(A) Cartoon of a hypothetical T. brucei minicircle showing the typical positions of the major cassettes relative to the conserved sequence region (CSR). The CSR contains the highly conserved CSB-1 and CSB-3 motifs and the less well conserved CSB-2. Upstream of the CSR are four to five A-tracts, each ∼5 bp long and positioned roughly in phase with the helical repeat. The major cassettes are labeled (in 5′ to 3′ order) I, II, IV, and V and are on average 142 nt long. A minor cassette III exists between cassettes II and IV, on a few minicircles. Cassettes are defined by a forward and reverse pair of imperfect 18 bp repeats (dark gray segments). Many cassettes encode canonical gRNA genes that can identifiably align with edited mRNA. Guide RNAs are usually encoded on the sense strand (e.g., in cassettes I, IV, and orphan) or rarely on the antisense strand (e.g., in cassette II). Cassettes also encode noncanonical genes of unknown, if any, function that do not identifiably match edited mRNA without gaps (e.g., in cassettes IV and V). Canonical genes have an ∼11 nt anchor region (magenta segments) and a ∼31 nt guiding region (green segments) that direct U-insertion/deletion. Some gRNA genes are not encoded within a cassette. These so-called orphan gRNA genes have only been found between cassettes II and IV on the sense strand. Sequencing and alignment of small RNA transcripts allow determination of canonical and noncanonical gRNA gene expression status and identification of 5 nt transcription initiation sites (blue segments in cassettes II, IV, and the orphan). Initiation sequences are 30–32 nt downstream from the forward 18 bp repeat of sense strand encoded genes and 30–32 nt upstream of the reverse 18 bp repeat of antisense strand encoded genes. Gene extent (shown as orange rectangles) is either determined by alignment of transcripts for expressed genes (e.g., in cassettes II, IV, and orphan), or the complementarity to edited mRNA for nonexpressed canonical genes (e.g., in cassette I) or is not defined for nonexpressed noncanonical genes (e.g., in cassette V). (B) Structure of 398 assembled minicircles ordered by length (rightmost brown line). Magenta, brown, and purple represent conserved sequence blocks CSB-1, CSB-2, and CSB-3, respectively. A cassette is shown as light gray, with flanking 18 bp inverted repeats as dark gray. Cassette-associated and orphan expressed canonical and noncanonical gRNAs genes are shown in orange. The labels for the five gRNA cassette positions, I–V, are located at each cassette's median center position. Black shows A-tracts of the bend region. Reproduced from Cooper et al. (2019) with minor modifications.
