Genome-Wide Association between Branch Point Properties and Alternative Splicing

The branch point (BP) is one of the three obligatory signals required for pre-mRNA splicing. In mammals, the degeneracy of the motif combined with the lack of a large set of experimentally verified BPs complicates the task of modeling it in silico, and therefore of predicting the location of natural BPs. Consequently, BPs have been disregarded in a considerable fraction of the genome-wide studies on the regulation of splicing in mammals. We present a new computational approach for mammalian BP prediction. Using sequence conservation and positional bias we obtained a set of motifs with good agreement with U2 snRNA binding stability. Using a Support Vector Machine algorithm, we created a model complemented with polypyrimidine tract features, which considerably improves the prediction accuracy over previously published methods. Applying our algorithm to human introns, we show that BP position is highly dependent on the presence of AG dinucleotides in the 3′ end of introns, with distance to the 3′ splice site and BP strength strongly correlating with alternative splicing. Furthermore, experimental BP mapping for five exons preceded by long AG-dinucleotide exclusion zones revealed that, for a given intron, more than one BP can be chosen throughout the course of splicing. Finally, the comparison between exons of different evolutionary ages and pseudo exons suggests a key role of the BP in the pathway of exon creation in human. Our computational and experimental analyses suggest that BP recognition is more flexible than previously assumed, and it appears highly dependent on the presence of downstream polypyrimidine tracts. The reported association between BP features and the splicing outcome suggests that this, so far disregarded but yet crucial, element buries information that can complement current acceptor site models

The Expanding Landscape of Alternative Splicing Variation in Human Populations.

Alternative splicing is a tightly regulated biological process by which the number of gene products for any given gene can be greatly expanded. Genomic variants in splicing regulatory sequences can disrupt splicing and cause disease. Recent developments in sequencing technologies and computational biology have allowed researchers to investigate alternative splicing at an unprecedented scale and resolution. Population-scale transcriptome studies have revealed many naturally occurring genetic variants that modulate alternative splicing and consequently influence phenotypic variability and disease susceptibility in human populations. Innovations in experimental and computational tools such as massively parallel reporter assays and deep learning have enabled the rapid screening of genomic variants for their causal impacts on splicing. In this review, we describe technological advances that have greatly increased the speed and scale at which discoveries are made about the genetic variation of alternative splicing. We summarize major findings from population transcriptomic studies of alternative splicing and discuss the implications of these findings for human genetics and medicine

Genome-Wide Profiling of Circular RNAs in the Rapidly Growing Shoots of Moso Bamboo (Phyllostachys edulis).

Circular RNAs, including circular exonic RNAs (circRNA), circular intronic RNAs (ciRNA) and exon-intron circRNAs (EIciRNAs), are a new type of noncoding RNAs. Growing shoots of moso bamboo (Phyllostachys edulis) represent an excellent model of fast growth and their circular RNAs have not been studied yet. To understand the potential regulation of circular RNAs, we systematically characterized circular RNAs from eight different developmental stages of rapidly growing shoots. Here, we identified 895 circular RNAs including a subset of mutually inclusive circRNA. These circular RNAs were generated from 759 corresponding parental coding genes involved in cellulose, hemicellulose and lignin biosynthetic process. Gene co-expression analysis revealed that hub genes, such as DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1), MAINTENANCE OF METHYLATION (MOM), dicer-like 3 (DCL3) and ARGONAUTE 1 (AGO1), were significantly enriched giving rise to circular RNAs. The expression level of these circular RNAs presented correlation with its linear counterpart according to transcriptome sequencing. Further protoplast transformation experiments indicated that overexpressing circ-bHLH93 generating from transcription factor decreased its linear transcript. Finally, the expression profiles suggested that circular RNAs may have interplay with miRNAs to regulate their cognate linear mRNAs, which was further supported by overexpressing miRNA156 decreasing the transcript of circ-TRF-1 and linear transcripts of TRF-1. Taken together, the overall profile of circular RNAs provided new insight into an unexplored category of long noncoding RNA regulation in moso bamboo

Control of human papillomavirus gene expression by alternative splicing

Human papillomaviruses possess circular double stranded DNA genomes of around 8 kb in size from which multiple mRNAs are synthesized during an infectious life cycle. Although at least three viral promoters are used to initiate transcription, viral mRNAs are largely the product of processing of pre-mRNAs by alternative splicing and polyadenylation. The HPV life cycle and viral gene expression are tightly linked to differentiation of the epithelium the virus infects: there is an orchestrated production of viral mRNAs and proteins. In this review we describe viral mRNA expression and the roles of the SR and hnRNP proteins that respectively positively and negatively regulate splicing. We discuss HPV regulation of splicing factors and detail the evidence that the papillomavirus E2 protein has splicing-related activities. We highlight the possibility that HPV-mediated control of splicing in differentiating epithelial cells may be necessary to accomplish the viral replication cycle

Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome.

Introns are a prevalent feature of eukaryotic genomes, yet their origins and contributions to genome function and evolution remain mysterious. In budding yeast, repression of the highly transcribed intron-containing ribosomal protein genes (RPGs) globally increases splicing of non-RPG transcripts through reduced competition for the spliceosome. We show that under these "hungry spliceosome" conditions, splicing occurs at more than 150 previously unannotated locations we call protointrons that do not overlap known introns. Protointrons use a less constrained set of splice sites and branchpoints than standard introns, including in one case AT-AC in place of GT-AG. Protointrons are not conserved in all closely related species, suggesting that most are not under positive selection and are fated to disappear. Some are found in non-coding RNAs (e. g. CUTs and SUTs), where they may contribute to the creation of new genes. Others are found across boundaries between noncoding and coding sequences, or within coding sequences, where they offer pathways to the creation of new protein variants, or new regulatory controls for existing genes. We define protointrons as (1) nonconserved intron-like sequences that are (2) infrequently spliced, and importantly (3) are not currently understood to contribute to gene expression or regulation in the way that standard introns function. A very few protointrons in S. cerevisiae challenge this classification by their increased splicing frequency and potential function, consistent with the proposed evolutionary process of "intronization", whereby new standard introns are created. This snapshot of intron evolution highlights the important role of the spliceosome in the expansion of transcribed genomic sequence space, providing a pathway for the rare events that may lead to the birth of new eukaryotic genes and the refinement of existing gene function

De novo prediction of PTBP1 binding and splicing targets reveals unexpected features of its RNA recognition and function.

The splicing regulator Polypyrimidine Tract Binding Protein (PTBP1) has four RNA binding domains that each binds a short pyrimidine element, allowing recognition of diverse pyrimidine-rich sequences. This variation makes it difficult to evaluate PTBP1 binding to particular sites based on sequence alone and thus to identify target RNAs. Conversely, transcriptome-wide binding assays such as CLIP identify many in vivo targets, but do not provide a quantitative assessment of binding and are informative only for the cells where the analysis is performed. A general method of predicting PTBP1 binding and possible targets in any cell type is needed. We developed computational models that predict the binding and splicing targets of PTBP1. A Hidden Markov Model (HMM), trained on CLIP-seq data, was used to score probable PTBP1 binding sites. Scores from this model are highly correlated (ρ = -0.9) with experimentally determined dissociation constants. Notably, we find that the protein is not strictly pyrimidine specific, as interspersed Guanosine residues are well tolerated within PTBP1 binding sites. This model identifies many previously unrecognized PTBP1 binding sites, and can score PTBP1 binding across the transcriptome in the absence of CLIP data. Using this model to examine the placement of PTBP1 binding sites in controlling splicing, we trained a multinomial logistic model on sets of PTBP1 regulated and unregulated exons. Applying this model to rank exons across the mouse transcriptome identifies known PTBP1 targets and many new exons that were confirmed as PTBP1-repressed by RT-PCR and RNA-seq after PTBP1 depletion. We find that PTBP1 dependent exons are diverse in structure and do not all fit previous descriptions of the placement of PTBP1 binding sites. Our study uncovers new features of RNA recognition and splicing regulation by PTBP1. This approach can be applied to other multi-RRM domain proteins to assess binding site degeneracy and multifactorial splicing regulation

Somatic Mutational Landscape of Splicing Factor Genes and Their Functional Consequences across 33 Cancer Types

Hotspot mutations in splicing factor genes have been recently reported at high frequency in hematological malignancies, suggesting the importance of RNA splicing in cancer. We analyzed whole-exome sequencing data across 33 tumor types in The Cancer Genome Atlas (TCGA), and we identified 119 splicing factor genes with significant non-silent mutation patterns, including mutation over-representation, recurrent loss of function (tumor suppressor-like), or hotspot mutation profile (oncogene-like). Furthermore, RNA sequencing analysis revealed altered splicing events associated with selected splicing factor mutations. In addition, we were able to identify common gene pathway profiles associated with the presence of these mutations. Our analysis suggests that somatic alteration of genes involved in the RNA-splicing process is common in cancer and may represent an underappreciated hallmark of tumorigenesis

Expand+Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors

Despite the critical role of pre-mRNA splicing in generating proteomic diversity and regulating gene expression, the sequence composition and function of intronic splicing regulatory elements (ISREs) have not been well elucidated. Here, we employed a high-throughput in vivo Screening PLatform for Intronic Control Elements (SPLICE) to identify 125 unique ISRE sequences from a random nucleotide library in human cells. Bioinformatic analyses reveal consensus motifs that resemble splicing regulatory elements and binding sites for characterized splicing factors and that are enriched in the introns of naturally occurring spliced genes, supporting their biological relevance. In vivo characterization, including an RNAi silencing study, demonstrate that ISRE sequences can exhibit combinatorial regulatory activity and that multiple trans-acting factors are involved in the regulatory effect of a single ISRE. Our work provides an initial examination into the sequence characteristics and function of ISREs, providing an important contribution to the splicing code

Computational Identification of Four Spliceosomal snRNAs from the Deep-Branching Eukaryote Giardia intestinalis

Funding: Marsden Fund New Zealand Allan Wilson Centre The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.RNAs processing other RNAs is very general in eukaryotes, but is not clear to what extent it is ancestral to eukaryotes. Here we focus on pre-mRNA splicing, one of the most important RNA-processing mechanisms in eukaryotes. In most eukaryotes splicing is predominantly catalysed by the major spliceosome complex, which consists of five uridine-rich small nuclear RNAs (U-snRNAs) and over 200 proteins in humans. Three major spliceosomal introns have been found experimentally in Giardia; one Giardia U-snRNA (U5) and a number of spliceosomal proteins have also been identified. However, because of the low sequence similarity between the Giardia ncRNAs and those of other eukaryotes, the other U-snRNAs of Giardia had not been found. Using two computational methods, candidates for Giardia U1, U2, U4 and U6 snRNAs were identified in this study and shown by RT-PCR to be expressed. We found that identifying a U2 candidate helped identify U6 and U4 based on interactions between them. Secondary structural modelling of the Giardia U-snRNA candidates revealed typical features of eukaryotic U-snRNAs. We demonstrate a successful approach to combine computational and experimental methods to identify expected ncRNAs in a highly divergent protist genome. Our findings reinforce the conclusion that spliceosomal small-nuclear RNAs existed in the last common ancestor of eukaryotes