ABSTRACT
In this issue of Cell, Kornblihtt and colleagues report a strategy to improve antisense oligonucleotide spinal muscular atrophy therapy. They discover that the oligonucleotide drug nusinersen, which induces exon inclusion, also promotes repressive chromatin modifications, which in turn work against exon inclusion. Notably, co-administration of histone deacetylase inhibitors counteracted this effect to augment exon inclusion.
Subject(s)
Muscular Atrophy, Spinal , Oligonucleotides, Antisense , DNA , Exons , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Humans , Muscular Atrophy, Spinal/drug therapy , Muscular Atrophy, Spinal/genetics , Oligonucleotides, Antisense/pharmacology , Oligonucleotides, Antisense/therapeutic useABSTRACT
Spinal muscular atrophy (SMA) is a motor-neuron disease caused by mutations of the SMN1 gene. The human paralog SMN2, whose exon 7 (E7) is predominantly skipped, cannot compensate for the lack of SMN1. Nusinersen is an antisense oligonucleotide (ASO) that upregulates E7 inclusion and SMN protein levels by displacing the splicing repressors hnRNPA1/A2 from their target site in intron 7. We show that by promoting transcriptional elongation, the histone deacetylase inhibitor VPA cooperates with a nusinersen-like ASO to promote E7 inclusion. Surprisingly, the ASO promotes the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to RNA polymerase II elongation that inhibits E7 inclusion. By removing the roadblock, VPA counteracts the chromatin effects of the ASO, resulting in higher E7 inclusion without large pleiotropic effects. Combined administration of the nusinersen-like ASO and VPA in SMA mice strongly synergizes SMN expression, growth, survival, and neuromuscular function.
Subject(s)
Muscular Atrophy, Spinal , Oligonucleotides, Antisense , Animals , Chromatin , Exons , Mice , Muscular Atrophy, Spinal/drug therapy , Muscular Atrophy, Spinal/genetics , Oligonucleotides, Antisense/pharmacology , Oligonucleotides, Antisense/therapeutic use , RNA SplicingABSTRACT
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.
Subject(s)
RNA Precursors/genetics , RNA Splice Sites/physiology , Transcription, Genetic , Exons/genetics , Humans , Introns/genetics , RNA Precursors/metabolism , RNA Splice Sites/genetics , RNA Splicing/genetics , RNA Splicing/physiology , RNA, Messenger/metabolism , Spliceosomes/metabolism , TranscriptomeABSTRACT
The spliceosome removes introns from messenger RNA precursors (pre-mRNA). Decades of biochemistry and genetics combined with recent structural studies of the spliceosome have produced a detailed view of the mechanism of splicing. In this review, we aim to make this mechanism understandable and provide several videos of the spliceosome in action to illustrate the intricate choreography of splicing. The U1 and U2 small nuclear ribonucleoproteins (snRNPs) mark an intron and recruit the U4/U6.U5 tri-snRNP. Transfer of the 5' splice site (5'SS) from U1 to U6 snRNA triggers unwinding of U6 snRNA from U4 snRNA. U6 folds with U2 snRNA into an RNA-based active site that positions the 5'SS at two catalytic metal ions. The branch point (BP) adenosine attacks the 5'SS, producing a free 5' exon. Removal of the BP adenosine from the active site allows the 3'SS to bind, so that the 5' exon attacks the 3'SS to produce mature mRNA and an excised lariat intron.
Subject(s)
DEAD-box RNA Helicases/genetics , RNA Splicing Factors/genetics , RNA Splicing , RNA, Small Nuclear/genetics , Saccharomyces cerevisiae/genetics , Spliceosomes/metabolism , Catalytic Domain , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/metabolism , Exons , Humans , Introns , Models, Molecular , Nucleic Acid Conformation , Protein Binding , Protein Structure, Secondary , RNA Helicases/chemistry , RNA Helicases/genetics , RNA Helicases/metabolism , RNA Precursors/chemistry , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing Factors/chemistry , RNA Splicing Factors/metabolism , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Spliceosomes/genetics , Spliceosomes/ultrastructureABSTRACT
Splicing of the precursor messenger RNA, involving intron removal and exon ligation, is mediated by the spliceosome. Together with biochemical and genetic investigations of the past four decades, structural studies of the intact spliceosome at atomic resolution since 2015 have led to mechanistic delineation of RNA splicing with remarkable insights. The spliceosome is proven to be a protein-orchestrated metalloribozyme. Conserved elements of small nuclear RNA (snRNA) constitute the splicing active site with two catalytic metal ions and recognize three conserved intron elements through duplex formation, which are delivered into the splicing active site for branching and exon ligation. The protein components of the spliceosome stabilize the conformation of the snRNA, drive spliceosome remodeling, orchestrate the movement of the RNA elements, and facilitate the splicing reaction. The overall organization of the spliceosome and the configuration of the splicing active site are strictly conserved between human and yeast.
Subject(s)
RNA Splicing Factors/genetics , RNA Splicing , RNA-Binding Proteins/genetics , Ribonucleoprotein, U4-U6 Small Nuclear/genetics , Ribonucleoprotein, U5 Small Nuclear/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Spliceosomes/metabolism , Catalytic Domain , Conserved Sequence , Exons , Humans , Introns , Models, Molecular , Nucleic Acid Conformation , Protein Structure, Secondary , RNA Helicases/chemistry , RNA Helicases/genetics , RNA Helicases/metabolism , RNA Precursors/chemistry , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing Factors/chemistry , RNA Splicing Factors/metabolism , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/genetics , RNA, Small Nuclear/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Ribonucleoprotein, U4-U6 Small Nuclear/chemistry , Ribonucleoprotein, U4-U6 Small Nuclear/metabolism , Ribonucleoprotein, U5 Small Nuclear/chemistry , Ribonucleoprotein, U5 Small Nuclear/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Spliceosomes/genetics , Spliceosomes/ultrastructureABSTRACT
Removal of introns from eukaryotic messenger RNA precursors often occurs co-transcriptionally. In this issue of Cell, Fiszbein et al. report that evolutionary or tissue-specific activation of an internal exon can enhance gene expression by promoting the use of alternative transcription initiation sites.
Subject(s)
RNA Precursors , RNA Splicing , Exons , Introns , RNA, MessengerABSTRACT
The processing of RNA transcripts from mammalian genes occurs in proximity to their transcription. Here, we describe a phenomenon affecting thousands of genes that we call exon-mediated activation of transcription starts (EMATS), in which the splicing of internal exons impacts promoter choice and the expression level of the gene. We observed that evolutionary gain of internal exons is associated with gain of new transcription start sites (TSSs) nearby and increased gene expression. Inhibiting exon splicing reduced transcription from nearby promoters, and creation of new spliced exons activated transcription from cryptic promoters. The strongest effects occurred for weak promoters located proximal and upstream of efficiently spliced exons. Together, our findings support a model in which splicing recruits transcription machinery locally to influence TSS choice and identify exon gain, loss, and regulatory change as major contributors to the evolution of alternative promoters and gene expression in mammals.
Subject(s)
Exons , Promoter Regions, Genetic , Transcriptional Activation/genetics , 3T3 Cells , Animals , Evolution, Molecular , HeLa Cells , Humans , Mice , RNA Splicing , Transcription Initiation SiteABSTRACT
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.
Subject(s)
Spliceosomes/physiology , Spliceosomes/ultrastructure , Catalytic Domain/physiology , Cryoelectron Microscopy/methods , Exons , Introns , Nuclear Proteins/metabolism , RNA Precursors/metabolism , RNA Splice Sites/genetics , RNA Splicing/physiology , RNA Splicing Factors/metabolism , RNA, Small Nuclear/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Spliceosomes/metabolismABSTRACT
The splicing of pre-mRNAs into mature transcripts is remarkable for its precision, but the mechanisms by which the cellular machinery achieves such specificity are incompletely understood. Here, we describe a deep neural network that accurately predicts splice junctions from an arbitrary pre-mRNA transcript sequence, enabling precise prediction of noncoding genetic variants that cause cryptic splicing. Synonymous and intronic mutations with predicted splice-altering consequence validate at a high rate on RNA-seq and are strongly deleterious in the human population. De novo mutations with predicted splice-altering consequence are significantly enriched in patients with autism and intellectual disability compared to healthy controls and validate against RNA-seq in 21 out of 28 of these patients. We estimate that 9%-11% of pathogenic mutations in patients with rare genetic disorders are caused by this previously underappreciated class of disease variation.
Subject(s)
Forecasting/methods , RNA Precursors/genetics , RNA Splicing/genetics , Algorithms , Alternative Splicing/genetics , Autistic Disorder/genetics , Deep Learning , Exons/genetics , Humans , Intellectual Disability/genetics , Introns/genetics , Neural Networks, Computer , RNA Precursors/metabolism , RNA Splice Sites/genetics , RNA Splice Sites/physiologyABSTRACT
Despite a wealth of molecular knowledge, quantitative laws for accurate prediction of biological phenomena remain rare. Alternative pre-mRNA splicing is an important regulated step in gene expression frequently perturbed in human disease. To understand the combined effects of mutations during evolution, we quantified the effects of all possible combinations of exonic mutations accumulated during the emergence of an alternatively spliced human exon. This revealed that mutation effects scale non-monotonically with the inclusion level of an exon, with each mutation having maximum effect at a predictable intermediate inclusion level. This scaling is observed genome-wide for cis and trans perturbations of splicing, including for natural and disease-associated variants. Mathematical modeling suggests that competition between alternative splice sites is sufficient to cause this non-linearity in the genotype-phenotype map. Combining the global scaling law with specific pairwise interactions between neighboring mutations allows accurate prediction of the effects of complex genotype changes involving >10 mutations.
Subject(s)
Alternative Splicing/genetics , RNA Splicing/genetics , fas Receptor/genetics , Animals , Exons/genetics , Genetic Techniques , Genetics , Genotype , Humans , Introns/genetics , Mice , Models, Theoretical , Mutation/genetics , Phenotype , RNA Precursors/metabolism , RNA Splice Sites/genetics , RNA, Messenger/metabolismABSTRACT
Stochastic activation of clustered Protocadherin (Pcdh) α, ß, and γ genes generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here, we show that Pcdhα gene choice involves the activation of an antisense promoter located in the first exon of each Pcdhα alternate gene. Transcription of an antisense long noncoding RNA (lncRNA) from this antisense promoter extends through the sense promoter, leading to DNA demethylation of the CTCF binding sites proximal to each promoter. Demethylation-dependent CTCF binding to both promoters facilitates cohesin-mediated DNA looping with a distal enhancer (HS5-1), locking in the transcriptional state of the chosen Pcdhα gene. Uncoupling DNA demethylation from antisense transcription by Tet3 overexpression in mouse olfactory neurons promotes CTCF binding to all Pcdhα promoters, resulting in proximity-biased DNA looping of the HS5-1 enhancer. Thus, antisense transcription-mediated promoter demethylation functions as a mechanism for distance-independent enhancer/promoter DNA looping to ensure stochastic Pcdhα promoter choice.
Subject(s)
Cadherins/genetics , DNA Demethylation , RNA, Antisense/metabolism , RNA, Long Noncoding/genetics , Animals , Binding Sites , CCCTC-Binding Factor/chemistry , CCCTC-Binding Factor/metabolism , Cadherins/metabolism , Cell Line , Enhancer Elements, Genetic , Exons , Female , Humans , Mice , Mice, Transgenic , Multigene Family , Neurons/cytology , Neurons/metabolism , Promoter Regions, Genetic , RNA Polymerase II/metabolism , RNA, Antisense/genetics , Transcription, GeneticABSTRACT
FOXP2, initially identified for its role in human speech, contains two nonsynonymous substitutions derived in the human lineage. Evidence for a recent selective sweep in Homo sapiens, however, is at odds with the presence of these substitutions in archaic hominins. Here, we comprehensively reanalyze FOXP2 in hundreds of globally distributed genomes to test for recent selection. We do not find evidence of recent positive or balancing selection at FOXP2. Instead, the original signal appears to have been due to sample composition. Our tests do identify an intronic region that is enriched for highly conserved sites that are polymorphic among humans, compatible with a loss of function in humans. This region is lowly expressed in relevant tissue types that were tested via RNA-seq in human prefrontal cortex and RT-PCR in immortalized human brain cells. Our results represent a substantial revision to the adaptive history of FOXP2, a gene regarded as vital to human evolution.
Subject(s)
Forkhead Transcription Factors/genetics , Brain/cytology , Brain/metabolism , Cell Line , Databases, Genetic , Exons , Female , Genome, Human , Haplotypes , Humans , Introns , Male , Markov Chains , Polymorphism, Single Nucleotide , Prefrontal Cortex/metabolismABSTRACT
Long mammalian introns make it challenging for the RNA processing machinery to identify exons accurately. We find that LINE-derived sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs) to introns. This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites within LINEs. Both RBPs repress splicing and 3' end processing within and around LINEs. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are located far from exons. These RBPs insulate the LINEs and the surrounding intronic regions from RNA processing. Upon evolutionary divergence, changes in RNA motifs within LINEs lead to gradual loss of their insulation. Hence, older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for the assembly of repressive RBPs and also contribute to the evolution of new, lineage-specific transcripts in mammals. VIDEO ABSTRACT.
Subject(s)
Heterogeneous-Nuclear Ribonucleoproteins/chemistry , Long Interspersed Nucleotide Elements , Nuclear Matrix-Associated Proteins/chemistry , Polyadenylation , Polypyrimidine Tract-Binding Protein/chemistry , RNA-Binding Proteins/chemistry , RNA/chemistry , Alternative Splicing , Animals , Binding Sites , Exons , HeLa Cells , Humans , Introns , Mice , Mutation , Nucleotide Motifs , Phylogeny , Protein Binding , Protein Interaction Mapping , RNA SplicingABSTRACT
The DNA-binding protein REST forms complexes with histone deacetylases (HDACs) to repress neuronal genes in non-neuronal cells. In differentiating neurons, REST is downregulated predominantly by transcriptional silencing. Here we report that post-transcriptional inactivation of REST by alternative splicing is required for hearing in humans and mice. We show that, in the mechanosensory hair cells of the mouse ear, regulated alternative splicing of a frameshift-causing exon into the Rest mRNA is essential for the derepression of many neuronal genes. Heterozygous deletion of this alternative exon of mouse Rest causes hair cell degeneration and deafness, and the HDAC inhibitor SAHA (Vorinostat) rescues the hearing of these mice. In humans, inhibition of the frameshifting splicing event by a novel REST variant is associated with dominantly inherited deafness. Our data reveal the necessity for alternative splicing-dependent regulation of REST in hair cells, and they identify a potential treatment for a group of hereditary deafness cases.
Subject(s)
Deafness/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Alternative Splicing/genetics , Animals , Cell Line , Exons , Gene Expression Regulation/genetics , HEK293 Cells , Hair Cells, Auditory/physiology , Hearing/genetics , Hearing/physiology , Histone Deacetylase Inhibitors/metabolism , Histone Deacetylases/metabolism , Humans , Mice , Mice, Inbred C57BL , Neurons , RNA Splicing/genetics , Repressor Proteins/physiology , Transcription Factors , Vorinostat/pharmacologyABSTRACT
Low-grade gliomas almost invariably progress into secondary glioblastoma (sGBM) with limited therapeutic option and poorly understood mechanism. By studying the mutational landscape of 188 sGBMs, we find significant enrichment of TP53 mutations, somatic hypermutation, MET-exon-14-skipping (METex14), PTPRZ1-MET (ZM) fusions, and MET amplification. Strikingly, METex14 frequently co-occurs with ZM fusion and is present in â¼14% of cases with significantly worse prognosis. Subsequent studies show that METex14 promotes glioma progression by prolonging MET activity. Furthermore, we describe a MET kinase inhibitor, PLB-1001, that demonstrates remarkable potency in selectively inhibiting MET-altered tumor cells in preclinical models. Importantly, this compound also shows blood-brain barrier permeability and is subsequently applied in a phase I clinical trial that enrolls MET-altered chemo-resistant glioma patients. Encouragingly, PLB-1001 achieves partial response in at least two advanced sGBM patients with rarely significant side effects, underscoring the clinical potential for precisely treating gliomas using this therapy.
Subject(s)
Brain Neoplasms , Exons , Glioblastoma , Mutation , Protein Kinase Inhibitors , Proto-Oncogene Proteins c-met , Animals , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/pathology , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Drug Delivery Systems , Female , Glioblastoma/drug therapy , Glioblastoma/genetics , Glioblastoma/metabolism , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins c-met/metabolism , Rats, Sprague-Dawley , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Many protein-coding genes in higher eukaryotes can produce circular RNAs (circRNAs) through back-splicing of exons. CircRNAs differ from mRNAs in their production, structure and turnover and thereby have unique cellular functions and potential biomedical applications. In this Review, I discuss recent progress in our understanding of the biogenesis of circRNAs and the regulation of their abundance and of their biological functions, including in transcription and splicing, sequestering or scaffolding of macromolecules to interfere with microRNA activities or signalling pathways, and serving as templates for translation. I further discuss the emerging roles of circRNAs in regulating immune responses and cell proliferation, and the possibilities of applying circRNA technologies in biomedical research.
Subject(s)
RNA, Circular/genetics , RNA, Circular/metabolism , RNA, Circular/physiology , Alternative Splicing/genetics , Animals , Exons/genetics , Gene Expression/genetics , Gene Expression Regulation/genetics , Humans , MicroRNAs/metabolism , RNA/genetics , RNA Splicing/genetics , RNA, Messenger/metabolismABSTRACT
Patterns of daily human activity are controlled by an intrinsic circadian clock that promotes â¼24 hr rhythms in many behavioral and physiological processes. This system is altered in delayed sleep phase disorder (DSPD), a common form of insomnia in which sleep episodes are shifted to later times misaligned with the societal norm. Here, we report a hereditary form of DSPD associated with a dominant coding variation in the core circadian clock gene CRY1, which creates a transcriptional inhibitor with enhanced affinity for circadian activator proteins Clock and Bmal1. This gain-of-function CRY1 variant causes reduced expression of key transcriptional targets and lengthens the period of circadian molecular rhythms, providing a mechanistic link to DSPD symptoms. The allele has a frequency of up to 0.6%, and reverse phenotyping of unrelated families corroborates late and/or fragmented sleep patterns in carriers, suggesting that it affects sleep behavior in a sizeable portion of the human population.
Subject(s)
Cryptochromes/metabolism , Sleep Disorders, Circadian Rhythm/genetics , Circadian Rhythm , Cryptochromes/genetics , Exons , Female , Gene Deletion , Humans , Male , Middle Aged , Pedigree , Sleep Disorders, Circadian Rhythm/physiopathologyABSTRACT
Chromosome conformation capture technologies have revealed important insights into genome folding. Yet, how spatial genome architecture is related to gene expression and cell fate remains unclear. We comprehensively mapped 3D chromatin organization during mouse neural differentiation in vitro and in vivo, generating the highest-resolution Hi-C maps available to date. We found that transcription is correlated with chromatin insulation and long-range interactions, but dCas9-mediated activation is insufficient for creating TAD boundaries de novo. Additionally, we discovered long-range contacts between gene bodies of exon-rich, active genes in all cell types. During neural differentiation, contacts between active TADs become less pronounced while inactive TADs interact more strongly. An extensive Polycomb network in stem cells is disrupted, while dynamic interactions between neural transcription factors appear in vivo. Finally, cell type-specific enhancer-promoter contacts are established concomitant to gene expression. This work shows that multiple factors influence the dynamics of chromatin interactions in development.
Subject(s)
Chromatin/metabolism , Genome , Neurogenesis , Animals , CCCTC-Binding Factor , Embryonic Stem Cells/metabolism , Enhancer Elements, Genetic , Exons , Gene Expression , Gene Regulatory Networks , Mice , Promoter Regions, Genetic , Repressor Proteins/metabolism , Transcription Factors/metabolismABSTRACT
Mechanistic understanding of pre-mRNA splicing requires detailed structural information on various states of the spliceosome. Here we report the cryo electron microscopy (cryo-EM) structure of the human spliceosome just before exon ligation (the C∗ complex) at an average resolution of 3.76 Å. The splicing factor Prp17 stabilizes the active site conformation. The step II factor Slu7 adopts an extended conformation, binds Prp8 and Cwc22, and is poised for selection of the 3'-splice site. Remarkably, the intron lariat traverses through a positively charged central channel of RBM22; this unusual organization suggests mechanisms of intron recruitment, confinement, and release. The protein PRKRIP1 forms a 100-Å α helix linking the distant U2 snRNP to the catalytic center. A 35-residue fragment of the ATPase/helicase Prp22 latches onto Prp8, and the quaternary exon junction complex (EJC) recognizes upstream 5'-exon sequences and associates with Cwc22 and the GTPase Snu114. These structural features reveal important mechanistic insights into exon ligation.
Subject(s)
RNA Precursors/metabolism , Spliceosomes/chemistry , Spliceosomes/ultrastructure , Base Sequence , Cryoelectron Microscopy , DEAD-box RNA Helicases/metabolism , Exons , Humans , Introns , Models, Molecular , RNA Splicing , RNA Splicing Factors/metabolism , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Spliceosomes/metabolismABSTRACT
The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal â¼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.