Dynamic imaging of nascent RNA reveals general principles of transcription dynamics and stochastic splice site selection.

The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the dynamics of these megadalton enzymatic complexes working in concert on endogenous genes have not been described. Here, we establish a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time. We find that all observed genes show transcriptional bursting. We also observe large kinetic variation in intron removal for single introns in single cells, which is inconsistent with deterministic splice site selection. Transcriptome-wide footprinting of the U2AF complex, nascent RNA profiling, long-read sequencing, and lariat sequencing further reveal widespread stochastic recursive splicing within introns. We propose and validate a unified theoretical model to explain the general features of transcription and pervasive stochastic splice site selection.

SnapShot: Splicing Alterations in Cancer.

RNA splicing, the spliceosome-catalyzed process by which pre-messenger RNA (pre-mRNA) is processed to mature mRNA, is altered in a number of ways in cancer. Tumor-specific splicing alterations are created by mutations that disrupt splicing-regulatory elements within genes and impair splicing recognition or by altering the RNA-binding preferences of individual splicing factors. This SnapShot summarizes our current understanding of splicing-factor alterations in cancers. To view this SnapShot, open or download the PDF.

Initiation of Parental Genome Reprogramming in Fertilized Oocyte by Splicing Kinase SRPK1-Catalyzed Protamine Phosphorylation.

The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.

Structures of the Catalytically Activated Yeast Spliceosome Reveal the Mechanism of Branching.

Pre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B∗) is pivotal for understanding the branching reaction. In this study, we assembled the B∗ complexes on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B∗ complexes at overall resolutions of 2.9-3.8 Å. The duplex between U2 small nuclear RNA (snRNA) and the branch point sequence (BPS) is discretely away from the 5'-splice site (5'SS) in the three B∗ complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5'SS, with the BPS nucleophile positioned 4 Å away from the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in branching. These structures on different pre-mRNAs reveal substrate-specific conformations of the spliceosome in a major functional state.

Mechanistic insights into precursor messenger RNA splicing by the spliceosome.

Precursor messenger RNA (pre-mRNA) splicing is an essential step in the flow of information from DNA to protein in all eukaryotes. Research over the past four decades has molecularly delineated the splicing pathway, including characterization of the detailed splicing reaction, definition of the spliceosome and identification of its components, and biochemical analysis of the various splicing complexes and their regulation. Structural information is central to mechanistic understanding of pre-mRNA splicing by the spliceosome. X-ray crystallography of the spliceosomal components and subcomplexes is complemented by electron microscopy of the intact spliceosome. In this Review, I discuss recent atomic-resolution structures of the intact spliceosome at different stages of the splicing cycle. These structures have provided considerable mechanistic insight into pre-mRNA splicing and have corroborated and explained a large body of genetic and biochemical data. Together, the structural data have proved that the spliceosome is a protein-directed metalloribozyme.

The oxidoreductase PYROXD1 uses NAD(P)+ as an antioxidant to sustain tRNA ligase activity in pre-tRNA splicing and unfolded protein response.

The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism.

Termination of pre-mRNA splicing requires that the ATPase and RNA unwindase Prp43p acts on the catalytic snRNA U6.

The termination of pre-mRNA splicing functions to discard suboptimal substrates, thereby enhancing fidelity, and to release excised introns in a manner coupled to spliceosome disassembly, thereby allowing recycling. The mechanism of termination, including the RNA target of the DEAH-box ATPase Prp43p, remains ambiguous. We discovered a critical role for nucleotides at the 3' end of the catalytic U6 small nuclear RNA in splicing termination. Although conserved sequence at the 3' end is not required, 2' hydroxyls are, paralleling requirements for Prp43p biochemical activities. Although the 3' end of U6 is not required for recruiting Prp43p to the spliceosome, the 3' end cross-links directly to Prp43p in an RNA-dependent manner. Our data indicate a mechanism of splicing termination in which Prp43p translocates along U6 from the 3' end to disassemble the spliceosome and thereby release suboptimal substrates or excised introns. This mechanism reveals that the spliceosome becomes primed for termination at the same stage it becomes activated for catalysis, implying a requirement for stringent control of spliceosome activity within the cell.

Exon Junction Complex Shapes the Transcriptome by Repressing Recursive Splicing.

Recursive splicing (RS) starts by defining an "RS-exon," which is then spliced to the preceding exon, thus creating a recursive 5' splice site (RS-5ss). Previous studies focused on cryptic RS-exons, and now we find that the exon junction complex (EJC) represses RS of hundreds of annotated, mainly constitutive RS-exons. The core EJC factors, and the peripheral factors PNN and RNPS1, maintain RS-exon inclusion by repressing spliceosomal assembly on RS-5ss. The EJC also blocks 5ss located near exon-exon junctions, thus repressing inclusion of cryptic microexons. The prevalence of annotated RS-exons is high in deuterostomes, while the cryptic RS-exons are more prevalent in Drosophila, where EJC appears less capable of repressing RS. Notably, incomplete repression of RS also contributes to physiological alternative splicing of several human RS-exons. Finally, haploinsufficiency of the EJC factor Magoh in mice is associated with skipping of RS-exons in the brain, with relevance to the microcephaly phenotype and human diseases.

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.

Quantitative Activity Profile and Context Dependence of All Human 5' Splice Sites.

Pre-mRNA splicing is an essential step in the expression of most human genes. Mutations at the 5' splice site (5'ss) frequently cause defective splicing and disease due to interference with the initial recognition of the exon-intron boundary by U1 small nuclear ribonucleoprotein (snRNP), a component of the spliceosome. Here, we use a massively parallel splicing assay (MPSA) in human cells to quantify the activity of all 32,768 unique 5'ss sequences (NNN/GYNNNN) in three different gene contexts. Our results reveal that although splicing efficiency is mostly governed by the 5'ss sequence, there are substantial differences in this efficiency across gene contexts. Among other uses, these MPSA measurements facilitate the prediction of 5'ss sequence variants that are likely to cause aberrant splicing. This approach provides a framework to assess potential pathogenic variants in the human genome and streamline the development of splicing-corrective therapies.

Exon Junction Complexes Suppress Spurious Splice Sites to Safeguard Transcriptome Integrity.

Productive splicing of human precursor messenger RNAs (pre-mRNAs) requires the correct selection of authentic splice sites (SS) from the large pool of potential SS. Although SS consensus sequence and splicing regulatory proteins are known to influence SS usage, the mechanisms ensuring the effective suppression of cryptic SS are insufficiently explored. Here, we find that many aberrant exonic SS are efficiently silenced by the exon junction complex (EJC), a multi-protein complex that is deposited on spliced mRNA near the exon-exon junction. Upon depletion of EJC proteins, cryptic SS are de-repressed, leading to the mis-splicing of a broad set of mRNAs. Mechanistically, the EJC-mediated recruitment of the splicing regulator RNPS1 inhibits cryptic 5'SS usage, while the deposition of the EJC core directly masks reconstituted 3'SS, thereby precluding transcript disintegration. Thus, the EJC protects the transcriptome of mammalian cells from inadvertent loss of exonic sequences and safeguards the expression of intact, full-length mRNAs.

What connects splicing of transfer RNA precursor molecules with pontocerebellar hypoplasia?

Transfer RNAs (tRNAs) represent the most abundant class of RNA molecules in the cell and are key players during protein synthesis and cellular homeostasis. Aberrations in the extensive tRNA biogenesis pathways lead to severe neurological disorders in humans. Mutations in the tRNA splicing endonuclease (TSEN) and its associated RNA kinase cleavage factor polyribonucleotide kinase subunit 1 (CLP1) cause pontocerebellar hypoplasia (PCH), a heterogeneous group of neurodegenerative disorders, that manifest as underdevelopment of specific brain regions typically accompanied by microcephaly, profound motor impairments, and child mortality. Recently, we demonstrated that mutations leading to specific PCH subtypes destabilize TSEN in vitro and cause imbalances of immature to mature tRNA ratios in patient-derived cells. However, how tRNA processing defects translate to disease on a systems level has not been understood. Recent findings suggested that other cellular processes may be affected by mutations in TSEN/CLP1 and obscure the molecular mechanisms of PCH emergence. Here, we review PCH disease models linked to the TSEN/CLP1 machinery and discuss future directions to study neuropathogenesis.

Settling the m6A debate: methylation of mature mRNA is not dynamic but accelerates turnover.

Post-transcriptional modification of RNA nucleosides has been implicated as a pivotal regulator of mRNA biology. In this issue of Genes & Development, Ke and colleagues (pp. 990-1006) provide insights into the temporal and spatial distribution of N6-methyladenosine (m6A) in RNA transcripts by analyzing different subcellular fractions. Using a recently developed biochemical approach for detecting m6A, the researchers show that m6A methylations are enriched in exons and are added to transcripts prior to splicing. Although m6A addition is widely thought to be readily reversible, they demonstrate in HeLa cells that once RNA is released from chromatin, the modifications are surprisingly static. This study integrates data from previous publications to clarify conflicting conclusions regarding the role of m6A in mRNA biogenesis and function. Ke and colleagues found that m6A methylation levels negatively correlate with transcript half-life but are not required for most pre-mRNA splicing events.

Archaeal Kink-Turn Binding Protein Mediates Inhibition of Orthomyxovirus Splicing Biology.

Despite lacking a DNA intermediate, orthomyxoviruses complete their replication cycle in the nucleus and generate multiple transcripts by usurping the host splicing machinery. This biology results in dynamic changes of relative viral transcripts over time and dictates the replicative phase of the infection. Here, we demonstrate that the family of archaeal L7Ae proteins uniquely inhibit the splicing biology of influenza A virus, influenza B virus, and Salmon isavirus, revealing a common strategy utilized by Orthomyxoviridae members to achieve this dynamic. L7Ae-mediated inhibition of virus biology was lost with the generation of a splicing-independent strain of influenza A virus and attempts to select for an escape mutant resulted in variants that conformed to host splicing biology at significant cost to their overall fitness. As L7Ae recognizes conventional kink turns in various RNAs, these data implicate the formation of a similar structure as a shared strategy adopted by this virus family to coordinate their replication cycle. IMPORTANCE Here, we demonstrate that a family of proteins from archaea specifically inhibit this splicing biology of all tested members of the Orthomyxoviridae family. We show that this inhibition extends to influenza A virus, influenza B virus, and isavirus genera, while having no significant impact on the mammalian transcriptome or proteome. Attempts to generate an escape mutant against L7Ae-mediated inhibition resulted in mutations surrounding the viral splice sites and a significant loss of viral fitness. Together, these findings reveal a unique biology shared among diverse members of the Orthomyxoviridae family that may serve as a means to generate future universal therapeutics.

Distinct roles of hnRNPH1 low-complexity domains in splicing and transcription.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins that control key events in RNA biogenesis under both normal and diseased cellular conditions. The low-complexity (LC) domain of hnRNPs can become liquid-like droplets or reversible amyloid-like polymers by phase separation. Yet, whether phase separation of the LC domains contributes to physiological functions of hnRNPs remains unclear. hnRNPH1 contains two LC domains, LC1 and LC2. Here, we show that reversible phase separation of the LC1 domain is critical for both interaction with different kinds of RNA-binding proteins and control of the alternative-splicing activity of hnRNPH1. Interestingly, although not required for phase separation, the LC2 domain contributes to the robust transcriptional activation of hnRNPH1 when fused to the DNA-binding domain, as found recently in acute lymphoblastic leukemia. Our data suggest that the ability of the LC1 domain to phase-separate into reversible polymers or liquid-like droplets is essential for function of hnRNPH1 as an alternative RNA-splicing regulator, whereas the LC2 domain may contribute to the aberrant transcriptional activity responsible for cancer transformation.

The DROL1 subunit of U5 snRNP in the spliceosome is specifically required to splice AT-AC-type introns in Arabidopsis.

An Arabidopsis mutant named defective repression of OLE3::LUC 1 (drol1) was originally isolated as a mutant with defects in the repression of OLEOSIN3 (OLE3) after seed germination. In this study, we show that DROL1 is an Arabidopsis homolog of yeast DIB1, a subunit of the U5 small nuclear ribonucleoprotein particle (snRNP) in the spliceosome. It is also part of a new subfamily that is specific to a certain class of eukaryotes. Comprehensive analysis of the intron splicing using RNA sequencing analysis of the drol1 mutants revealed that most of the minor introns with AT-AC dinucleotide termini had reduced levels of splicing. Only two nucleotide substitutions from AT-AC to GT-AG enabled AT-AC-type introns to be spliced in drol1 mutants. Forty-eight genes, including those having important roles in abiotic stress responses and cell proliferation, exhibited reduced splicing of AT-AC-type introns in the drol1 mutants. Additionally, drol1 mutant seedlings showed retarded growth, similar to that caused by the activation of abscisic acid signaling, possibly as a result of reduced AT-AC-type intron splicing in the endosomal Na+ /H+ antiporters and plant-specific histone deacetylases. These results indicate that DROL1 is specifically involved in the splicing of minor introns with AT-AC termini and that this plays an important role in plant growth and development.

Back-spliced RNA from retrotransposon binds to centromere and regulates centromeric chromatin loops in maize.

In most plants, centromeric DNA contains highly repetitive sequences, including tandem repeats and retrotransposons; however, the roles of these sequences in the structure and function of the centromere are unclear. Here, we found that multiple RNA sequences from centromeric retrotransposons (CRMs) were enriched in maize (Zea mays) centromeres, and back-spliced RNAs were generated from CRM1. We identified 3 types of CRM1-derived circular RNAs with the same back-splicing site based on the back-spliced sequences. These circular RNAs bound to the centromere through R-loops. Two R-loop sites inside a single circular RNA promoted the formation of chromatin loops in CRM1 regions. When RNA interference (RNAi) was used to target the back-splicing site of the circular CRM1 RNAs, the levels of R-loops and chromatin loops formed by these circular RNAs decreased, while the levels of R-loops produced by linear RNAs with similar binding sites increased. Linear RNAs with only one R-loop site could not promote chromatin loop formation. Higher levels of R-loops and lower levels of chromatin loops in the CRM1 regions of RNAi plants led to a reduced localization of the centromeric H3 variant (CENH3). Our work reveals centromeric chromatin organization by circular CRM1 RNAs via R-loops and chromatin loops, which suggested that CRM1 elements might help build a suitable chromatin environment during centromere evolution. These results highlight that R-loops are integral components of centromeric chromatin and proper centromere structure is essential for CENH3 localization.

Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer.

Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5' exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5' exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites.

Different roles for the adjoining and structurally similar A-rich and poly(A) domains of oskar mRNA: Only the A-rich domain is required for oskar noncoding RNA function, which includes MTOC positioning.

Drosophila oskar (osk) mRNA has both coding and noncoding functions, with the latter required for progression through oogenesis. Noncoding activity is mediated by the osk 3' UTR. Three types of cis elements act most directly and are clustered within the final ~120 nucleotides of the 3' UTR: multiple binding sites for the Bru1 protein, a short highly conserved region, and A-rich sequences abutting the poly(A) tail. Here we extend the characterization of these elements and their functions, providing new insights into osk noncoding RNA function and the makeup of the cis elements. We show that all three elements are required for correct positioning of the microtubule organizing center (MTOC), a defect not previously reported for any osk mutant. Normally, the MTOC is located at the posterior of the oocyte during previtellogenic stages of oogenesis, and this distribution underlies the strong posterior enrichment of many mRNAs transported into the oocyte from the nurse cells. When osk noncoding function was disrupted the MTOC was dispersed in the oocyte and osk mRNA failed to be enriched at the posterior, although transport to the oocyte was not affected. A previous study did not detect loss of posterior enrichment for certain osk mutants lacking noncoding activity (Kanke et al., 2015). This discrepancy may be due to use of imaging aimed at monitoring transport to the oocyte rather than posterior enrichment. Involvement in MTOC positioning suggests that the osk noncoding function may act in conjunction with genes whose loss has similar effects, and that osk function may extend to other processes requiring those genes. Further characterization of the cis elements required for osk noncoding function included completion of saturation mutagenesis of the most highly conserved region, providing critical information for evaluating the possible contribution of candidate binding factors. The 3'-most cis element is a cluster of A-rich sequences, the ARS. The close juxtaposition and structural similarity of the ARS and poly(A) tail raised the possibility that they comprise an extended A-rich element required for osk noncoding function. We found that absence of the poly(A) tail did not mimic the effects of mutation of the ARS, causing neither arrest of oogenesis nor mispositioning of osk mRNA in previtellogenic stage oocytes. Thus, the ARS and the poly(A) tail are not interchangeable for osk noncoding RNA function, suggesting that the role of the ARS is not in recruitment of Poly(A) binding protein (PABP), the protein that binds the poly(A) tail. Furthermore, although PABP has been implicated in transport of osk mRNA from the nurse cells to the oocyte, mutation of the ARS in combination with loss of the poly(A) tail did not disrupt transport of osk mRNA into the oocyte. We conclude that PABP acts indirectly in osk mRNA transport, or is associated with osk mRNA independent of an A-rich binding site. Although the poly(A) tail was not required for osk mRNA transport into the oocyte, its absence was associated with a novel osk mRNA localization defect later in oogenesis, potentially revealing a previously unrecognized step in the localization process.

The Physcomitrella patens chromatin adaptor PpMRG1 interacts with H3K36me3 and regulates light-responsive alternative splicing.

Plants perceive dynamic light conditions and optimize their growth and development accordingly by regulating gene expression at multiple levels. Alternative splicing (AS), a widespread mechanism in eukaryotes that post-transcriptionally generates two or more messenger RNAs (mRNAs) from the same pre-mRNA, is rapidly controlled by light. However, a detailed mechanism of light-regulated AS is still not clear. In this study, we demonstrate that histone 3 lysine 36 trimethylation (H3K36me3) rapidly and differentially responds to light at specific gene loci with light-regulated intron retention (IR) of their transcripts in the moss Physcomitrella patens. However, the level of H3K36me3 following exposure to light is inversely related to that of IR events. Physcomitrella patens MORF-related gene 1 (PpMRG1), a chromatin adaptor, bound with higher affinity to H3K36me3 in light conditions than in darkness and was differentially targeted to gene loci showing light-responsive IR. Transcriptome analysis indicated that PpMRG1 functions in the regulation of light-mediated AS. Furthermore, PpMRG1 was also involved in red light-mediated phototropic responses. Our results suggest that light regulates histone methylation, which leads to alterations of AS patterns. The chromatin adaptor PpMRG1 potentially participates in light-mediated AS, revealing that chromatin-coupled regulation of pre-mRNA splicing is an important aspect of the plant's response to environmental changes.