Pre-mRNA splicing and its cotranscriptional connections.

Transcription of eukaryotic genes by RNA polymerase II (Pol II) yields RNA precursors containing introns that must be spliced out and the flanking exons ligated together. Splicing is catalyzed by a dynamic ribonucleoprotein complex called the spliceosome. Recent evidence has shown that a large fraction of splicing occurs cotranscriptionally as the RNA chain is extruded from Pol II at speeds of up to 5 kb/minute. Splicing is more efficient when it is tethered to the transcription elongation complex, and this linkage permits functional coupling of splicing with transcription. We discuss recent progress that has uncovered a network of connections that link splicing to transcript elongation and other cotranscriptional RNA processing events.

Exon definitive regions for MPC1 microexon splicing and its usage for splicing modulation.

Alternative splicing of microexons (3-30 base pairs [bp]) is involved in important biological processes in brain development and human cancers. However, understanding a splicing process of non-3x bp microexons is scarce. We showed that 4 bp microexon of mitochondrial pyruvate carrier1 (MPC1) is constitutively included in mRNA. Based on our studies with minigene and exon island constructs, we found the strong exon definition region in the proximal introns bordering MPC1 microexon. Ultimately, we defined a nucleotide fragment from the 3'ss 67 bp of MPC1 microexon to the 5'ss consensus sequence, as a core exon island, which can concatenate its microexon and neighboring exons by splicing. Furthermore, we showed that insertion of the core exon island into a target exon or intron induced skip the target exon or enhance the splicing of an adjacent exon, respectively. Collectively, we suggest that the exon island derived from MPC1 microexon modifies genuine splicing patterns depending on its position, thereby providing insights on strategies for splicing-mediated gene correction.

Profiling lariat intermediates reveals genetic determinants of early and late co-transcriptional splicing.

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.

Transcriptome-Wide Detection of Intron/Exon Definition in the Endogenous Pre-mRNA Transcripts of Mammalian Cells and Its Regulation by Depolarization.

Pairing of splice sites across an intron or exon is the central point of intron or exon definition in pre-mRNA splicing with the latter mode proposed for most mammalian exons. However, transcriptome-wide pairing within endogenous transcripts has not been examined for the prevalence of each mode in mammalian cells. Here we report such pairings in rat GH3 pituitary cells by measuring the relative abundance of nuclear RNA-Seq reads at the intron start or end (RISE). Interestingly, RISE indexes are positively correlated between 5' and 3' splice sites specifically across introns or exons but inversely correlated with the usage of adjacent exons. Moreover, the ratios between the paired indexes were globally modulated by depolarization, which was disruptible by 5-aza-Cytidine. The nucleotide matrices of the RISE-positive splice sites deviate significantly from the rat consensus, and short introns or exons are enriched with the cross-intron or -exon RISE pairs, respectively. Functionally, the RISE-positive genes cluster for basic cellular processes including RNA binding/splicing, or more specifically, hormone production if regulated by depolarization. Together, the RISE analysis identified the transcriptome-wide regulation of either intron or exon definition between weak splice sites of short introns/exons in mammalian cells. The analysis also provides a way to further track the splicing intermediates and intron/exon definition during the dynamic regulation of alternative splicing by extracellular factors.

Splice site proximity influences alternative exon definition.

Alternative splicing enables higher eukaryotes to expand mRNA diversity from a finite number of genes through highly combinatorial splice site selection mechanisms that are influenced by the sequence of competing splice sites, cis-regulatory elements binding trans-acting factors, the length of exons and introns harbouring alternative splice sites and RNA secondary structures at putative splice junctions. To test the hypothesis that the intron definition or exon definition modes of splice site recognition direct the selection of alternative splice patterns, we created a database of alternative splice site usage (ALTssDB). When alternative splice sites are embedded within short introns (intron definition), the 5' and 3' splice sites closest to each other across the intron preferentially pair, consistent with previous observations. However, when alternative splice sites are embedded within large flanking introns (exon definition), the 5' and 3' splice sites closest to each other across the exon are preferentially selected. Thus, alternative splicing decisions are influenced by the intron and exon definition modes of splice site recognition. The results demonstrate that the spliceosome pairs splice sites that are closest in proximity within the unit of initial splice site selection.

Gene architecture directs splicing outcome in separate nuclear spatial regions.

How the splicing machinery defines exons or introns as the spliced unit has remained a puzzle for 30 years. Here, we demonstrate that peripheral and central regions of the nucleus harbor genes with two distinct exon-intron GC content architectures that differ in the splicing outcome. Genes with low GC content exons, flanked by long introns with lower GC content, are localized in the periphery, and the exons are defined as the spliced unit. Alternative splicing of these genes results in exon skipping. In contrast, the nuclear center contains genes with a high GC content in the exons and short flanking introns. Most splicing of these genes occurs via intron definition, and aberrant splicing leads to intron retention. We demonstrate that the nuclear periphery and center generate different environments for the regulation of alternative splicing and that two sets of splicing factors form discrete regulatory subnetworks for the two gene architectures. Our study connects 3D genome organization and splicing, thus demonstrating that exon and intron definition modes of splicing occur in different nuclear regions.

Exon-independent recruitment of SRSF1 is mediated by U1 snRNP stem-loop 3.

SRSF1 protein and U1 snRNPs are closely connected splicing factors. They both stimulate exon inclusion, SRSF1 by binding to exonic splicing enhancer sequences (ESEs) and U1 snRNPs by binding to the downstream 5' splice site (SS), and both factors affect 5' SS selection. The binding of U1 snRNPs initiates spliceosome assembly, but SR proteins such as SRSF1 can in some cases substitute for it. The mechanistic basis of this relationship is poorly understood. We show here by single-molecule methods that a single molecule of SRSF1 can be recruited by a U1 snRNP. This reaction is independent of exon sequences and separate from the U1-independent process of binding to an ESE. Structural analysis and cross-linking data show that SRSF1 contacts U1 snRNA stem-loop 3, which is required for splicing. We suggest that the recruitment of SRSF1 to a U1 snRNP at a 5'SS is the basis for exon definition by U1 snRNP and might be one of the principal functions of U1 snRNPs in the core reactions of splicing in mammals.

At the Intersection of Major and Minor Spliceosomes: Crosstalk Mechanisms and Their Impact on Gene Expression.

Many eukaryotic species contain two separate molecular machineries for removing non-coding intron sequences from pre-mRNA molecules. The majority of introns (more than 99.5% in humans) are recognized and excised by the major spliceosome, which utilizes relatively poorly conserved sequence elements at the 5' and 3' ends of the intron that are used for intron recognition and in subsequent catalysis. In contrast, the minor spliceosome targets a rare group of introns (approximately 0.5% in humans) with highly conserved sequences at the 5' and 3' ends of the intron. Minor introns coexist in the same genes with major introns and while the two intron types are spliced by separate spliceosomes, the two splicing machineries can interact with one another to shape mRNA processing events in genes containing minor introns. Here, we review known cooperative and competitive interactions between the two spliceosomes and discuss the mechanistic basis of the spliceosome crosstalk, its regulatory significance, and impact on spliceosome diseases.

Transcription and splicing: A two-way street.

RNA synthesis by RNA polymerase II and RNA processing are closely coupled during the transcription cycle of protein-coding genes. This coupling affords opportunities for quality control and regulation of gene expression and the effects can go in both directions. For example, polymerase speed can affect splice site selection and splicing can increase transcription and affect the chromatin landscape. Here we review the many ways that transcription and splicing influence one another, including how splicing "talks back" to transcription. We will also place the connections between transcription and splicing in the context of other RNA processing events that define the exons that will make up the final mRNA. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing.

Splicing Kinetics and Coordination Revealed by Direct Nascent RNA Sequencing through Nanopores.

Understanding how splicing events are coordinated across numerous introns in metazoan RNA transcripts requires quantitative analyses of transient RNA processing events in living cells. We developed nanopore analysis of co-transcriptional processing (nano-COP), in which nascent RNAs are directly sequenced through nanopores, exposing the dynamics and patterns of RNA splicing without biases introduced by amplification. Long nano-COP reads reveal that, in human and Drosophila cells, splicing occurs after RNA polymerase II transcribes several kilobases of pre-mRNA, suggesting that metazoan splicing transpires distally from the transcription machinery. Inhibition of the branch-site recognition complex SF3B rapidly diminished global co-transcriptional splicing. We found that splicing order does not strictly follow the order of transcription and is associated with cis-acting elements, alternative splicing, and RNA-binding factors. Further, neighboring introns in human cells tend to be spliced concurrently, implying that splicing of these introns occurs cooperatively. Thus, nano-COP unveils the organizational complexity of RNA processing.

Alternative Splicing Regulatory Networks: Functions, Mechanisms, and Evolution.

High-throughput sequencing-based methods and their applications in the study of transcriptomes have revolutionized our understanding of alternative splicing. Networks of functionally coordinated and biologically important alternative splicing events continue to be discovered in an ever-increasing diversity of cell types in the context of physiologically normal and disease states. These studies have been complemented by efforts directed at defining sequence codes governing splicing and their cognate trans-acting factors, which have illuminated important combinatorial principles of regulation. Additional studies have revealed critical roles of position-dependent, multivalent protein-RNA interactions that direct splicing outcomes. Investigations of evolutionary changes in RNA binding proteins, splice variants, and associated cis elements have further shed light on the emergence, mechanisms, and functions of splicing networks. Progress in these areas has emphasized the need for a coordinated, community-based effort to systematically address the functions of individual splice variants associated with normal and disease biology.

Genome-wide CRISPR-Cas9 Interrogation of Splicing Networks Reveals a Mechanism for Recognition of Autism-Misregulated Neuronal Microexons.

Alternative splicing is crucial for diverse cellular, developmental, and pathological processes. However, the full networks of factors that control individual splicing events are not known. Here, we describe a CRISPR-based strategy for the genome-wide elucidation of pathways that control splicing and apply it to microexons with important functions in nervous system development and that are commonly misregulated in autism. Approximately 200 genes associated with functionally diverse regulatory layers and enriched in genetic links to autism control neuronal microexons. Remarkably, the widely expressed RNA binding proteins Srsf11 and Rnps1 directly, preferentially, and frequently co-activate these microexons. These factors form critical interactions with the neuronal splicing regulator Srrm4 and a bi-partite intronic splicing enhancer element to promote spliceosome formation. Our study thus presents a versatile system for the identification of entire splicing regulatory pathways and further reveals a common mechanism for the definition of neuronal microexons that is disrupted in autism.

RNA Polymerase II Phosphorylated on CTD Serine 5 Interacts with the Spliceosome during Co-transcriptional Splicing.

The highly intronic nature of protein coding genes in mammals necessitates a co-transcriptional splicing mechanism as revealed by mNET-seq analysis. Immunoprecipitation of MNase-digested chromatin with antibodies against RNA polymerase II (Pol II) shows that active spliceosomes (both snRNA and proteins) are complexed to Pol II S5P CTD during elongation and co-transcriptional splicing. Notably, elongating Pol II-spliceosome complexes form strong interactions with nascent transcripts, resulting in footprints of approximately 60 nucleotides. Also, splicing intermediates formed by cleavage at the 5' splice site are associated with nearly all spliced exons. These spliceosome-bound intermediates are frequently ligated to upstream exons, implying a sequential, constitutive, and U12-dependent splicing process. Finally, lack of detectable spliced products connected to the Pol II active site in human HeLa or murine lymphoid cells suggests that splicing does not occur immediately following 3' splice site synthesis. Our results imply that most mammalian splicing requires exon definition for completion.

Behind the scenes of HIV-1 replication: Alternative splicing as the dependency factor on the quiet.

Alternative splicing plays a key role in the HIV-1 life cycle and is essential to maintain an equilibrium of mRNAs that encode viral proteins and polyprotein-isoforms. In particular, since all early HIV-1 proteins are expressed from spliced intronless and late enzymatic and structural proteins from intron containing, i.e. splicing repressed viral mRNAs, cellular splicing factors and splicing regulatory proteins are crucial for the replication capacity. In this review, we will describe the complex network of cis-acting splicing regulatory elements (SREs), which are mainly localized in the neighbourhoods of all HIV-1 splice sites and warrant the proper ratio of individual transcript isoforms. Since SREs represent binding sites for trans-acting cellular splicing factors interacting with the cellular spliceosomal apparatus we will review the current knowledge of interactions between viral RNA and cellular proteins as well as their impact on viral replication. Finally, we will discuss potential therapeutic approaches targeting HIV-1 alternative splicing.

A global survey of alternative splicing in allopolyploid cotton: landscape, complexity and regulation.

Alternative splicing (AS) is a crucial regulatory mechanism in eukaryotes, which acts by greatly increasing transcriptome diversity. The extent and complexity of AS has been revealed in model plants using high-throughput next-generation sequencing. However, this technique is less effective in accurately identifying transcript isoforms in polyploid species because of the high sequence similarity between coexisting subgenomes. Here we characterize AS in the polyploid species cotton. Using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq), we developed an integrated pipeline for Iso-Seq transcriptome data analysis (https://github.com/Nextomics/pipeline-for-isoseq). We identified 176 849 full-length transcript isoforms from 44 968 gene models and updated gene annotation. These data led us to identify 15 102 fibre-specific AS events and estimate that c. 51.4% of homoeologous genes produce divergent isoforms in each subgenome. We reveal that AS allows differential regulation of the same gene by miRNAs at the isoform level. We also show that nucleosome occupancy and DNA methylation play a role in defining exons at the chromatin level. This study provides new insights into the complexity and regulation of AS, and will enhance our understanding of AS in polyploid species. Our methodology for Iso-Seq data analysis will be a useful reference for the study of AS in other species.

The Output of Protein-Coding Genes Shifts to Circular RNAs When the Pre-mRNA Processing Machinery Is Limiting.

Many eukaryotic genes generate linear mRNAs and circular RNAs, but it is largely unknown how the ratio of linear to circular RNA is controlled or modulated. Using RNAi screening in Drosophila cells, we identify many core spliceosome and transcription termination factors that control the RNA outputs of reporter and endogenous genes. When spliceosome components were depleted or inhibited pharmacologically, the steady-state levels of circular RNAs increased while expression of their associated linear mRNAs concomitantly decreased. Upon inhibiting RNA polymerase II termination via depletion of the cleavage/polyadenylation machinery, circular RNA levels were similarly increased. This is because readthrough transcripts now extend into downstream genes and are subjected to backsplicing. In total, these results demonstrate that inhibition or slowing of canonical pre-mRNA processing events shifts the steady-state output of protein-coding genes toward circular RNAs. This is in part because nascent RNAs become directed into alternative pathways that lead to circular RNA production.

The power of fission: yeast as a tool for understanding complex splicing.

Pre-mRNA splicing is an essential component of eukaryotic gene expression. Many metazoans, including humans, regulate alternative splicing patterns to generate expansions of their proteome from a limited number of genes. Importantly, a considerable fraction of human disease causing mutations manifest themselves through altering the sequences that shape the splicing patterns of genes. Thus, understanding the mechanistic bases of this complex pathway will be an essential component of combating these diseases. Dating almost to the initial discovery of splicing, researchers have taken advantage of the genetic tractability of budding yeast to identify the components and decipher the mechanisms of splicing. However, budding yeast lacks the complex splicing machinery and alternative splicing patterns most relevant to humans. More recently, many researchers have turned their efforts to study the fission yeast, Schizosaccharomyces pombe, which has retained many features of complex splicing, including degenerate splice site sequences, the usage of exonic splicing enhancers, and SR proteins. Here, we review recent work using fission yeast genetics to examine pre-mRNA splicing, highlighting its promise for modeling the complex splicing seen in higher eukaryotes.

Cutting a Long Intron Short: Recursive Splicing and Its Implications.

Over time eukaryotic genomes have evolved to host genes carrying multiple exons separated by increasingly larger intronic, mostly non-protein-coding, sequences. Initially, little attention was paid to these intronic sequences, as they were considered not to contain regulatory information. However, advances in molecular biology, sequencing, and computational tools uncovered that numerous segments within these genomic elements do contribute to the regulation of gene expression. Introns are differentially removed in a cell type-specific manner to produce a range of alternatively-spliced transcripts, and many span tens to hundreds of kilobases. Recent work in human and fruitfly tissues revealed that long introns are extensively processed cotranscriptionally and in a stepwise manner, before their two flanking exons are spliced together. This process, called "recursive splicing," often involves non-canonical splicing elements positioned deep within introns, and different mechanisms for its deployment have been proposed. Still, the very existence and widespread nature of recursive splicing offers a new regulatory layer in the transcript maturation pathway, which may also have implications in human disease.

Stable tri-snRNP integration is accompanied by a major structural rearrangement of the spliceosome that is dependent on Prp8 interaction with the 5' splice site.

Exon definition is the predominant initial spliceosome assembly pathway in higher eukaryotes, but it remains much less well-characterized compared to the intron-defined assembly pathway. Addition in trans of an excess of 5'ss containing RNA to a splicing reaction converts a 37S exon-defined complex, formed on a single exon RNA substrate, into a 45S B-like spliceosomal complex with stably integrated U4/U6.U5 tri-snRNP. This 45S complex is compositonally and structurally highly similar to an intron-defined spliceosomal B complex. Stable tri-snRNP integration during B-like complex formation is accompanied by a major structural change as visualized by electron microscopy. The changes in structure and stability during transition from a 37S to 45S complex can be induced in affinity-purified cross-exon complexes by adding solely the 5'ss RNA oligonucleotide. This conformational change does not require the B-specific proteins, which are recruited during this stabilization process, or site-specific phosphorylation of hPrp31. Instead it is triggered by the interaction of U4/U6.U5 tri-snRNP components with the 5'ss sequence, most importantly between Prp8 and nucleotides at the exon-intron junction. These studies provide novel insights into the conversion of a cross-exon to cross-intron organized spliceosome and also shed light on the requirements for stable tri-snRNP integration during B complex formation.

Splicing of designer exons informs a biophysical model for exon definition.

Pre-mRNA molecules in humans contain mostly short internal exons flanked by longer introns. To explain the removal of such introns, exon recognition instead of intron recognition has been proposed. We studied this exon definition using designer exons (DEs) made up of three prototype modules of our own design: an exonic splicing enhancer (ESE), an exonic splicing silencer (ESS), and a Reference Sequence (R) predicted to be neither. Each DE was examined as the central exon in a three-exon minigene. DEs made of R modules showed a sharp size dependence, with exons shorter than 14 nt and longer than 174 nt splicing poorly. Changing the strengths of the splice sites improved longer exon splicing but worsened shorter exon splicing, effectively displacing the curve to the right. For the ESE we found, unexpectedly, that its enhancement efficiency was independent of its position within the exon. For the ESS we found a step-wise positional increase in its effects; it was most effective at the 3' end of the exon. To apply these results quantitatively, we developed a biophysical model for exon definition of internal exons undergoing cotranscriptional splicing. This model features commitment to inclusion before the downstream exon is synthesized and competition between skipping and inclusion fates afterward. Collision of both exon ends to form an exon definition complex was incorporated to account for the effect of size; ESE/ESS effects were modeled on the basis of stabilization/destabilization. This model accurately predicted the outcome of independent experiments on more complex DEs that combined ESEs and ESSs.