RESUMEN
BACKGROUND: The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) can modulate transcript abundance. Previous studies have identified several examples of such a regulation in developing neurons. However, the systems-level effects of AS-NMD in this context are poorly understood. RESULTS: We developed an R package, factR2, which offers a comprehensive suite of AS-NMD analysis functions. Using this tool, we conducted a longitudinal analysis of gene expression in pluripotent stem cells undergoing induced neuronal differentiation. Our analysis uncovers hundreds of AS-NMD events with significant potential to regulate gene expression. Notably, this regulation is significantly overrepresented in specific functional groups of developmentally downregulated genes. Particularly strong association with gene downregulation is detected for alternative cassette exons stimulating NMD upon their inclusion into mature mRNA. By combining bioinformatic analyses with CRISPR/Cas9 genome editing and other experimental approaches we show that NMD-stimulating cassette exons regulated by the RNA-binding protein PTBP1 dampen the expression of their genes in developing neurons. We also provided evidence that the inclusion of NMD-stimulating cassette exons into mature mRNAs is temporally coordinated with NMD-independent gene repression mechanisms. CONCLUSIONS: Our study provides an accessible workflow for the discovery and prioritization of AS-NMD targets. It further argues that the AS-NMD pathway plays a widespread role in developing neurons by facilitating the downregulation of functionally related non-neuronal genes.
Asunto(s)
Empalme Alternativo , Regulación hacia Abajo , Neuronas , Degradación de ARNm Mediada por Codón sin Sentido , Proteína de Unión al Tracto de Polipirimidina , Animales , Ratones , Neuronas/metabolismo , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Exones , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Diferenciación Celular/genética , Neurogénesis/genéticaRESUMEN
Many spliceosomal introns are excised from nascent transcripts emerging from RNA polymerase II (RNA Pol II). The extent of cell-type-specific regulation and possible functions of such co-transcriptional events remain poorly understood. We examined the role of the RNA-binding protein PTBP1 in this process using an acute depletion approach followed by the analysis of chromatin- and RNA Pol II-associated transcripts. We show that PTBP1 activates the co-transcriptional excision of hundreds of introns, a surprising effect given that this protein is known to promote intron retention. Importantly, some co-transcriptionally activated introns fail to complete their splicing without PTBP1. In a striking example, retention of a PTBP1-dependent intron triggers nonsense-mediated decay of transcripts encoding DNA methyltransferase DNMT3B. We provide evidence that this regulation facilitates the natural decline in DNMT3B levels in developing neurons and protects differentiation-specific genes from ectopic methylation. Thus, PTBP1-activated co-transcriptional splicing is a widespread phenomenon mediating epigenetic control of cellular identity.
Asunto(s)
Células Madre Pluripotentes , ARN Polimerasa II , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Empalme del ARN/genética , Empalmosomas/metabolismo , Intrones/genética , Células Madre Pluripotentes/metabolismo , Epigénesis Genética , Empalme AlternativoRESUMEN
Regulation of pre-mRNA splicing and polyadenylation plays a profound role in neurons by diversifying the proteome and modulating gene expression in response to physiological cues. Although most of the pre-mRNA processing is thought to occur in the nucleus, numerous splicing regulators are also found in neurites. Here, we show that U1-70K/SNRNP70, a component of the major spliceosome, localizes in RNA-associated granules in zebrafish axons. We identify the extra-nuclear SNRNP70 as an important regulator of motor axonal growth, nerve-dependent acetylcholine receptor (AChR) clustering, and neuromuscular synaptogenesis. This cytoplasmic pool has a protective role for a limited number of transcripts regulating their abundance and trafficking inside axons. Moreover, non-nuclear SNRNP70 regulates splice variants of transcripts such as agrin, thereby controlling synapse formation. Our results point to an unexpected, yet essential, function of non-nuclear SNRNP70 in axonal development, indicating a role of spliceosome proteins in cytoplasmic RNA metabolism during neuronal connectivity.
Asunto(s)
Precursores del ARN , Pez Cebra , Animales , Pez Cebra/genéticaRESUMEN
Loss of SFPQ is a hallmark of motor degeneration in ALS and prevents maturation of motor neurons when occurring during embryogenesis. Here, we show that in zebrafish, developing motor neurons lacking SFPQ exhibit axon extension, branching and synaptogenesis defects, prior to degeneration. Subcellular transcriptomics reveals that loss of SFPQ in neurons produces a complex set of aberrant intron-retaining (IR) transcripts coding for neuron-specific proteins that accumulate in neurites. Some of these local IR mRNAs are prematurely terminated within the retained intron (PreT-IR). PreT-IR mRNAs undergo intronic polyadenylation, nuclear export, and localise to neurites in vitro and in vivo. We find these IR and PreT-IR mRNAs enriched in RNAseq datasets of tissue from patients with familial and sporadic ALS. This shared signature, between SFPQ-depleted neurons and ALS, functionally implicates SFPQ with the disease and suggests that neurite-centred perturbation of alternatively spliced isoforms drives the neurodegenerative process.
Asunto(s)
Esclerosis Amiotrófica Lateral , Animales , Intrones/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Axones/metabolismo , Neuronas Motoras/metabolismoRESUMEN
Neurons use local protein synthesis to support their morphological complexity, which requires independent control across multiple subcellular compartments up to the level of individual synapses. We identify a signaling pathway that regulates the local synthesis of proteins required to form excitatory synapses on parvalbumin-expressing (PV+) interneurons in the mouse cerebral cortex. This process involves regulation of the TSC subunit 2 (Tsc2) by the Erb-B2 receptor tyrosine kinase 4 (ErbB4), which enables local control of messenger RNA {mRNA} translation in a cell type-specific and synapse type-specific manner. Ribosome-associated mRNA profiling reveals a molecular program of synaptic proteins downstream of ErbB4 signaling required to form excitatory inputs on PV+ interneurons. Thus, specific connections use local protein synthesis to control synapse formation in the nervous system.
Asunto(s)
Corteza Cerebral , Interneuronas , Biosíntesis de Proteínas , Receptor ErbB-4 , Sinapsis , Proteína 2 del Complejo de la Esclerosis Tuberosa , Animales , Ratones , Corteza Cerebral/metabolismo , Interneuronas/metabolismo , Receptor ErbB-4/genética , Receptor ErbB-4/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sinapsis/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismoRESUMEN
Many eukaryotic genes can give rise to different alternative transcripts depending on stage of development, cell type, and physiological cues. Current transcriptome-wide sequencing technologies highlight the remarkable extent of this regulation in metazoans and allow for RNA isoforms to be profiled in increasingly small biological samples and with a growing confidence. Understanding biological functions of sample-specific transcripts is a major challenge in genomics and RNA processing fields. Here we describe simple bioinformatics workflows that facilitate this task by streamlining reference-guided annotation of novel transcripts. A key part of our protocol is the R package factR that rapidly matches custom-assembled transcripts to their likely host genes, deduces the sequence and domain structure of novel protein products, and predicts sensitivity of newly identified RNA isoforms to nonsense-mediated decay.
Asunto(s)
Isoformas de ARN , Transcriptoma , Empalme Alternativo , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Anotación de Secuencia Molecular , Degradación de ARNm Mediada por Codón sin Sentido , Isoformas de ARN/genética , Análisis de Secuencia de ARNRESUMEN
The RNA-binding protein SFPQ plays an important role in neuronal development and has been associated with several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease. Here, we report that loss of sfpq leads to premature termination of multiple transcripts due to widespread activation of previously unannotated cryptic last exons (CLEs). These SFPQ-inhibited CLEs appear preferentially in long introns of genes with neuronal functions and can dampen gene expression outputs and/or give rise to short peptides interfering with the normal gene functions. We show that one such peptide encoded by the CLE-containing epha4b mRNA isoform is responsible for neurodevelopmental defects in the sfpq mutant. The uncovered CLE-repressive activity of SFPQ is conserved in mouse and human, and SFPQ-inhibited CLEs are found expressed across ALS iPSC-derived neurons. These results greatly expand our understanding of SFPQ function and uncover a gene regulation mechanism with wide relevance to human neuropathologies.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Codón sin Sentido , Exones/genética , Factor de Empalme Asociado a PTB/genética , Animales , Secuencia de Bases , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Hibridación in Situ/métodos , Intrones/genética , Ratones , Neuronas/metabolismo , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
Many RNA-binding proteins including a master regulator of splicing in developing brain and muscle, polypyrimidine tract-binding protein 1 (PTBP1), can either activate or repress alternative exons depending on the pre-mRNA recruitment position. When bound upstream or within regulated exons PTBP1 tends to promote their skipping, whereas binding to downstream sites often stimulates inclusion. How this switch is orchestrated at the molecular level is poorly understood. Using bioinformatics and biochemical approaches we show that interaction of PTBP1 with downstream intronic sequences can activate natural cassette exons by promoting productive docking of the spliceosomal U1 snRNP to a suboptimal 5' splice site. Strikingly, introducing upstream PTBP1 sites to this circuitry leads to a potent splicing repression accompanied by the assembly of an exonic ribonucleoprotein complex with a tightly bound U1 but not U2 snRNP. Our data suggest a molecular mechanism underlying the transition between a better-known repressive function of PTBP1 and its role as a bona fide splicing activator. More generally, we argue that the functional outcome of individual RNA contacts made by an RNA-binding protein is subject to extensive context-specific modulation.
Asunto(s)
Empalme Alternativo , Ribonucleoproteínas Nucleares Heterogéneas/fisiología , Modelos Genéticos , Proteína de Unión al Tracto de Polipirimidina/fisiología , Empalme Alternativo/genética , Animales , Línea Celular Tumoral , Biología Computacional , Proteínas de Unión al ADN/genética , Exones/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Intrones/genética , Ratones , Neuroblastoma , Proteína de Unión al Tracto de Polipirimidina/genética , ARN Interferente Pequeño/farmacología , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U1 , Ubiquitina-Proteína LigasasRESUMEN
Eukaryotic gene expression is extensively controlled at the level of mRNA stability and the mechanisms underlying this regulation are markedly different from their archaeal and bacterial counterparts. We propose that two such mechanisms, nonsense-mediated decay (NMD) and motif-specific transcript destabilization by CCCH-type zinc finger RNA-binding proteins, originated as a part of cellular defense against RNA pathogens. These branches of the mRNA turnover pathway might have been used by primeval eukaryotes alongside RNA interference to distinguish their own messages from those of RNA viruses and retrotransposable elements. We further hypothesize that the subsequent advent of "professional" innate and adaptive immunity systems allowed NMD and the motif-triggered mechanisms to be efficiently repurposed for regulation of endogenous cellular transcripts. This scenario explains the rapid emergence of archetypical mRNA destabilization pathways in eukaryotes and argues that other aspects of post-transcriptional gene regulation in this lineage might have been derived through a similar exaptation route.
Asunto(s)
Eucariontes/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Animales , Eucariontes/genética , HumanosRESUMEN
Many eukaryotic organisms encode more than one RNA-dependent RNA polymerase (RdRP) that probably emerged as a result of gene duplication. Such RdRP paralogs often participate in distinct RNA silencing pathways and show characteristic repertoires of enzymatic activities in vitro However, to what extent members of individual paralogous groups can undergo functional changes during speciation remains an open question. We show that orthologs of QDE-1, an RdRP component of the quelling pathway in Neurospora crassa, have rapidly diverged in evolution at the amino acid sequence level. Analyses of purified QDE-1 polymerases from N. crassa (QDE-1(Ncr)) and related fungi, Thielavia terrestris (QDE-1(Tte)) and Myceliophthora thermophila (QDE-1(Mth)), show that all three enzymes can synthesize RNA, but the precise modes of their action differ considerably. Unlike their QDE-1(Ncr) counterpart favoring processive RNA synthesis, QDE-1(Tte) and QDE-1(Mth) produce predominantly short RNA copies via primer-independent initiation. Surprisingly, a 3.19 Å resolution crystal structure of QDE-1(Tte) reveals a quasisymmetric dimer similar to QDE-1(Ncr) Further electron microscopy analyses confirm that QDE-1(Tte) occurs as a dimer in solution and retains this status upon interaction with a template. We conclude that divergence of orthologous RdRPs can result in functional innovation while retaining overall protein fold and quaternary structure.
Asunto(s)
Evolución Molecular , Proteínas Fúngicas , Neurospora crassa , Multimerización de Proteína/fisiología , ARN Polimerasa Dependiente del ARN , Cristalografía por Rayos X , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Neurospora crassa/enzimología , Neurospora crassa/genética , Estructura Cuaternaria de Proteína , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismoRESUMEN
Alternative splicing (AS) provides a potent mechanism for increasing protein diversity and modulating gene expression levels. How alternate splice sites are selected by the splicing machinery and how AS is integrated into gene regulation networks remain important questions of eukaryotic biology. Here we report that polypyrimidine tract-binding protein 1 (Ptbp1/PTB/hnRNP-I) controls alternate 5' and 3' splice site (5'ss and 3'ss) usage in a large set of mammalian transcripts. A top scoring event identified by our analysis was the choice between competing upstream and downstream 5'ss (u5'ss and d5'ss) in the exon 18 of the Hps1 gene. Hps1 is essential for proper biogenesis of lysosome-related organelles and loss of its function leads to a disease called type 1 Hermansky-Pudlak Syndrome (HPS). We show that Ptbp1 promotes preferential utilization of the u5'ss giving rise to stable mRNAs encoding a full-length Hps1 protein, whereas bias towards d5'ss triggered by Ptbp1 down-regulation generates transcripts susceptible to nonsense-mediated decay (NMD). We further demonstrate that Ptbp1 binds to pyrimidine-rich sequences between the u5'ss and d5'ss and activates the former site rather than repressing the latter. Consistent with this mechanism, u5'ss is intrinsically weaker than d5'ss, with a similar tendency observed for other genes with Ptbp1-induced u5'ss bias. Interestingly, the brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5'ss utilization but with a considerably lower efficiency than Ptbp1. This may account for the tight correlation between Hps1 with Ptbp1 expression levels observed across mammalian tissues. More generally, these data expand our understanding of AS regulation and uncover a post-transcriptional strategy ensuring co-expression of a subordinate gene with its master regulator through an AS-NMD tracking mechanism.
Asunto(s)
Empalme Alternativo/genética , Síndrome de Hermanski-Pudlak/genética , Proteína de Unión al Tracto de Polipirimidina/genética , ARN Mensajero/genética , Animales , Exones , Regulación de la Expresión Génica , Células HeLa , Síndrome de Hermanski-Pudlak/patología , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Proteína de Unión al Tracto de Polipirimidina/biosíntesis , Sitios de Empalme de ARN/genéticaRESUMEN
Higher eukaryotes rely on AS (alternative splicing) of pre-mRNAs (mRNA precursors) to generate more than one protein product from a single gene and to regulate mRNA stability and translational activity. An important example of the latter function involves an interplay between AS and NMD (nonsense-mediated decay), a cytoplasmic quality control mechanism eliminating mRNAs containing PTCs (premature translation termination codons). Although originally identified as an error surveillance process, AS-NMD additionally provides an efficient strategy for deterministic regulation of gene expression outputs. In this review, we discuss recently published examples of AS-NMD and delineate functional contexts where recurrent use of this mechanism orchestrates expression of important genes.