Splice Site Usage Research Articles

Identification and analysis of short indels inducing exon extension/shrinkage events.

The search for genetic variants that act as causative factors in human diseases by disrupting the normal splicing process has primarily focused on single nucleotide variants (SNVs). It is worth noting that insertions or deletions (indels) have also been sporadically reported as causative disease variants through their potential impact on the splicing process. In this study, to perform identification of indels inducing exon extension/shrinkage events, we used individual-specific genomes and RNA sequencing (RNA-seq) data pertaining to the corresponding individuals and identified 12 exon extension/shrinkage events that were potentially induced by indels that disrupted authentic splice sites or created novel splice sites in 235 normal individuals. By evaluating the impact of these abnormal splicing events on the resulting transcripts, we found that five events led to the generation of premature termination codons (PTCs), including those occurring within genes associated with genetic disorders. Our analysis revealed that the potential functions of indels have been underexamined, and it is worth considering the possibility that indels may affect splice site usage, using RNA-seq data to discover novel potentially disease-associated mutations.

The interferon-regulated host factor hnRNPA0 modulates HIV-1 production by interference with LTR activity, mRNA trafficking, and programmed ribosomal frameshifting.

The interplay between host factors and viral components impacts viral replication efficiency profoundly. Members of the cellular heterogeneous nuclear ribonucleoprotein family (hnRNPs) have been extensively studied as HIV-1 host dependency factors, but whether they play a role in innate immunity is currently unknown. This study aimed to identify hnRNPA0 as a type I interferon (IFN)-repressed host factor in HIV-1-infected cells. Knockdown of hnRNPA0, a situation that mirrors conditions under IFN stimulation, increased LTR activity, export of unspliced HIV-1 mRNA, viral particle production, and thus, increased infectivity. Conversely, hnRNPA0 overexpression primarily reduced plasmid-driven and integrated HIV-1 long terminal repeat (LTR) activity, significantly decreasing total viral mRNA and protein levels. In addition, high levels of hnRNPA0 significantly reduced the HIV-1 programmed ribosomal frameshifting efficiency, resulting in a shift in the HIV-1 p55/p15 ratio. The HIV-1 alternative splice site usage remained largely unaffected by altered hnRNPA0 levels suggesting that the synergistic inhibition of the LTR activity and viral mRNA transcription, as well as impaired ribosomal frameshifting efficiency, are critical factors for efficient HIV-1 replication regulated by hnRNPA0. The pleiotropic dose-dependent effects under high or low hnRNPA0 levels were further confirmed in HIV-1-infected Jurkat cells. Finally, our study revealed that hnRNPA0 levels in PBMCs were lower in therapy-naive HIV-1-infected individuals compared to healthy controls. Our findings highlight a significant role for hnRNPA0 in HIV-1 replication and suggest that its IFN-I-regulated expression levels are critical for viral fitness allowing replication in an antiviral environment.IMPORTANCERNA-binding proteins, in particular, heterogeneous nuclear ribonucleoproteins (hnRNPs), have been extensively studied. Some act as host dependency factors for HIV-1 since they are involved in multiple cellular gene expression processes. Our study revealed hnRNPA0 as an IFN-regulated host factor, that is differently expressed after IFN-I treatment in HIV-1 target cells and lower expressed in therapy-naïve HIV-1-infected individuals. Our findings demonstrate the significant pleiotropic role of hnRNPA0 in viral replication: In high concentrations, hnRNPA0 limits viral replication by negatively regulating Tat-LTR transcription, retaining unspliced mRNA in the nucleus, and significantly impairing programmed ribosomal frameshifting. Low hnRNPA0 levels as observed in IFN-treated THP-1 cells, particularly facilitate HIV LTR activity and unspliced mRNA export, suggesting a role in innate immunity in favor of HIV replication. Understanding the mode of action between hnRNPA0 and HIV-1 gene expression might help to identify novel therapeutically strategies against HIV-1 and other viruses.

Reference-informed prediction of alternative splicing and splicing-altering mutations from sequences.

Alternative splicing plays a crucial role in protein diversity and gene expression regulation in higher eukaryotes, and mutations causing dysregulated splicing underlie a range of genetic diseases. Computational prediction of alternative splicing from genomic sequences not only provides insight into gene-regulatory mechanisms but also helps identify disease-causing mutations and drug targets. However, the current methods for the quantitative prediction of splice site usage still have limited accuracy. Here, we present DeltaSplice, a deep neural network model optimized to learn the impact of mutations on quantitative changes in alternative splicing from the comparative analysis of homologous genes. The model architecture enables DeltaSplice to perform "reference-informed prediction" by incorporating the known splice site usage of a reference gene sequence to improve its prediction on splicing-altering mutations. We benchmarked DeltaSplice and several other state-of-the-art methods on various prediction tasks, including evolutionary sequence divergence on lineage-specific splicing and splicing-altering mutations in human populations and neurodevelopmental disorders, and demonstrated that DeltaSplice outperformed consistently. DeltaSplice predicted ∼15% of splicing quantitative trait loci (sQTLs) in the human brain as causal splicing-altering variants. It also predicted splicing-altering de novo mutations outside the splice sites in a subset of patients affected by autism and other neurodevelopmental disorders (NDDs), including 19 genes with recurrent splicing-altering mutations. Integration of splicing-altering mutations with other types of de novo mutation burdens allowed the prediction of eight novel NDD-risk genes. Our work expanded the capacity of in silico splicing models with potential applications in genetic diagnosis and the development of splicing-based precision medicine.

HnRNPM protects against the dsRNA-mediated interferon response by repressing LINE-associated cryptic splicing

RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.

A systematic assessment of the impact of rare canonical splice site variants on splicing using functional and in silico methods

Background/ObjectivesCanonical splice site variants (CSSVs) are often presumed to cause loss-of-function (LoF) and are assigned very strong evidence of pathogenicity (according to ACMG criterion PVS1). The exact nature and predictability of splicing effects of unselected rare CSSVs in blood-expressed genes is poorly understood. Methods168 rare CSSVs in unselected blood-expressed genes were identified by genome sequencing in 112 individuals, and their impact on splicing was interrogated manually in RNA sequencing (RNA-seq) data. Blind to these RNA-seq data, we attempted to predict the precise impact of CSSVs by applying in silico tools and the ClinGen Sequence Variant Interpretation Working Group 2018 guidelines for applying PVS1 criterion. ResultsThere was no evidence of a frameshift nor of reduced expression consistent with nonsense-mediated decay for 25.6% of CSSVs: 17.9% had wildtype splicing only and normal junction depths, 3.6% resulted in cryptic splice site usage and in-frame indels, 3.6% resulted in full exon skipping (in-frame), and 0.6% resulted in full intron inclusion (in-frame). The predicted impact on splicing using (i) SpliceAI, (ii) MaxEntScan, and (iii) AutoPVS1, an automatic classification tool for PVS1 interpretation of null variants that utilizes Ensembl Variant Effect Predictor and MaxEntScan, was concordant with RNA-seq analyses for 65%, 63% and 61% of CSSVs, respectively. ConclusionApproximately 1 in 4 rare CSSVs may not cause LoF based on analysis of RNA-seq data. Predictions from in silico methods were often discordant with findings from RNA-seq. More caution may be warranted in applying PVS1-level evidence to CSSVs in the absence of functional data.

Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

Here, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of U11/U12-65K, a known unique component of the U11/U12 di-snRNP. Both proteins use their highly similar C-terminal RRMs to bind to 3'-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with U11/U12-65K, which uses its N-terminal region to interact with U11 snRNP during intron recognition. Finally, while RBM41 knockout cells are viable, they show alterations in U12-type 3' splice site usage. Together, our results highlight the role of the 3'-terminal stem-loop of U12 snRNA as a dynamic binding platform for the U11/U12-65K and RBM41 proteins, which function at distinct stages of the assembly/disassembly cycle.

A role for SNU66 in maintaining 5' splice site identity during spliceosome assembly.

In spliceosome assembly, the 5' splice site is initially recognized by U1 snRNA. U1 leaves the spliceosome during the assembly process, therefore other factors contribute to the maintenance of 5' splice site identity as it is loaded into the catalytic site. Recent structural data suggest that human tri-snRNP 27K (SNRP27) M141 and SNU66 H734 interact to stabilize the U4/U6 quasi-pseudo knot at the base of the U6 snRNA ACAGAGA box in pre-B complex. Previously, we found that mutations in Caenorhabditis elegans at SNRP-27 M141 promote changes in alternative 5'ss usage. We tested whether the potential interaction between SNRP-27 M141 and SNU-66 H765 (the C. elegans equivalent position to human SNU66 H734) contributes to maintaining 5' splice site identity during spliceosome assembly. We find that SNU-66 H765 mutants promote alternative 5' splice site usage. Many of the alternative 5' splicing events affected by SNU-66(H765G) overlap with those affected SNRP-27(M141T). Double mutants of snrp-27(M141T) and snu-66(H765G) are homozygous lethal. We hypothesize that mutations at either SNRP-27 M141 or SNU-66 H765 allow the spliceosome to load alternative 5' splice sites into the active site. Tests with mutant U1 snRNA and swapped 5' splice sites indicate that the ability of SNRP-27 M141 and SNU-66 H765 mutants to affect a particular 5' splice alternative splicing event is dependent on both the presence of a weaker consensus 5'ss nearby and potentially nearby splicing factor binding sites. Our findings confirm a new role for the C terminus of SNU-66 in maintenance of 5' splice site identity during spliceosome assembly.

An ATP-independent role for Prp16 in promoting aberrant splicing.

The spliceosome is assembled through a step-wise process of binding and release of its components to and from the pre-mRNA. The remodeling process is facilitated by eight DExD/H-box RNA helicases, some of which have also been implicated in splicing fidelity control. In this study, we unveil a contrasting role for the prototypic splicing proofreader, Prp16, in promoting the utilization of aberrant 5' splice sites and mutated branchpoints. Prp16 is not essential for the branching reaction in wild-type pre-mRNA. However, when a mutation is present at the 5' splice site or if Cwc24 is absent, Prp16 facilitates the reaction and encourages aberrant 5' splice site usage independently of ATP. Prp16 also promotes the utilization of mutated branchpoints while preventing the use of nearby cryptic branch sites. Our study demonstrates that Prp16 can either enhance or impede the utilization of faulty splice sites by stabilizing or destabilizing interactions with other splicing components. Thus, Prp16 exerts dual roles in 5' splice site and branch site selection, via ATP-dependent and ATP-independent activities. Furthermore, we provide evidence that these functions of Prp16 are mediated through the step-one factor Cwc25.

Biochemical and genetic evidence supports Fyv6 as a second-step splicing factor in Saccharomyces cerevisiae.

Precursor mRNA (pre-mRNA) splicing is an essential process for gene expression in eukaryotes catalyzed by the spliceosome in two transesterification steps. The spliceosome is a large, highly dynamic complex composed of five small nuclear RNAs and dozens of proteins, some of which are needed throughout the splicing reaction while others only act during specific stages. The human protein FAM192A was recently proposed to be a splicing factor that functions during the second transesterification step, exon ligation, based on analysis of cryo-electron microscopy (cryo-EM) density. It was also proposed that Fyv6 might be the Saccharomyces cerevisiae functional and structural homolog of FAM192A; however, no biochemical or genetic data has been reported to support this hypothesis. Herein, we show that Fyv6 is a splicing factor and acts during exon ligation. Deletion of FYV6 results in genetic interactions with the essential splicing factors Prp8, Prp16, and Prp22 and decreases splicing in vivo of reporter genes harboring intron substitutions that limit the rate of exon ligation. When splicing is assayed in vitro, whole-cell extracts lacking Fyv6 accumulate first-step products and exhibit a defect in exon ligation. Moreover, loss of Fyv6 causes a change in 3' splice site (SS) selection in both a reporter gene and the endogenous SUS1 transcript in vivo. Together, these data suggest that Fyv6 is a component of the yeast spliceosome that influences 3' SS usage and the potential homolog of human FAM192A.

AAV-vectored base editor trans-splicing delivers dystrophin repair

AAV-vectored base editor trans-splicing delivers dystrophin repair

The regulation of expression and splicing of transcription factors are related to the muscle damage caused by radiation in tree shrews

Radiotherapy-induced muscle injury (RIMI) is a major complication of radiotherapy for nasopharyngeal carcinoma. Transcription factor (TF) expression and alternative splicing are crucial events in transcriptional and posttranscriptional regulation, respectively, and are known to be involved in key signaling pathways contributing to a variety of human disorders, including radiation injury. To investigate the TFs and alternative splicing events involved in RIMI, we constructed a tree shrew model as described previously in which the RIMI group received 20 Gy of irradiation on the tensor veli palatini (TVP) muscles. The irradiated muscles were evaluated by RNA sequencing (RNA-seq) 6 months later, and the results compared with those for normal TVP muscles. The alt5p and alt3p events were the two main types of differentially regulated alternative splicing events (RASEs) identified via the Splice sites Usage Variation Analysis (SUVA) software, and these RASEs were highly conserved in RIMI. According to functional enrichment analysis, the differentially RASEs were primarily enriched in pathways related to transcriptional regulation. Furthermore, we identified 16 alternative splicing TFs (ASTFs) in ASTF–differentially expressed gene (DEG) networks based on co-expression analysis, and the regulatory networks were chiefly enriched in pathways linked to cell proliferation and differentiation. This study revealed that RASEs and ASTF–DEG networks may both play important regulatory roles in gene expression network alteration in RIMI. Future studies on the targeting mechanisms and early interventions directed at RASEs and ASTF–DEG networks may aid in the treatment of RIMI.

Aberrant splicing prediction across human tissues.

Aberrant splicing is a major cause of genetic disorders but its direct detection in transcriptomes is limited to clinically accessible tissues such as skin or body fluids. While DNA-based machine learning models can prioritize rare variants for affecting splicing, their performance in predicting tissue-specific aberrant splicing remains unassessed. Here we generated an aberrant splicing benchmark dataset, spanning over 8.8 million rare variants in 49 human tissues from the Genotype-Tissue Expression (GTEx) dataset. At 20% recall, state-of-the-art DNA-based models achieve maximum 12% precision. By mapping and quantifying tissue-specific splice site usage transcriptome-wide and modeling isoform competition, we increased precision by threefold at the same recall. Integrating RNA-sequencing data of clinically accessible tissues into our model, AbSplice, brought precision to 60%. These results, replicated in two independent cohorts, substantially contribute to noncoding loss-of-function variant identification and to genetic diagnostics design and analytics.

Diverse targets of SMN2-directed splicing-modulating small molecule therapeutics for spinal muscular atrophy.

Designing an RNA-interacting molecule that displays high therapeutic efficacy while retaining specificity within a broad concentration range remains a challenging task. Risdiplam is an FDA-approved small molecule for the treatment of spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. Branaplam is another small molecule which has undergone clinical trials. The therapeutic merit of both compounds is based on their ability to restore body-wide inclusion of Survival Motor Neuron 2 (SMN2) exon 7 upon oral administration. Here we compare the transcriptome-wide off-target effects of these compounds in SMA patient cells. We captured concentration-dependent compound-specific changes, including aberrant expression of genes associated with DNA replication, cell cycle, RNA metabolism, cell signaling and metabolic pathways. Both compounds triggered massive perturbations of splicing events, inducing off-target exon inclusion, exon skipping, intron retention, intron removal and alternative splice site usage. Our results of minigenes expressed in HeLa cells provide mechanistic insights into how these molecules targeted towards a single gene produce different off-target effects. We show the advantages of combined treatments with low doses of risdiplam and branaplam. Our findings are instructive for devising better dosing regimens as well as for developing the next generation of small molecule therapeutics aimed at splicing modulation.

Arg-tRNA synthetase links inflammatory metabolism to RNA splicing and nuclear trafficking via SRRM2.

Cells respond to perturbations such as inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Aminoacyl-tRNA synthetases, enzymes that catalyse the first step of protein synthesis, can also mediate cell signalling. Here we show that depletion of arginine during inflammation decreased levels of nuclear-localized arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts and co-localizes with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal and nuclear speckle protein, and that decreased levels of nuclear ArgRS correlated with changes in condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in the synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a mechanism whereby an aminoacyl-tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery.

Regulation of 3' splice site selection after step 1 of splicing by spliceosomal C* proteins.

Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3'ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3' region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3'ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3'ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3'ss choices.

Building a quantitative and predictive model of 5'SS selection by human U1 snRNP using RNA-map.

Alternative splicing of pre-mRNA greatly expands the protein coding potential of our genome and accounts for extensive cell and tissue-specific variation in gene expression. Spliceosome assembly depends on the recognition of the 5′ splice site (5′SS) at the exon-intron boundary by the U1 small nuclear ribonucleoprotein (U1 snRNP). Critically, mutations in either U1 or the 5′SS can lead to human disease, including cancer. Despite decades of investigation, accurately predicting 5′SS selection and alternative splicing outcomes is difficult, suggesting new approaches are needed. Here, we present our progress in developing a quantitative and predictive model of 5′SS selection by human U1 snRNP grounded in biochemical understanding. We used the RNA on a massively parallel array (RNA-MaP) platform to measure equilibrium binding constants of reconstituted human U1 snRNP to >60,000 unique RNAs. Our designed RNA library features all possible GU and GC variants in addition to thousands of identified mutations, allowing us to build a comprehensive and predictive model of 5′SS/U1 interactions at the transcriptome scale. Overall, our results will provide a first principles understanding of 5′SS selection and will serve as a foundation for future work to systematically determine the effects of other splicing factors and splice site usage in vivo.

BAY61-3606 Alters snRNP Composition and Enhances Usage of Suboptimal Splice Acceptor Site.

Intron recognition by the spliceosome mainly depends on conserved intronic sequences such as 5' splice sites, 3' splice sites, and branch sites. Therefore, even substitution of just a single nucleotide in a 5' or 3' splice site abolishes the splicing at the mutated site and leads to cryptic splice site usage. A number of disease-causative mutations have been found in 5' and 3' splice sites, but the genes with these mutations still maintain the correct protein-coding sequence, so recovery of splicing at the mutated splice site may produce a normal protein. Mutations in the spliceosome components have been shown to change the balance between the conformational transition and disassembly of the spliceosome, which affects the decision about whether the reaction of the incorporated substrate will proceed. In addition, the lower disassembly rate caused by such mutations induces splicing of the mutated splice site. We hypothesized that small compounds targeting the spliceosome may include a compound mimicking the effect of those mutations. Thus, we screened a small-compound library and identified a compound, BAY61-3606, that changed the cellular small nuclear ribonucleoprotein composition and also showed activity of enhancing splicing at the mutated 3' splice site of the reporter gene, as well as splicing at the suboptimal 3' splice site of endogenous cassette exons. These results indicate that further analysis of the mechanism of action of BAY61-3606 could enable modulation of the fidelity of splicing.

MAEL gene contributes to bovine testicular development through the m5C-mediated splicing

Knowledge of RNA molecules regulating testicular development and spermatogenesis in bulls is essential for elite bull selection and an ideal breeding program. Herein, we performed direct RNA sequencing (DRS) to explore the functional characterization of RNA molecules produced in the testicles of 9 healthy Simmental bulls at three testicular development stages (prepuberty, puberty, and postpuberty). We identified 5,043 differentially expressed genes associated with testicular weight. These genes exhibited more alternative splicing at sexual maturity, particularly alternative 3' (A3) and 5' (A5) splice sites usage and exon skipping (SE). The expression of hub genes in testicular developmental stages was also affected by both m6A and m5C RNA modifications. We found m5C-mediated splicing events significantly (p<0.05) increased MAEL gene expression at the isoform level, likely promoting spermatogenesis. Our findings highlight the complexity of RNA processing and expression as well as the regulation of transcripts involved in testicular development and spermatogenesis.

Abnormal Pre-mRNA Splicing in Exonic Fabry Disease-Causing GLA Mutations.

Fabry disease (FD) is a rare X-linked disease due to a multiverse of disrupting mutations within the GLA gene encoding lysosomal α-galactosidase A (AGAL). Absent AGAL activity causes the accumulation of complex glycosphingolipids inside of lysosomes in a variety of cell types and results in a progressive multisystem disease. Known disease-associated point mutations in protein-coding gene regions usually cause translational perturbations and result in premature chain termination, punctual amino acid sequence alterations or overall altered sequence alterations downstream of the mutation site. However, nucleotide exchanges at the border between introns and exons can affect splicing behavior and lead to abnormal pre-mRNA processing. Prediction with the Human Splicing Finder (HSF) revealed an indication of a significant change in splicing-relevant information for some known FD-associated GLA mutations. To experimentally determine the extent of the change, we made use of a minigene reporter assay and verified alternative splicing events for the exonic mutations c.194G>T and c.358C>G, which led to the usage of alternative donor splice sites at exon 1 and exon 2, respectively. In addition, the mutations c.548G>T and c.638A>T led to significant exon 4 skipping. We conclude that splicing phenotype analysis should be employed in the in vitro analysis of exonic GLA gene mutations, since abnormal splicing may result in a reduction of enzyme activity and alter the amenability for treatment with pharmacological chaperone (PC).

M6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site.

Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5' splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the ACAGA box is modified by N6 methylation, but the role of this m6A modification is poorly understood. Here, we show that m6A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIONA1, is required for Arabidopsis U6 snRNA m6A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5' splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m6A influences 3' splice site usage. We generalise these findings to reveal two major classes of 5' splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA ACAGA box. We conclude that U6 snRNA m6A modification contributes to the selection of degenerate 5' splice sites crucial to alternative splicing.