated in cancer cells, often driven by somatic mutations in core splicing factors such as SF3B1. Pedro Bak-Gordon and James Manley explain how SF3B1 mutations disrupt splicing fidelity, resulting in aberrant transcripts that drive cancer development. The authors highlight that not all SF3B1 mutations have the same impact, emphasizing distinct prognostic outcomes based on how the mutations alter SF3B1's interaction with another splicing factor, SUGP1. This example illustrates the close connections that can occur between disease etiology and specific molecular interactions within highly complex RNA processing machineries.

Both alternative splicing and nonsense or frameshift mutations can generate premature termination codons (PTCs) in mRNAs, influencing physiological processes or contributing to disease by altering protein expression. Mary McMahon and Lynne Maquat explore the therapeutic potential of targeting nonsense-mediated mRNA decay (NMD), a cellular quality control mechanism that detects and degrades mRNAs containing PTCs. Strategies under development include splicing modulation to circumvent disease-causing mutations that introduce PTCs, bypassing translation termination at PTCs, damping NMD activity, and engineering tRNAs for nonsense suppression.

Mirroring the concept of protein gain-of-function mutations, where an abnormal gene product acquires new or enhanced activity that contributes to disease, RNA gain of function involves the RNA itself becoming pathogenic by acquiring novel properties or interacting aberrantly with cellular components. Mackenzie Davenport and Maurice Swanson offer insights into RNA gain of function associated with the abnormal expansion of short tandem repeats, a mechanism implicated in multiple developmental and degenerative diseases, including myotonic dystrophy.

After transcription, the nucleotide sequence of RNA molecules can be altered by RNA editing, resulting in transcripts that differ from the original DNA template. Kasra Tamizkar and Michael Jantsch present recent advances in understanding RNA editing, particularly the adenosine-to-inosine (A-to-I) conversion mediated by ADAR enzymes. The discovery that ADAR1-mediated editing is essential for distinguishing self from non-self RNA spurred numerous studies linking dysregulated ADAR1 activity to immune disorders and inflammatory diseases. ADAR1 has also emerged as a promising target for cancer immunotherapy. Despite the development of multiple pipelines to detect inosine residues across transcriptomes, the frequency and extent of editing events at the single-molecule level remain poorly characterized. Additionally, the question of whether altered RNA editing is a driver or a consequence of disease continues to be a subject of debate.

RNA sensors have evolved as components of innate immune responses to detect viral replication intermediates and activate the expression of inflammatory cytokines and type I interferon-stimulated genes. Sandra G. Williams, Soyeong Sim, and Sandy Wolin provide a comprehensive perspective of how aberrant RNA sensing by a complex network of Toll-like, RIG-I-like receptors, and other sensor molecules that recognize foreign RNAs, breaks immune tolerance to host RNAs and leads to both autoimmune (involving the adaptive immune system) and autoinflammatory (involving the innate immune system) diseases such as systemic lupus erythematosus or Aicardi Goutières syndrome. Alterations in the components of these sensor systems, which include not only germline mutations but also somatic mutations in immune cells, may be behind a broad spectrum of diseases that reflect the dense communication between the innate and adaptive immune systems.

Alike the impact that histone modifications have had to understand all aspects of DNA metabolism, the discovery of over 170 chemical modifications in RNA molecules (the epitranscriptome) is transforming our views of RNA metabolism, as recently discussed in a Special Issue of RNA coordinated by Kate Meyer and Tao Pan (May 2024). Chujo and Tomizawa discuss key roles of chemical modifications on the stability and decoding properties of mitochondrial tRNAs (mt-tRNAs), some of which—along the enzymes involved—have become understood in great detail. Such modifications are particularly important for the expression of essential ATP-producing respiratory complex proteins, and loss of mt-tRNA modifications leads to a variety of disease conditions in tissues with high ATP consumption, for example, encephalopathies and cardiomyopathies. Envisioned therapeutic approaches range from targeted deletion of mutant mtDNA by nucleases (thus increasing the ratio of wild type versus mutant mt-tRNAs over the pathological threshold), to taurine supplementation in diseases associated with decreased 5-taurinomethyluridine modifications in the wobble position of certain mt-tRNAs. Development of a humanized mouse model, however, remains an urgent need to progress toward cures for these pathologies.

The epitranscriptome paradigm is also powerfully illustrated by Liangliang Wang, Ralph R. Weichselbaum, and Chuan He's perspective article, which focuses on the functions and alterations of members of a family of proteins (YTHDF) that recognize the most abundant internal (non-cap) RNA modification, N⁶-methyladenosine (m⁶A). YTHDF2, the first identified m⁶A reader, helps to degrade mRNAs by recruiting components of the decay machineries and is responsible for key transcriptome switches occurring during development, stem cell and neural differentiation. YTHDF2 also coordinates the function of multiple types of immune cells and its role as suppressor of antitumor immunity by myeloid-derived suppressor cells has become a focus of attention. Upregulated upon cancer treatment, its levels correlate with poor prognosis and YTHDF2 inhibition might help to overcome resistance to radioimmunotherapy and prevent metastatic disease, two of the main roadblocks in cancer therapeutics. The identification of an inhibitor of m⁶A recognition specific for YTHDF2 suggests that it can indeed become a pharmaceutical target.

Along similar lines, Mercedes Álvarez and Raúl Méndez provide a comprehensive overview of the multitude of biological functions, pathological alterations, and opportunities for therapeutical intervention provided by proteins of the CPEB family. These proteins recognize cytoplasmic polyadenylation element (CPE) sequences at the 3′ UTR of mRNAs and thus control the length of poly(A) tails, which in turn influences mRNA decay and translation, promoting or repressing gene expression depending on the cellular context. Alterations in the fine balance between CPEB-mediated regulation and other factors impact on a variety of neurological conditions spanning from neurodegenerative disorders to autism, cancer, immune regulation, inflammation, liver, and metabolic disease. This variety of CPEBs’ functions may reflect the evolutionary need to overcome physiological constraints of transcriptional regulation. This complexity represents challenges but can also offer opportunities for therapeutic intervention by precisely targeting the functional antagonism between CPEB isoforms.

mRNA translation is also finely tuned by signaling pathways that target core components of the translation machinery. Mehdi Amiri, Niaz Mahmood, Soroush Tahmasebi, and Nahum Sonenberg dive into these intricate axes to provide exciting examples of how leveraging detailed knowledge of these (often ambidextrous) regulatory events can help to design effective anticancer therapies. Translational control is indeed key for virtually every cancer hallmark, from the modulation of cancer cell metabolism, heterogeneity, or microenvironment to influencing chemotherapy resistance or immune evasion in a variety of tumor types. Strategic targeting of components of the eIF4F complex, which channels the 43S preinitiation complex to the mRNA 5′ cap structure, can enhance the effects of conventional treatments and overcome resistance, particularly when combined with inhibitors of mTOR or MEK pathways. Identification of critical mRNA targets using ribosome profiling and CRISPR-based screens should help to develop more specific and efficacious combinatorial therapies.

RNA viruses are a classical example of pathogenic effects mediated by RNA molecules. Esteban Domingo and colleagues discuss persuasive evidence that any virus isolate is in fact an assembly of different RNA sequence variants (quasispecies). This nonequilibrium population diversity is maintained (indeed, selected) by the relatively high rate of nucleotide misincorporation of RNA-dependent RNA polymerases, as well as by the generation of insertion and deletion mutants that lead to pools of defective genomes. This is the case even for viral species, such as SARS-CoV-2, that count on replication proofreading mechanisms. This intrinsic feature is fundamental for all aspects of the viral life cycle, evolution, and pathogenesis, including the infection of new hosts, cell tropism, evading the host's immune response, vaccine failures, or the emergence of pandemic outbreaks. Such concepts may well go beyond understanding the epidemiology of viral infections and be expanded to other pathological contexts such as cancer evolution. They have also inspired therapeutic approaches, such as viral extinction, by enhancing variability beyond the limit of error catastrophe predicted by quasispecies theory.

An example of the sophisticated arms race between host cells and RNA viruses is illustrated in the article by Alfredo Castello and Wael Kamel focusing on mechanisms that control nuclear-cytoplasmic communication. Thus, cytoplasmic viruses hamper the function of the nuclear pore complexes to avoid the import of transcription factors and export of mRNAs involved in antiviral responses, while interferon γ antagonizes these effects by enhancing the expression of proteins (e.g., nucleoporins) targeted by the virus. Recent publications have revealed the antiviral effects of relocation of nuclear RNA-binding proteins to the cytoplasm, including DDX39A helicase and the splicing complex U2 snRNP. While the molecular basis for the antiviral effects of these factors remains to be determined, their interactions with viral RNAs in cytoplasmic factories are likely to interfere with viral replication, as illustrated by massive enhancement of certain viral infections in cells treated with U2 snRNP inhibitors.

The collective message of the articles in this Special Issue is that detailed pathogenic mechanisms of RNA-related diseases can be understood to the point of explaining diverse etiologies and allowing the rational design of innovative therapeutics. The authors provide abundant evidence that investments in basic research, in terms of resources, time, and scholar depth, often in areas orthogonal to those directly involved in disease generation, are the most fruitful avenues to advance toward effective cures.

We sincerely thank all the colleagues who generously contributed articles to this Special Issue, and we are particularly grateful to Ann Marie Micenmacher for her exceptional efficiency and gracious support throughout the process.

We are grateful to Tim Nilsen for his vision and encouragement to put together a Special Issue on RNA and Disease. We are sad to note that Tim very recently passed away, and we dedicate this Special Issue to his memory. Tim was a visionary and pillar of the RNA research community. As the founder of RNA journal 30 years ago and its long-standing Editor-in-Chief, Tim's tireless dedication and leadership profoundly shaped and inspired the field. We owe Tim an immense debt of gratitude for decades of support and insightful guidance.

• © 2025 Carmo-Fonseca and Valcárcel; Published by Cold Spring Harbor Laboratory Press for the RNA Society