Unannotated Splice Research Articles

Female germline expression of OVO transcription factor bridges Drosophila generations.

OVO is required for female germ cell viability but has no known function in the male germline in Drosophila. ovo is autoregulated by two antagonistic isoforms, OVO-A and OVO-B. All ovo- alleles were created as partial revertants of the antimorphic ovoD1 allele. Creation of new targeted alleles in an ovo+ background indicated that disrupting the germline-specific exon extension of ovo-B leads to an arrested egg chamber phenotype, rather than germ cell death. RNA-seq analysis, including >1K full length cDNAs, indicates that ovo has several unannotated splice variations in the extended exon and a minor population of ovo-B transcripts have an alternative splice. This indicates that classical ovo alleles such as ovoD1rv23, are not truly null for ovo, and are likely to be weak antimorphs. To generate bonafide nulls, we deleted the ovo-A and ovo-B promoters showing that only ovo-B is required for female germ cell viability and there is an early and continual developmental requirement for ovo-B in the female germline. To visualize OVO expression and localization, we endogenously tagged ovo and found nuclear OVO in all differentiating female germ cells throughout oogenesis in adults. We also found that OVO is maternally deposited into the embryo, where it showed nuclear localization in newly formed pole cells. Maternal OVO persisted in embryonic germ cells until zygotic OVO expression was detectable, suggesting that there is continuous nuclear OVO expression in the female germline in the transition from one generation to the next.

Scalable and unsupervised discovery from raw sequencing reads using SPLASH2.

We introduce SPLASH2, a fast, scalable implementation of SPLASH based on an efficient k-mer counting approach for regulated sequence variation detection in massive datasets from a wide range of sequencing technologies and biological contexts. We demonstrate biological discovery by SPLASH2 in single-cell RNA sequencing (RNA-seq) data and in bulk RNA-seq data from the Cancer Cell Line Encyclopedia, including unannotated alternative splicing in cancer transcriptomes and sensitive detection of circular RNA.

RNA isoform expression landscape of the human dorsal root ganglion generated from long-read sequencing.

Splicing is a posttranscriptional RNA processing mechanism that enhances genomic complexity by creating multiple isoforms from the same gene. We aimed to characterize the isoforms expressed in the human peripheral nervous system, with the goal of creating a resource to identify novel isoforms of functionally relevant genes associated with somatosensation and nociception. We used long-read sequencing to document isoform expression in the human dorsal root ganglia from 3 organ donors and validated in silico by confirming expression in short-read sequencing from 3 independent organ donors. Nineteen thousand five hundred forty-seven isoforms of protein-coding genes were detected and validated. We identified 763 isoforms with at least one previously undescribed splice junction. Previously unannotated isoforms of multiple pain-associated genes, including ASIC3, MRGPRX1, and HNRNPK, were identified. In the novel isoforms of ASIC3, a region comprising approximately 35% of the 5'UTR was excised. By contrast, a novel splice junction was used in isoforms of MRGPRX1 to include an additional exon upstream of the start codon, consequently adding a region to the 5'UTR. Novel isoforms of HNRNPK were identified, which used previously unannotated splice sites to both excise exon 14 and include a sequence in the 3' end of exon 13. This novel insertion is predicted to introduce a tyrosine phosphorylation site potentially phosphorylated by SRC. We also independently confirm a recently reported DRG-specific splicing event in WNK1 that gives insight into how painless peripheral neuropathy occurs when this gene is mutated. Our findings give a clear overview of mRNA isoform diversity in the human dorsal root ganglia obtained using long-read sequencing.

MAJIQlopedia: an encyclopedia of RNA splicing variations in human tissues and cancer.

Quantification of RNA splicing variations based on RNA-Sequencing can reveal tissue- and disease-specific splicing patterns. To study such splicing variations, we introduce MAJIQlopedia, an encyclopedia of splicing variations that encompasses 86 human tissues and 41 cancer datasets. MAJIQlopedia reports annotated and unannotated splicing events for a total of 486175 alternative splice junctions in normal tissues and 338317 alternative splice junctions in cancer. This database, available at https://majiq.biociphers.org/majiqlopedia/, includes a user-friendly interface that provides graphical representations of junction usage quantification for each junction across all tissue or cancer types. To demonstrate case usage of MAJIQlopedia, we review splicing variations in genes WT1, MAPT and BIN1, which all have known tissue or cancer-specific splicing variations. We also use MAJIQlopedia to highlight novel splicing variations in FDX1 and MEGF9 in normal tissues, and we uncover a novel exon inclusion event in RPS6KA6 that only occurs in two cancer types. Users can download the database, request the addition of data to the webtool, or install a MAJIQlopedia server to integrate proprietary data. MAJIQlopedia can serve as a reference database for researchers seeking to understand what splicing variations exist in genes of interest, and those looking to understand tissue- or cancer-specific splice isoform usage.

Comparative analysis of nascent RNA sequencing methods and their applications in studies of cotranscriptional splicing dynamics.

High-throughput detection of nascent RNA is critical for studies of transcription and much more challenging than that of mRNA. Recently, several massively parallel nascent RNA sequencing methods were established in eukaryotic cells. Here, we systematically compared 3 classes of methods on the same pure or crude nuclei preparations: GRO-seq for sequence nuclear run-on RNAs, pNET-seq for sequence RNA polymerase II-associated RNAs, and CB RNA-seq for sequence chromatin-bound (CB) RNAs in Arabidopsis (Arabidopsis thaliana). To improve the resolution of CB RNAs, 3'CB RNA-seq was established to sequence the 3' ends of CB RNAs. In addition, we modified pNET-seq to establish the Chromatin Native Elongation Transcript sequencing (ChrNET) method using chromatin as the starting material for RNA immunoprecipitation. Reproducibility, sensitivity and accuracy in detecting nascent transcripts, experimental procedures, and costs were analyzed, which revealed the strengths and weaknesses of each method. We found that pNET and GRO methods best detected active RNA polymerase II. CB RNA-seq is a simple and cost-effective alternative for nascent RNA studies, due to its high correlation with pNET-seq and GRO-seq. Compared with pNET, ChrNET has higher specificity for nascent RNA capture and lower sequencing cost. 3'CB is sensitive to transcription-coupled splicing. Using these methods, we identified 1,404 unknown transcripts, 4,482 unannotated splicing events, and 60 potential recursive splicing events. This comprehensive comparison of different nascent/chromatin RNA sequencing methods highlights the strengths of each method and serves as a guide for researchers aiming to select a method that best meets their study goals.

RNA splicing analysis using heterogeneous and large RNA-seq datasets

The ubiquity of RNA-seq has led to many methods that use RNA-seq data to analyze variations in RNA splicing. However, available methods are not well suited for handling heterogeneous and large datasets. Such datasets scale to thousands of samples across dozens of experimental conditions, exhibit increased variability compared to biological replicates, and involve thousands of unannotated splice variants resulting in increased transcriptome complexity. We describe here a suite of algorithms and tools implemented in the MAJIQ v2 package to address challenges in detection, quantification, and visualization of splicing variations from such datasets. Using both large scale synthetic data and GTEx v8 as benchmark datasets, we assess the advantages of MAJIQ v2 compared to existing methods. We then apply MAJIQ v2 package to analyze differential splicing across 2,335 samples from 13 brain subregions, demonstrating its ability to offer insights into brain subregion-specific splicing regulation.

SpliceVault predicts the precise nature of variant-associated mis-splicing

Even for essential splice-site variants that are almost guaranteed to alter mRNA splicing, no current method can reliably predict whether exon-skipping, cryptic activation or multiple events will result, greatly complicating clinical interpretation of pathogenicity. Strikingly, ranking the four most common unannotated splicing events across 335,663 reference RNA-sequencing (RNA-seq) samples (300K-RNA Top-4) predicts the nature of variant-associated mis-splicing with 92% sensitivity. The 300K-RNA Top-4 events correctly identify 96% of exon-skipping events and 86% of cryptic splice sites for 140 clinical cases subject to RNA testing, showing higher sensitivity and positive predictive value than SpliceAI. Notably, RNA re-analyses showed we had missed 300K-RNA Top-4 events for several clinical cases tested before the development of this empirical predictive method. Simply, mis-splicing events that happen around a splice site in RNA-seq data are those most likely to be activated by a splice-site variant. The SpliceVault web portal allows users easy access to 300K-RNA for informed splice-site variant interpretation and classification.

Reversal of splicing infidelity is a pre-activation step in B cell differentiation.

B cell activation and differentiation is central to the adaptive immune response. Changes in exon usage can have major impacts on cellular signaling and differentiation but have not been systematically explored in differentiating B cells. We analyzed exon usage and intron retention in RNA-Seq data from subsets of human B cells at various stages of differentiation, and in an in vitro laboratory model of B cell activation and differentiation (Epstein Barr virus infection). Blood naïve B cells were found to have an unusual splicing profile, with unannotated splicing events in over 30% of expressed genes. Splicing changed substantially upon naïve B cell entry into secondary lymphoid tissue and before activation, involving significant increases in exon commitment and reductions in intron retention. These changes preferentially involved short introns with weak splice sites and were likely mediated by an overall increase in splicing efficiency induced by the lymphoid environment. The majority of transcripts affected by splicing changes showed restoration of encoded conserved protein domains and/or reduced targeting to the nonsense-mediated decay pathway. Affected genes were enriched in functionally important immune cell activation pathways such as antigen-mediated signaling, cell cycle control and mRNA processing and splicing. Functional observations from donor B cell subsets in progressive states of differentiation and from timecourse experiments using the in vitro model suggest that these widespread changes in mRNA splicing play a role in preparing naïve B cells for the decisive step of antigen-mediated activation and differentiation.

Specific splice junction detection in single cells with SICILIAN

Precise splice junction calls are currently unavailable in scRNA-seq pipelines such as the 10x Chromium platform but are critical for understanding single-cell biology. Here, we introduce SICILIAN, a new method that assigns statistical confidence to splice junctions from a spliced aligner to improve precision. SICILIAN is a general method that can be applied to bulk or single-cell data, but has particular utility for single-cell analysis due to that data’s unique challenges and opportunities for discovery. SICILIAN’s precise splice detection achieves high accuracy on simulated data, improves concordance between matched single-cell and bulk datasets, and increases agreement between biological replicates. SICILIAN detects unannotated splicing in single cells, enabling the discovery of novel splicing regulation through single-cell analysis workflows.

Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved alternative splicing nonsense-mediated decay pathways

BackgroundAlternative splicing, which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing splicing pathways cannot be determined by traditional RNA-Seq, however, because different mRNA isoforms can have widely differing decay rates. Indeed, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses.ResultsRNA immunoprecipitation in tandem (RIPiT) of exon junction complex components allows for purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and, therefore, translation-dependent mRNA decay. Here we compare exon junction complex RIPiT-Seq to whole cell RNA-Seq data from HEK293 cells. Consistent with expectation, the flux through known AS-NMD pathways is substantially higher than that captured by RNA-Seq. Our RIPiT-Seq also definitively demonstrates that the splicing machinery itself has no ability to detect reading frame. We identify thousands of previously unannotated splicing events; while many can be attributed to splicing noise, others are evolutionarily conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis.ConclusionsDeep sequencing of RNAs in post-splicing, pre-translational mRNPs provides a means to identify and quantify splicing events without the confounding influence of differential mRNA decay. For many known AS-NMD targets, the nonsense-mediated decay-linked alternative splicing pathway predominates. Exon junction complex RIPiT-Seq also revealed numerous conserved but previously unannotated AS-NMD events.

Unannotated splicing regulatory elements in deep intron space.

Deep intron space harbors a diverse array of splicing regulatory elements that cooperate with better-known exon-proximal elements to enforce proper tissue-specific and development-specific pre-mRNA processing. Many deep intron elements have been highly conserved through vertebrate evolution, yet remain poorly annotated in the human genome. Recursive splicing exons (RS-exons) and intraexons promote noncanonical, multistep resplicing pathways in long introns, involving transient intermediate structures that are greatly underrepresented in RNA-seq datasets. Decoy splice sites and decoy exons act at a distance to inhibit splicing catalysis at annotated splice sites, with functional consequences such as exon skipping and intron retention. RNA:RNA bridges can juxtapose distant sequences within or across introns to activate deep intron splicing enhancers and silencers, to loop out exons to be skipped, or to select one member of a mutually exclusive set of exons. Similarly, protein bridges mediated by interactions among transcript-bound RNA binding proteins (RBPs) can modulate splicing outcomes. Experimental disruption of deep intron elements serving any of these functions can abrogate normal splicing, strongly suggesting that natural mutations of deep intron elements can do likewise to cause human disease. Understanding noncanonical splicing pathways and discovering deep intron regulatory signals, many of which map hundreds to many thousands of nucleotides from annotated splice junctions, is of great academic interest for basic scientists studying alternative splicing mechanisms. Hopefully, this knowledge coupled with increased analysis of deep intron sequences will also have important medical applications, as better interpretation of deep intron mutations may reveal new disease mechanisms and suggest new therapies. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing.

NCK-associated protein 1 like (nckap1l) minor splice variant regulates intrahepatic biliary network morphogenesis.

Impaired formation of the intrahepatic biliary network leads to cholestatic liver diseases, which are frequently associated with autoimmune disorders. Using a chemical mutagenesis strategy in zebrafish combined with computational network analysis, we screened for novel genes involved in intrahepatic biliary network formation. We positionally cloned a mutation in the nckap1l gene, which encodes a cytoplasmic adaptor protein for the WAVE regulatory complex. The mutation is located in the last exon after the stop codon of the primary splice isoform, only disrupting a previously unannotated minor splice isoform, which indicates that the minor splice isoform is responsible for the intrahepatic biliary network phenotype. CRISPR/Cas9-mediated nckap1l deletion, which disrupts both the primary and minor isoforms, showed the same defects. In the liver of nckap1l mutant larvae, WAVE regulatory complex component proteins are degraded specifically in biliary epithelial cells, which line the intrahepatic biliary network, thus disrupting the actin organization of these cells. We further show that nckap1l genetically interacts with the Cdk5 pathway in biliary epithelial cells. These data together indicate that although nckap1l was previously considered to be a hematopoietic cell lineage-specific protein, its minor splice isoform acts in biliary epithelial cells to regulate intrahepatic biliary network formation.

Overlapping Activities of ELAV/Hu Family RNA Binding Proteins Specify the Extended Neuronal 3′ UTR Landscape in Drosophila

The tissue-specific deployment of highly extended neural 3' UTR isoforms, generated by alternative polyadenylation (APA), is a broad and conserved feature of metazoan genomes. However, the factors and mechanisms that control neural APA isoforms are not well understood. Here, we show that three ELAV/Hu RNA binding proteins (Elav, Rbp9, and Fne) have similar capacities to induce a lengthened 3' UTR landscape in an ectopic setting. These factors promote accumulation of chromatin-associated, 3' UTR-extended, nascent transcripts, through inhibition of proximal polyadenylation site (PAS) usage. Notably, Elav represses an unannotated splice isoform of fne, switching the normally cytoplasmic Fne toward the nucleus in elav mutants. We use genomic profiling to reveal strong and broad loss of neural APA in elav/fne double mutant CNS, the first genetic background to largely abrogate this distinct APA signature. Overall, we demonstrate how regulatory interplay and functionally overlapping activities of neural ELAV/Hu RBPs drives the neural APA landscape.

The Genetic Regulation of Alternative Splicing in Populus deltoides.

Alternative splicing (AS) is a mechanism of regulation of the proteome via enabling the production of multiple mRNAs from a single gene. To date, the dynamics of AS and its effects on the protein sequences of individuals in a large and genetically unrelated population of trees have not been investigated. Here we describe the diversity of AS events within a previously genotyped population of 268 individuals of Populus deltoides and their putative downstream functional effects. Using a robust bioinformatics pipeline, the AS events and resulting transcript isoforms were discovered and quantified for each individual in the population. Analysis of the AS revealed that, as expected, most AS isoforms are conserved. However, we also identified a substantial collection of new, unannotated splice junctions and transcript isoforms. Heritability estimates for the expression of transcript isoforms showed that approximately half of the isoforms are heritable. The genetic regulators of these AS isoforms and splice junction usage were then identified using a genome-wide association analysis. The expression of AS isoforms was predominately cis regulated while splice junction usage was generally regulated in trans. Additionally, we identified 696 genes encoding alternatively spliced isoforms that changed putative protein domains relative to the longest protein coding isoform of the gene, and 859 genes exhibiting this same phenomenon relative to the most highly expressed isoform. Finally, we found that 748 genes gained or lost micro-RNA binding sites relative to the longest protein coding isoform of a given gene, while 940 gained or lost micro-RNA binding sites relative to the most highly expressed isoform. These results indicate that a significant fraction of AS events are genetically regulated and that this isoform usage can result in protein domain architecture changes.

ASCOT identifies key regulators of neuronal subtype-specific splicing

Public archives of next-generation sequencing data are growing exponentially, but the difficulty of marshaling this data has led to its underutilization by scientists. Here, we present ASCOT, a resource that uses annotation-free methods to rapidly analyze and visualize splice variants across tens of thousands of bulk and single-cell data sets in the public archive. To demonstrate the utility of ASCOT, we identify novel cell type-specific alternative exons across the nervous system and leverage ENCODE and GTEx data sets to study the unique splicing of photoreceptors. We find that PTBP1 knockdown and MSI1 and PCBP2 overexpression are sufficient to activate many photoreceptor-specific exons in HepG2 liver cancer cells. This work demonstrates how large-scale analysis of public RNA-Seq data sets can yield key insights into cell type-specific control of RNA splicing and underscores the importance of considering both annotated and unannotated splicing events.

Deep profiling and custom databases improve detection of proteoforms generated by alternative splicing.

Alternative pre-mRNA splicing has long been proposed to contribute greatly to proteome complexity. However, the extent to which mature mRNA isoforms are successfully translated into protein remains controversial. Here, we used high-throughput RNA sequencing and mass spectrometry (MS)–based proteomics to better evaluate the translation of alternatively spliced mRNAs. To increase proteome coverage and improve protein quantitation, we optimized cell fractionation and sample processing steps at both the protein and peptide level. Furthermore, we generated a custom peptide database trained on analysis of RNA-seq data with MAJIQ, an algorithm optimized to detect and quantify differential and unannotated splice junction usage. We matched tandem mass spectra acquired by data-dependent acquisition (DDA) against our custom RNA-seq based database, as well as SWISS-PROT and RefSeq databases to improve identification of splicing-derived proteoforms by 28% compared with use of the SWISS-PROT database alone. Altogether, we identified peptide evidence for 554 alternate proteoforms corresponding to 274 genes. Our increased depth and detection of proteins also allowed us to track changes in the transcriptome and proteome induced by T-cell stimulation, as well as fluctuations in protein subcellular localization. In sum, our data here confirm that use of generic databases in proteomic studies underestimates the number of spliced mRNA isoforms that are translated into protein and provides a workflow that improves isoform detection in large-scale proteomic experiments.

RNA Splicing Defects in Cancer-Linked Genes Indicate Mutation or Focal Gene Deletion and Are Associated with TKI Resistance in CML

Background Mutated cancer genes in patients (pts) with TKI failure and blast crisis (BC) CML have recently been described. RUNX1 mutations, namely single nucleotide variants (SNVs) and indels, were the most frequently detected besides BCR-ABL1 [reviewed in Branford, Kim Leuk 2019]. They were found in ~18% of pts, although splice variants were rarely described. RNA splicing events were associated with focal deletion of IKZF1 and RUNX1 in TKI resistant pts that were identified by copy number analysis and RNAseq [Branford Blood 2018]. Novel splicing associated with mutation of cancer genes is an unexplored area of study in resistance. RNA sequencing can assess the functional effect of splice site variants, which lead to splicing errors due to the use of alternative or cryptic splice sites and cause alterations to protein function. Aim We determined whether novel splicing can identify cancer genes with potential altered function. Methods RNAseq analysis was performed for 48 pts at diagnosis and 33 at BC using a protocol that preserved intron-retaining precursor RNA. Coverage of intron-exon borders was sufficient to detect intronic splice region variants. The STAR aligner was used to bioinformatically collate unannotated RNA splice junctions. 54 cancer genes were assessed and aberrant splice events were filtered based on the number of samples in which a splice junction occurred. Manual inspection of the splice junctions was performed using the Integrative Genomics Viewer. This approach identified previously verified aberrant splicing associated with IKZF1 and RUNX1 deletions. Results Ten previously undetected novel splice junctions were revealed in 9/33 pts (27%) in BC within key tumor suppressor genes CDKN2A/B (5), RB1 (1), ATM (1), and RUNX1 (3). The aberrant splicing pattern of CDKN2A and RB1 (Fig A/B) in 6 pts suggested large deletions, as previously described in our cohort with IKZF1 and RUNX1 deletions. Breakpoints associated with deletions ranging from 53 to 181 Kb were detected in the 5 pts with CDKN2A aberrant splicing. Similarly, a 90 Kb deletion of exons 18-27 of the RB1 gene led to the aberrant splicing. The pts transformed to lymphoid BC (median 5 months). 4 of these 6 pts were tested at diagnosis and the deletions were not detected, indicating they were gained at resistance. The aberrant splicing patterns of ATM and RUNX1 did not predict large deletions. These were related to somatic SNVs at canonical splice sites in ATM and in 2 of the pts with RUNX1 aberrant splicing. A splice acceptor site SNV in ATM resulted in skipping of exon 61 (Fig C) and protein truncation. This novel SNV has not been reported in any population or somatic variant database. Two pts in myeloid BC at 28 and 48 months after diagnosis had an identical somatic RUNX1 mutation at the canonical splice donor site of exon 5. The SNV was not detectable prior to imatinib treatment in both pts. The splice site SNV led to activation of a cryptic splice site within exon 5 in both pts (Fig D), which predicted premature termination. While this mutation is novel, an adjacent intronic SNV occurs in familial platelet disorder, leading to activation of the same cryptic splice site. The atypical RUNX1 splicing of the 3rd patient was associated with retention of 55 bp of intron 6 as a cryptic exon (Fig E), leading to protein truncation. A deep intronic SNV identified at lymphoid BC at 6 months of imatinib was detected near the cryptic exon by RNAseq and verified as somatic by DNA Sanger sequencing. This was predicted to activate cryptic RNA splicing elements and lead to intron sequence retention in a RUNX1 transcript. We sequenced the diagnosis sample using an RNA-based gene panel method under development that provides enhanced sensitivity of variant detection. The same pattern of atypical splicing was observed and the intronic SNV was present at low level. The RUNX1 mutation at diagnosis may have contributed to early BC. To our knowledge this is the first report of a RUNX1 truncating variant in CML involving a cryptic exon. Conclusion Enhanced bioinformatic analysis of RNAseq data has revealed a high proportion of pts with truncating mutations in cancer genes indicated by novel RNA splicing (27% pts in BC). Using RNA-based sequencing allows an evaluation of the potential functional effect of variants that are not apparent by DNA-based mutation analysis. We suggest that future studies include RNA sequencing to detect a broader spectrum of mutations associated with TKI resistance. Disclosures Shanmuganathan: Gilead: Other: Travel Support; Janssen: Other: Travel Support; Amgen: Other: Travel Support; Bristol-Myers Squibb: Honoraria, Other: Travel Support; Novartis: Honoraria, Other: Travel Support. Yeung:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. Scott:Celgene: Honoraria. Hughes:Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel. Branford:Cepheid: Consultancy, Honoraria; Qiagen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau.

Ultra-deep sequencing reveals pre-mRNA splicing as a sequence driven high-fidelity process.

Alternative splicing diversifies mRNA transcripts in human cells. While the spliceosome pairs exons with a high degree of accuracy, the rates of rare aberrant and non-canonical pre-mRNA splicing have not been evaluated at the nucleotide level to determine the quantity and identity of these events across splice junctions. Using ultra-deep sequencing the frequency of aberrant and non-canonical splicing events for three splice junctions flanking exon 7 of SMN1 were determined at single nucleotide resolution. After correction for background noise introduced by PCR amplification and sequencing steps, pre-mRNA splicing was shown to maintain a low overall rate of aberrant and non-canonically spliced events. Several previously unannotated splicing events across 3 exon|intron junctions in SMN1 were identified. Mutations within SMN exon 7 were shown to affect splicing fidelity by modulating RNA secondary structures, by altering the binding site of regulatory proteins and by changing the 5’ splice site strength. Mutations also create a truncated SMN1 exon 7 through the introduction of a de novo non-canonical 5’ splice site. The results from the ultra-deep sequencing approach highlight the impressive fidelity of pre-mRNA splicing and demonstrate that the immediate sequence context around splice sites is the main driving force behind non-canonical splice site pairing.

A time-resolved multi-omic atlas of the developing mouse stomach

The mammalian stomach is structurally highly diverse and its organ functionality critically depends on a normal embryonic development. Although there have been several studies on the morphological changes during stomach development, a system-wide analysis of the underlying molecular changes is lacking. Here, we present a comprehensive, temporal proteome and transcriptome atlas of the mouse stomach at multiple developmental stages. Quantitative analysis of 12,108 gene products allows identifying three distinct phases based on changes in proteins and RNAs and the gain of stomach functions on a longitudinal time scale. The transcriptome indicates functionally important isoforms relevant to development and identifies several functionally unannotated novel splicing junction transcripts that we validate at the peptide level. Importantly, many proteins differentially expressed in stomach development are also significantly overexpressed in diffuse-type gastric cancer. Overall, our study provides a resource to understand stomach development and its connection to gastric cancer tumorigenesis.

ASGAL: aligning RNA-Seq data to a splicing graph to detect novel alternative splicing events

BackgroundWhile the reconstruction of transcripts from a sample of RNA-Seq data is a computationally expensive and complicated task, the detection of splicing events from RNA-Seq data and a gene annotation is computationally feasible. This latter task, which is adequate for many transcriptome analyses, is usually achieved by aligning the reads to a reference genome, followed by comparing the alignments with a gene annotation, often implicitly represented by a graph: the splicing graph.ResultsWe present ASGAL (Alternative Splicing Graph ALigner): a tool for mapping RNA-Seq data to the splicing graph, with the specific goal of detecting novel splicing events, involving either annotated or unannotated splice sites. ASGAL takes as input the annotated transcripts of a gene and a RNA-Seq sample, and computes (1) the spliced alignments of each read in input, and (2) a list of novel events with respect to the gene annotation.ConclusionsAn experimental analysis shows that ASGAL allows to enrich the annotation with novel alternative splicing events even when genes in an experiment express at most one isoform. Compared with other tools which use the spliced alignment of reads against a reference genome for differential analysis, ASGAL better predicts events that use splice sites which are novel with respect to a splicing graph, showing a higher accuracy. To the best of our knowledge, ASGAL is the first tool that detects novel alternative splicing events by directly aligning reads to a splicing graph.AvailabilitySource code, documentation, and data are available for download at http://asgal.algolab.eu.