BACKGROUND: Nuclear introns in Euglenida have been understudied. This study aimed to investigate nuclear introns in Euglenida by identifying a large number of introns in Euglena gracilis (E. gracilis), including cis-spliced conventional and nonconventional introns, as well as trans-spliced outrons. We also examined the sequence characteristics of these introns.
RESULTS: A total of 28,337 introns and 11,921 outrons were identified. Conventional and nonconventional introns have distinct splice site features; the former harbour canonical GT/C-AG splice sites, whereas the latter are capable of forming structured motifs with their terminal sequences. We observed that short introns had a preference for canonical GT-AG introns. Notably, conventional introns and outrons in E. gracilis exhibited a distinct cytidine-rich polypyrimidine tract, in contrast to the thymidine-rich tracts observed in other organisms. Furthermore, the SL-RNAs in E. gracilis, as well as in other trans-splicing species, can form a recently discovered motif called the extended U6/5\' ss duplex with the respective U6s. We also describe a novel type of alternative splicing pattern in E. gracilis. The tandem repeat sequences of introns in this protist were determined, and their contents were comparable to those in humans.
CONCLUSIONS: Our findings highlight the unique features of E. gracilis introns and provide insights into the splicing mechanism of these introns, as well as the genomics and evolution of Euglenida.

Human pre-mRNA splicing requires the removal of introns with highly variable lengths, from tens to over a million nucleotides. Therefore, mechanisms of intron recognition and splicing are likely not universal. Recently, we reported that splicing in a subset of human short introns with truncated polypyrimidine tracts depends on RBM17 (SPF45), instead of the canonical splicing factor U2 auxiliary factor (U2AF) heterodimer. Here, we demonstrate that SAP30BP, a factor previously implicated in transcriptional control, is an essential splicing cofactor for RBM17. In vitro binding and nuclear magnetic resonance analyses demonstrate that a U2AF-homology motif (UHM) in RBM17 binds directly to a newly identified UHM-ligand motif in SAP30BP. We show that this RBM17-SAP30BP interaction is required to specifically recruit RBM17 to phosphorylated SF3B1 (SF3b155), a U2 small nuclear ribonucleoprotein (U2 snRNP) component in active spliceosomes. We propose a mechanism for splicing in a subset of short introns, in which SAP30BP guides RBM17 in the assembly of active spliceosomes.

Among eukaryotes, the major spliceosomal pathway is highly conserved. While long introns may contain additional regulatory sequences, the ones in short introns seem to be nearly exclusively related to splicing. Although these regulatory sequences involved in splicing are well-characterized, little is known about their evolution. At the 3\' end of introns, the splice signal nearly universally contains the dimer AG, which consists of purines, and the polypyrimidine tract upstream of this 3\' splice signal is characterized by over-representation of pyrimidines. If the over-representation of pyrimidines in the polypyrimidine tract is also due to avoidance of a premature splicing signal, we hypothesize that AG should be the most under-represented dimer. Through the use of DNA-strand asymmetry patterns, we confirm this prediction in fruit flies of the genus Drosophila and by comparing the asymmetry patterns to a presumably neutrally evolving region, we quantify the selection strength acting on each motif. Moreover, our inference and simulation method revealed that the best explanation for the base composition evolution of the polypyrimidine tract is the joint action of purifying selection against a spurious 3\' splice signal and the selection for pyrimidines. Patterns of asymmetry in other eukaryotes indicate that avoidance of premature splicing similarly affects the nucleotide composition in their polypyrimidine tracts.

Long introns with short exons in vertebrate genes are thought to require spliceosome assembly across exons (exon definition), rather than introns, thereby requiring transcription of an exon to splice an upstream intron. Here, we developed CoLa-seq (co-transcriptional lariat sequencing) to investigate the timing and determinants of co-transcriptional splicing genome wide. Unexpectedly, 90% of all introns, including long introns, can splice before transcription of a downstream exon, indicating that exon definition is not obligatory for most human introns. Still, splicing timing varies dramatically across introns, and various genetic elements determine this variation. Strong U2AF2 binding to the polypyrimidine tract predicts early splicing, explaining exon definition-independent splicing. Together, our findings question the essentiality of exon definition and reveal features beyond intron and exon length that are determinative for splicing timing.

Diverse elements within the 5\' untranslated region of an mRNA can influence the translation efficiency at the main AUG codon. We previously identified a core picornaviral like Y16X11-AUG motif with 16-nt polypyrimidine CU tract separated by an 11-nt spacer sequence from the 13th AUG codon, which is recognized as the preferred initiation site within the Triticum mosaic virus (TriMV) internal ribosome entry site (IRES) element. The motif is proposed to function as an internal ribosomal landing site at the designated start codon. Here, we exposed the cooperative role of multiple CU-rich segments flanking the TriMV YX-AUG motif to reach and drive internal initiation of translation at the preferred start site. We propose that these auxiliary domains may enhance the ribosome capacity and their delivery at proximity of the correct initiation site. These polypyrimidine tracts can be modulated with a cryptic AUG in a position-dependent manner to replace the native YX-AUG motif, and thus uncovering a new layer of control of start codon selection. In line with these observations, mass spectrometry analysis of proteins directly interacting with translationally impaired TriMV IRES mutants that bear these motifs indicated an enrichment in 40S and 60S ribosomal related proteins, revealing a new function of polypyrimidine tracts to regulate IRES-driven translation. Accessibility of these RNA regions for in trans interaction was validated by SHAPE analysis of the entire TriMV leader sequence and supported by the ability of anti-sense oligonucleotides designed to block the CU tracts accessibility to impair IRES activity. This is the first evidence that defines the core modular domains required for ribosomal recruitment and start codon selection in a complex, multi-AUG viral 5\' UTR for translation in plants.

SF3B1, an essential RNA splicing factor, is frequently mutated in various types of cancers, and the cancer-associated SF3B1 mutation causes aberrant RNA splicing. The aberrant splicing of several transcripts, including MAP3K7, promotes tumorigenesis. Here, we identify a premature termination codon in the aberrantly spliced transcript of MAP3K7. Treatment of HEK293T cells transfected with the K700E-mutated SF3B1 with cycloheximide leads to increased accumulation of the aberrant spliced transcript of MAP3K7, demonstrating that the aberrantly spliced transcript of MAP3K7 is targeted by nonsense-mediated decay. The aberrantly spliced MAP3K7 transcript uses an aberrant 3\' splice sites and an alternative branchpoint sequence. In addition, the aberrant splicing of MAP3K7 requires not only the polypyrimidine tract associated with normal splicing but also an alternative polypyrimidine tract upstream of the aberrant 3\' splice site. Other cancer-associated SF3B1 mutations also cause the aberrant splicing of MAP3K7, which depends on the same sequence features. Our data provide a further understanding of the mechanisms underlying aberrant splicing induced by cancer-associated SF3B1 mutation, and reveal an important role of alternative polypyrimidine tract in diseases.

Artificial insemination in pig (Sus scrofa domesticus) breeding involves the evaluation of the semen quality of breeding boars. Ejaculates that fulfill predefined quality requirements are processed, diluted and used for inseminations. Within short time, eight Swiss Large White boars producing immotile sperm that had multiple morphological abnormalities of the sperm flagella were noticed at a semen collection center. The eight boars were inbred on a common ancestor suggesting that the novel sperm flagella defect is a recessive trait. Transmission electron microscopy cross-sections revealed that the immotile sperm had disorganized flagellar axonemes. Haplotype-based association testing involving microarray-derived genotypes at 41,094 SNPs of six affected and 100 fertile boars yielded strong association (P = 4.22 × 10-15) at chromosome 12. Autozygosity mapping enabled us to pinpoint the causal mutation on a 1.11 Mb haplotype located between 3,473,632 and 4,587,759 bp. The haplotype carries an intronic 13-bp deletion (Chr12:3,556,401-3,556,414 bp) that is compatible with recessive inheritance. The 13-bp deletion excises the polypyrimidine tract upstream exon 56 of DNAH17 (XM_021066525.1: c.8510-17_8510-5del) encoding dynein axonemal heavy chain 17. Transcriptome analysis of the testis of two affected boars revealed that the loss of the polypyrimidine tract causes exon skipping which results in the in-frame loss of 89 amino acids from DNAH17. Disruption of DNAH17 impairs the assembly of the flagellar axoneme and manifests in multiple morphological abnormalities of the sperm flagella. Direct gene testing may now be implemented to monitor the defective allele in the Swiss Large White population and prevent the frequent manifestation of a sterilizing sperm tail disorder in breeding boars.

Clinical exome sequencing is frequently used to identify gene-disrupting variants in individuals with neurodevelopmental disorders. While splice-disrupting variants are known to contribute to these disorders, clinical interpretation of cryptic splice variants outside of the canonical splice site has been challenging. Here, we discuss papers that improve such detection.

RNA splicing is a critical mechanism by which to modify transcriptome, and its dysregulation is the underlying cause of many human diseases. It remains challenging, however, to genetically modulate a splicing event in its native context. Here, we demonstrate that a CRISPR-guided cytidine deaminase (i.e., targeted-AID mediated mutagenesis [TAM]) can efficiently modulate various forms of mRNA splicing. By converting invariant guanines to adenines at either 5\' or 3\' splice sites (SS), TAM induces exon skipping, activation of alternative SS, switching between mutually exclusive exons, or targeted intron retention. Conversely, TAM promotes downstream exon inclusion by mutating cytidines into thymines at the polypyrimidine tract. Applying this approach, we genetically restored the open reading frame and dystrophin function of a mutant DMD gene in patient-derived induced pluripotent stem cells (iPSCs). Thus, the CRISPR-guided cytidine deaminase provides a versatile genetic platform to modulate RNA splicing and to correct mutations associated with aberrant splicing in human diseases.

Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3\' splice site. In the nematode Caenorhabditis elegans, 3\' splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in Caenorhabditis elegans, we have isolated a strain containing a point mutation in the octamer motif of a 3\' splice site in the daf-12 gene. This mutation, a single base T-to-G transversion at the -5 position relative to the splice site, causes a strong daf-12 loss-of-function phenotype by abrogating splicing. The resulting transcript is predicted to encode a truncated DAF-12 protein generated by translation into the retained intron, which contains an in-frame stop codon. Other than the perfectly conserved AG dinucleotide at the site of splicing, G at the -5 position of the octamer motif is the most uncommon base in C. elegans 3\' splice sites, occurring at closely paired sites where the better match to the splicing consensus is a few bases downstream. Our results highlight both the biological importance of the highly conserved -5 uridine residue in the C. elegans 3\' splice site octamer motif as well as the utility of using ENU as a mutagen to study the function of polypyrimidine tracts and other AU- or AT-rich motifs in vivo.