Splicing Intermediates Research Articles

Branchpoints as potential targets of exon-skipping therapies for genetic disorders

Fukutin (FKTN) c.647+2084G>T creates a pseudo-exon with apremature stop codon, which causes Fukuyama congenital muscular dystrophy (FCMD). We aimed to ameliorate aberrant splicing of FKTN caused by this variant. We screened compounds focusing on splicing regulation using the c.647+2084G>T splicing reporter and discovered that the branchpoint, which is essential for splicing reactions, could be a potential therapeutic target. To confirm the effectiveness of branchpoints as targets for exon skipping, we designed branchpoint-targeted antisense oligonucleotides (BP-AONs). This restored normal FKTN mRNA and protein production in FCMD patient myotubes. We identified a functional BP by detecting splicing intermediates and creating BP mutations in the FKTN reporter gene; this BP was non-redundant and sufficiently blocked by BP-AONs. Next, a BP-AON was designed for a different FCMD-causing variant, which induces pathogenic exon trapping by a common SINE-VNTR-Alu-type retrotransposon. Notably, this BP-AON also restored normal FKTN mRNA and protein production in FCMD patient myotubes. Our findings suggest that BPs could be potential targets in exon-skipping therapeutic strategies for genetic disorders.

PacBio full-length transcriptome analysis provides new insights into transcription of chloroplast genomes

ABSTRACT Chloroplast and mitochondrial DNA (cpDNA and mtDNA) are apart from nuclear DNA (nuDNA) in a eukaryotic cell. The transcription system of chloroplasts differs from those of mitochondria and eukaryotes. In contrast to nuDNA and animal mtDNA, the transcription of cpDNA is still not well understood, primarily due to the unresolved identification of transcription initiation sites (TISs) and transcription termination sites (TTSs) on the genome scale. In the present study, we characterized the transcription of chloroplast (cp) genes with greater accuracy and comprehensive information using PacBio full-length transcriptome data from Arabidopsis thaliana. The major findings included the discovery of four types of artifacts, the validation and correction of cp gene annotations, the exact identification of TISs that start with G, and the discovery of polyA-like sites as TTSs. Notably, we proposed a new model to explain cp transcription initiation and termination at the whole-genome level. Four types of artifacts, degraded RNAs and splicing intermediates deserve the attention from researchers working with PacBio full-length transcriptome data, as these contaminant sequences can lead to incorrect downstream analysis. Cp transcription initiates at multiple promoters and terminates at polyA-like sites. Our study provides new insights into cp transcription and new clues to study the evolution of promoters, TISs, TTSs and polyA tails of eukaryotic genes.

Snapshots of the second-step self-splicing of Tetrahymena ribozyme revealed by cryo-EM

Group I introns are catalytic RNAs that coordinate two consecutive transesterification reactions for self-splicing. To understand how the group I intron promotes catalysis and coordinates self-splicing reactions, we determine the structures of L-16 Tetrahymena ribozyme in complex with a 5′-splice site analog product and a 3′-splice site analog substrate using cryo-EM. We solve six conformations from a single specimen, corresponding to different splicing intermediates after the first ester-transfer reaction. The structures reveal dynamics during self-splicing, including large conformational changes of the internal guide sequence and the J5/4 junction as well as subtle rearrangements of active-site metals and the hydrogen bond formed between the 2′-OH group of A261 and the N2 group of guanosine substrate. These results help complete a detailed structural and mechanistic view of this paradigmatic group I intron undergoing the second step of self-splicing.

Temporal-iCLIP captures co-transcriptional RNA-protein interactions

Dynamic RNA-protein interactions govern the co-transcriptional packaging of RNA polymerase II (RNAPII)-derived transcripts. Yet, our current understanding of this process in vivo primarily stems from steady state analysis. To remedy this, we here conduct temporal-iCLIP (tiCLIP), combining RNAPII transcriptional synchronisation with UV cross-linking of RNA-protein complexes at serial timepoints. We apply tiCLIP to the RNA export adaptor, ALYREF; a component of the Nuclear Exosome Targeting (NEXT) complex, RBM7; and the nuclear cap binding complex (CBC). Regardless of function, all tested factors interact with nascent RNA as it exits RNAPII. Moreover, we demonstrate that the two transesterification steps of pre-mRNA splicing temporally separate ALYREF and RBM7 binding to splicing intermediates, and that exon-exon junction density drives RNA 5′end binding of ALYREF. Finally, we identify underappreciated steps in snoRNA 3′end processing performed by RBM7. Altogether, our data provide a temporal view of RNA-protein interactions during the early phases of transcription.

Snapshots of the first-step self-splicing of Tetrahymena ribozyme revealed by cryo-EM.

Tetrahymena ribozyme is a group I intron, whose self-splicing is the result of two sequential ester-transfer reactions. To understand how it facilitates catalysis in the first self-splicing reaction, we used cryogenic electron microscopy (cryo-EM) to resolve the structures of L-16 Tetrahymena ribozyme complexed with a 11-nucleotide 5'-splice site analog substrate. Four conformations were achieved to 4.14, 3.18, 3.09 and 2.98 Å resolutions, respectively, corresponding to different splicing intermediates during the first enzymatic reaction. Comparison of these structures reveals structural alterations, including large conformational changes in IGS/IGSext (P1-P1ext duplex) and J5/4, as well as subtle local rearrangements in the G-binding site. These structural changes are required for the enzymatic activity of the Tetrahymena ribozyme. Our study demonstrates the ability of cryo-EM to capture dynamic RNA structural changes, ushering in a new era in the analysis of RNA structure-function by cryo-EM.

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.

Recursive splicing is a rare event in the mouse brain.

Recursive splicing (RS) is a splicing mechanism to remove long introns from messenger RNA precursors of long genes. Compared to the hundreds of RS events identified in humans and drosophila, only ten RS events have been reported in mice. To further investigate RS in mice, we analyzed RS in the mouse brain, a tissue that is enriched in the expression of long genes. We found that nuclear total RNA sequencing is an efficient approach to investigate RS events. We analyzed 1.15 billion uniquely mapped reads from the nuclear total RNA sequencing data in the mouse cerebral cortex. Unexpectedly, we only identified 20 RS sites, suggesting that RS is a rare event in the mouse brain. We also identified that RS is constitutive between excitatory and inhibitory neurons and between sexes in the mouse cerebral cortex. In addition, we found that the primary sequence context is associated with RS splicing intermediates and distinguishes RS AGGT site from non-RS AGGT sites, indicating the importance of the primary sequence context in RS sites. Moreover, we discovered that cryptic exons may use an RS-like mechanism for splicing. Overall, we provide novel findings about RS in long genes in the mouse brain.

Galectin-3-U1 snRNP Complexes Initiate Splicing Activity in U1-Depleted Nuclear Extracts.

Fractionation of HeLa cell nuclear extracts by glycerol gradient centrifugation separates endogenous uracil-rich small nuclear ribonucleoprotein complexes (U snRNP) into numerous particles sedimenting from 7S to greater than 60S. Complexes sedimenting at 10S contain a single U snRNP (U1 snRNP) and galectin-3. Addition of antibodies specific for galectin-3 to fractions containing these 10S complexes coprecipitates U1 snRNP, indicating that a fraction of the U1 snRNP is associated with this galectin. Galectin-3 has been shown by depletion-reconstitution studies to be an integral splicing component involved both in spliceosome assembly and splicing activity. The first step in initiation of spliceosome assembly is binding of U1 snRNP to the 5' splice site of the premessenger RNA substrate. The finding that U1 snRNP and galectin-3 are associated in splicing extracts hints that this complex affords a potential entry point for galectin-3 into the splicing pathway. Addition of U1 snRNP-galectin-3 complexes immunoselected from the 10S region of glycerol gradients to a U1-depleted nuclear extract initiates splicing activity with the formation of splicing intermediates and mature mRNA. This chapter describes the materials and methods for these experiments that document galectin-3-U1 snRNP complexes initiate the splicing reaction in a U1-depleted nuclear extract.

Detecting circRNA in purified spliceosomal P complex.

Circular RNAs (circRNAs) generated from back-splicing of exons have been found in a wide range of eukaryotic species and exert a variety of biological functions. Unlike canonical splicing, the mechanism of back-splicing has long remained elusive. We recently determined the cryo-EM structure of the yeast spliceosomal E complex assembled on introns, leading us to hypothesize that the same E complex can assemble across an exon forming the exon-definition complex. This complex, when assembled on long exons, goes through the splicing cycle and catalyzes back-splicing to generate circRNAs. Supporting this hypothesis, we purified the yeast post-catalytic spliceosomal P complex (the best complex in the splicing cycle to trap splicing products and intermediates) and detected canonical and back-splicing products as well as splicing intermediates. Here we describe in detail this procedure, which may be applied to other organisms to facilitate research on the biogenesis and regulation of circRNA.

Co-transcriptional splicing regulates 3′ end cleavage during mammalian erythropoiesis

Pre-mRNA processing steps are tightly coordinated with transcription in many organisms. To determine how co-transcriptional splicing is integrated with transcription elongation and 3' end formation in mammalian cells, we performed long-read sequencing of individual nascent RNAs and precision run-on sequencing (PRO-seq) during mouse erythropoiesis. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (Pol II) was within 75-300 nucleotides of 3' splice sites (3'SSs), often during transcription of the downstream exon. Interestingly, several hundred introns displayed abundant splicing intermediates, suggesting that splicing delays can take place between the two catalytic steps. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 3' end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 3'SS improved both splicing and 3' end cleavage of individual β-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.

A spliceosomal twin intron (stwintron) participates in both exon skipping and evolutionary exon loss

Spliceosomal twin introns (stwintrons) are introns where any of the three consensus sequences involved in splicing is interrupted by another intron (internal intron). In Aspergillus nidulans, a donor-disrupted stwintron (intron-1) is extant in the transcript encoding a reticulon-like protein. The orthologous transcript of Aspergillus niger can be alternatively spliced; the exon downstream the stwintron could be skipped by excising a sequence that comprises this stwintron, the neighbouring intron-2, and the exon bounded by these. This process involves the use of alternative 3′ splice sites for the internal intron, the resulting alternative intervening sequence being a longer 3′-extended stwintron. In 29 species of Onygenales, a multi-step splicing process occurs in the orthologous transcript, in which a complex intervening sequence including the stwintron and neigbouring intron-2, generates by three splicing reactions a “second order intron” which must then be excised with a fourth splicing event. The gene model in two species can be envisaged as one canonical intron (intron-1) evolved from this complex intervening sequence of nested canonical introns found elsewhere in Onygenales. Postulated splicing intermediates were experimentally verified in one or more species. This work illustrates a role of stwintrons in both alternative splicing and the evolution of intron structure.

Robust mapping of polyadenylated and non-polyadenylated RNA 3' ends at nucleotide resolution by 3'-end sequencing.

3'-end poly(A)+ sequencing is an efficient and economical method for global measurement of mRNA levels and alternative poly(A) site usage. A common method involves oligo(dT)19V reverse-transcription (RT)-based library preparation and high-throughput sequencing with a custom primer ending in (dT)19. While the majority of library products have the first sequenced nucleotide reflect the bona fide poly(A) site (pA), a substantial fraction of sequencing reads arise from various mis-priming events. These can result in incorrect pA site calls anywhere from several nucleotides downstream to several kilobases upstream from the bona fide pA site. While these mis-priming events can be mitigated by increasing annealing stringency (e.g. increasing temperature from 37 °C to 42 °C), they still persist at an appreciable level (∼10%) and computational methods must be used to prevent artifactual calls. Here we present a bioinformatics workflow for precise mapping of poly(A)+ 3' ends and handling of artifacts due to oligo(dT) mis-priming and sample polymorphisms. We test pA site calling with three different read mapping programs (STAR, BWA, and BBMap), and show that the way in which each handles terminal mismatches and soft clipping has a substantial impact on identifying correct pA sites, with BWA requiring the least post-processing to correct artifacts. We demonstrate the use of this pipeline for mapping pA sites in the model eukaryote S. cerevisiae, and further apply this technology to non-polyadenylated transcripts by employing in vitro polyadenylation prior to library prep (IVP-seq). As proof of principle, we show that a fraction of tRNAs harbor CCU 3' tails instead of the canonical CCA tail, and globally identify 3' ends of splicing intermediates arising from inefficiently spliced transcripts.

Multiplexed primer extension sequencing: A targeted RNA-seq method that enables high-precision quantitation of mRNA splicing isoforms and rare pre-mRNA splicing intermediates.

The study of pre-mRNA splicing has been greatly aided by the advent of RNA sequencing (RNA-seq), which enables the genome-wide detection of discrete splice isoforms. Quantification of these splice isoforms requires analysis of splicing informative sequencing reads, those that unambiguously map to a single splice isoform, including exon-intron spanning alignments corresponding to retained introns, as well as exon-exon junction spanning alignments corresponding to either canonically- or alternatively-spliced isoforms. Because most RNA-seq experiments are designed to produce sequencing alignments that uniformly cover the entirety of transcripts, only a comparatively small number of splicing informative alignments are generated for any given splice site, leading to a decreased ability to detect and/or robustly quantify many splice isoforms. To address this problem, we have recently described a method termed Multiplexed Primer Extension sequencing, or MPE-seq, which uses pools of reverse transcription primers to target sequencing to user selected loci. By targeting reverse transcription to pre-mRNA splice junctions, this approach enables a dramatic enrichment in the fraction of splicing informative alignments generated per splicing event, yielding an increase in both the precision with which splicing efficiency can be measured, and in the detection of splice isoforms including rare splicing intermediates. Here we provide a brief review of the shortcomings associated with RNA-seq that drove our development of MPE-seq, as well as a detailed protocol for implementation of MPE-seq.

Multiple decay events target HAC1 mRNA during splicing to regulate the unfolded protein response.

In the unfolded protein response (UPR), stress in the endoplasmic reticulum (ER) activates a large transcriptional program to increase ER folding capacity. During the budding yeast UPR, Ire1 excises an intron from the HAC1 mRNA and the exon products of cleavage are ligated, and the translated protein induces hundreds of stress-response genes. Using cells with mutations in RNA repair and decay enzymes, we show that phosphorylation of two different HAC1 splicing intermediates is required for their degradation by the 5'→3' exonuclease Xrn1 to enact opposing effects on the UPR. We also found that ligated but 2'-phosphorylated HAC1 mRNA is cleaved, yielding a decay intermediate with both 5'- and 2'-phosphates at its 5'-end that inhibit 5'→3' decay and suggesting that Ire1 degrades incompletely processed HAC1. These decay events expand the scope of RNA-based regulation in the budding yeast UPR and have implications for the control of the metazoan UPR.

Detection of splice isoforms and rare intermediates using multiplexed primer extension sequencing.

Targeted RNA-sequencing aims to focus coverage on areas of interest that are inadequately sampled in standard RNA-sequencing experiments. Here we present a novel approach for targeted RNA-sequencing that uses complex pools of reverse transcription primers to enable sequencing enrichment at user-selected locations across the genome. We demonstrate this approach by targeting hundreds to thousands of pre-mRNA splice junctions, revealing high-precision detection of splice isoforms, including rare pre-mRNA splicing intermediates.

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

SummaryThe 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.

Numerous recursive sites contribute to accuracy of splicing in long introns in flies.

Recursive splicing, a process by which a single intron is removed from pre-mRNA transcripts in multiple distinct segments, has been observed in a small subset of Drosophila melanogaster introns. However, detection of recursive splicing requires observation of splicing intermediates that are inherently unstable, making it difficult to study. Here we developed new computational approaches to identify recursively spliced introns and applied them, in combination with existing methods, to nascent RNA sequencing data from Drosophila S2 cells. These approaches identified hundreds of novel sites of recursive splicing, expanding the catalog of recursively spliced fly introns by 4-fold. A subset of recursive sites were validated by RT-PCR and sequencing. Recursive sites occur in most very long (> 40 kb) fly introns, including many genes involved in morphogenesis and development, and tend to occur near the midpoints of introns. Suggesting a possible function for recursive splicing, we observe that fly introns with recursive sites are spliced more accurately than comparably sized non-recursive introns.

Emerging Roles of Plant Circular RNAs

Circular RNAs (circRNAs) are covalently closed single-stranded loop RNA molecules with or without protein coding capability. CircRNAs were previously considered to be splicing intermediates or artifacts but are now found to be pervasively expressed in all eukaryotes studied with some demonstrated to have important molecular functions in various biological processes. CircRNA is now a hot study topic of molecular biology. In this review, we summarize the progress achieved so far on plant circRNAs, including identification and functional characterization, compare the similarities and differences of circRNAs between plants and animals, and discuss the challenges for confident detection and functional investigation of plant circRNAs. Similar to what have been found in animals, plant genomes contain a large number of circRNAs that potentially regulate a wide range of biological progresses related to plant development and biotic/abiotic responses. Despite only a few plant circRNAs have been functionally characterized, novel function/mechanism that has not been reported in animals was revealed, implying more exciting findings about plant circRNAs are expected in future studies.

Genetic bypass of essential RNA repair enzymes in budding yeast.

RNA repair enzymes catalyze rejoining of an RNA molecule after cleavage of phosphodiester linkages. RNA repair in budding yeast is catalyzed by two separate enzymes that process tRNA exons during their splicing and HAC1 mRNA exons during activation of the unfolded protein response (UPR). The RNA ligase Trl1 joins 2′,3′-cyclic phosphate and 5′-hydroxyl RNA fragments, creating a phosphodiester linkage with a 2′-phosphate at the junction. The 2′-phosphate is removed by the 2′-phosphotransferase Tpt1. We bypassed the essential functions of TRL1 and TPT1 in budding yeast by expressing “prespliced,” intronless versions of the 10 normally intron-containing tRNAs, indicating this repair pathway does not have additional essential functions. Consistent with previous studies, expression of intronless tRNAs failed to rescue the growth of cells with deletions in components of the SEN complex, implying an additional essential role for the splicing endonuclease. The trl1Δ and tpt1Δ mutants accumulate tRNA and HAC1 splicing intermediates indicative of RNA repair defects and are hypersensitive to drugs that inhibit translation. Failure to induce the unfolded protein response in trl1Δ cells grown with tunicamycin is lethal owing to their inability to ligate HAC1 after its cleavage by Ire1. In contrast, tpt1Δ mutants grow in the presence of tunicamycin despite reduced accumulation of spliced HAC1 mRNA. We optimized a PCR-based method to detect RNA 2′-phosphate modifications and show they are present on ligated HAC1 mRNA. These RNA repair mutants enable new studies of the role of RNA repair in cellular physiology.

Export of discarded, splicing intermediates provides insight into mRNA export

The precise removal of introns is essential for cell survival. To promote fidelity in nuclear pre‐‐mRNA splicing, the spliceosome rejects and discards suboptimal splicing substrates that have engaged the spliceosome. Although these discarded splicing substrates can be degraded in the nucleus, discarded splicing species, the 5′ exon and lariat intermediate, can be exported to the cytoplasm, as the discarded species can be translated and degraded by cytoplasmic nucleases. However, it is unknown how these suboptimal intermediates are exported.Here, we used single molecule RNA‐FISH in budding yeast to provide direct evidence that cells export suboptimal splicing intermediates, both the 5′ exon and lariat intermediate. We observed that the export of suboptimal splicing intermediates requires the nuclear basket and the general mRNA export receptor Mex67. Further, we found that two essential Mex67 adaptors, Yra1 and Nab2, are required for exporting suboptimal lariat intermediates, demonstrating that both mRNAs and lariat intermediates undergo a similar process to assemble export machinery. Surprisingly, we discovered that the export of lariat intermediates requires Mlp1, a factor implicated in retaining unspliced pre‐‐mRNA in the nucleus of budding yeast. Importantly, Mlp1 promoted the export of lariat intermediates through its interaction with Nab2. Together, our findings establish the importance of the mRNA export pathway in exporting discarded splicing intermediates and implicate a dual role for Mlp1 in not only retaining unspliced transcripts but also exporting partially spliced transcripts. We propose that this dual role of Mlp1 reflects a function in docking mRNAs at the nuclear basket during export.Support or Funding InformationNIH