Modeling of autosomal-dominant retinitis pigmentosa in Caenorhabditis elegans uncovers a nexus between global impaired functioning of certain splicing factors and cell type-specific apoptosis.
ABSTRACT: Retinitis pigmentosa (RP) is a rare genetic disease that causes gradual blindness through retinal degeneration. Intriguingly, seven of the 24 genes identified as responsible for the autosomal-dominant form (adRP) are ubiquitous spliceosome components whose impairment causes disease only in the retina. The fact that these proteins are essential in all organisms hampers genetic, genomic, and physiological studies, but we addressed these difficulties by using RNAi in Caenorhabditis elegans. Our study of worm phenotypes produced by RNAi of splicing-related adRP (s-adRP) genes functionally distinguishes between components of U4 and U5 snRNP complexes, because knockdown of U5 proteins produces a stronger phenotype. RNA-seq analyses of worms where s-adRP genes were partially inactivated by RNAi, revealed mild intron retention in developing animals but not in adults, suggesting a positive correlation between intron retention and transcriptional activity. Interestingly, RNAi of s-adRP genes produces an increase in the expression of atl-1 (homolog of human ATR), which is normally activated in response to replicative stress and certain DNA-damaging agents. The up-regulation of atl-1 correlates with the ectopic expression of the pro-apoptotic gene egl-1 and apoptosis in hypodermal cells, which produce the cuticle, but not in other cell types. Our model in C. elegans resembles s-adRP in two aspects: The phenotype caused by global knockdown of s-adRP genes is cell type-specific and associated with high transcriptional activity. Finally, along with a reduced production of mature transcripts, we propose a model in which the retina-specific cell death in s-adRP patients can be induced through genomic instability.
Project description:Purpose. The small nuclear ribonucleoprotein 200?kDa (SNRNP200) gene is a fundamental component for precursor message RNA (pre-mRNA) splicing and has been implicated in the etiology of autosomal dominant retinitis pigmentosa (adRP). This study aims to determine the consequences of knocking down Snrnp200 in zebrafish. Methods. Expression of the Snrnp200 transcript in zebrafish was determined via whole mount in situ hybridization. Morpholino oligonucleotide (MO) aiming to knock down the expression of Snrnp200 was injected into zebrafish embryos, followed by analyses of aberrant splicing and expression of the U4/U6-U5 tri-small nuclear ribonucleoproteins (snRNPs) components and retina-specific transcripts. Systemic changes and retinal phenotypes were further characterized by histological study and immunofluorescence staining. Results. Snrnp200 was ubiquitously expressed in zebrafish. Knocking down Snrnp200 in zebrafish triggered aberrant splicing of the cbln1 gene, upregulation of other U4/U6-U5 tri-snRNP components, and downregulation of a panel of retina-specific transcripts. Systemic defects were found correlated with knockdown of Snrnp200 in zebrafish. Only demorphogenesis of rod photoreceptors was detected in the initial stage, mimicking the disease characteristics of RP. Conclusions. We conclude that knocking down Snrnp200 in zebrafish could alter regular splicing and expression of a panel of genes, which may eventually trigger rod defects.
Project description:The accurate inheritance of genetic material is a basic necessity in all domains of life and an unexpectedly large number of RNA processing factors are required for mitotic progression and genome stability. NRDE2 (nuclear RNAi defective-2) is an evolutionarily conserved protein originally discovered for its role in nuclear RNA interference (RNAi) and heritable gene silencing in <i>Caenorhabditis elegans</i> (<i>C. elegans</i>). The function of the human <i>NRDE2</i> gene remains poorly understood. Here we show that human NRDE2 is an essential protein required for suppressing intron retention in a subset of pre-mRNAs containing short, GC-rich introns with relatively weak 5' and 3' splice sites. NRDE2 preferentially interacts with components of the U5 small nuclear ribonucleoprotein (snRNP), the exon junction complex, and the RNA exosome. Interestingly, <i>NRDE2-</i>depleted cells exhibit greatly increased levels of genomic instability and DNA damage, as well as defects in centrosome maturation and mitotic progression. We identify the essential centriolar satellite protein, CEP131, as a direct NRDE2-regulated target. NRDE2 specifically binds to and promotes the efficient splicing of <i>CEP131</i> pre-mRNA, and depleting <i>NRDE2</i> dramatically reduces CEP131 protein expression, contributing to impaired recruitment of critical centrosomal proteins (e.g., ?-tubulin and Aurora Kinase A) to the spindle poles during mitosis. Our work establishes a conserved role for human <i>NRDE2</i> in RNA splicing, characterizes the severe genomic instability phenotypes observed upon loss of <i>NRDE2</i>, and highlights the direct regulation of <i>CEP131</i> splicing as one of multiple mechanisms through which such phenotypes might be explained.
Project description:Mutations in genes associated with the U4/U6-U5 small nuclear ribonucleoprotein (snRNP) complex of the spliceosome are implicated in autosomal-dominant retinitis pigmentosa (adRP), a group of progressive retinal degenerative disorders leading to visual impairment, loss of visual field, and even blindness. We recently assigned a locus (RP33) for adRP to 2cen-q12.1, a region that harbors the SNRNP200 gene encoding hBrr2, another U4/U6-U5 snRNP component that is required for unwinding of U4/U6 snRNAs during spliceosome activation and for disassembly of the spliceosome. Here, we report the identification of a missense mutation, c.3260C>T (p.S1087L), in exon 25 of the SNRNP200 gene in an RP33-linked family. The c.3260C>T substitution showed complete cosegregation with the retinitis pigmentosa (RP) phenotype over four generations, but was absent in a panel of 400 controls. The p.S1087L mutation and p.R1090L, another adRP-associated allele, reside in the "ratchet" helix of the first of two Sec63 domains implicated in the directionality and processivity of nucleic acid unwinding. Indeed, marked defects in U4/U6 unwinding, but not U4/U6-U5 snRNP assembly, were observed in budding yeast for the analogous mutations (N1104L and R1107L) of the corresponding Brr2p residues. The linkage of hBrr2 to adRP suggests that the mechanism of pathogenesis for splicing-factor-related RP may fundamentally derive from a defect in hBrr2-dependent RNA unwinding and a consequent defect in spliceosome activation.
Project description:High oxygen consumption and cyclical changes related to dark-adaptation are characteristic of the outer retina. Oxygenation changes may contribute to the selective vulnerability of the retina in retinitis pigmentosa (RP) patients, especially for those forms involving genes with global cellular functions. Genes coding for components of the U4/U6.U5 tri small nuclear ribonucleoprotein (tri-snRNP) complex of the spliceosome stand out, because mutations in four genes cause RP, i.e., RP9 (PAP1), RP11 (PRPF31), RP13 (PRPF8), and RP18 (PRPF3), while there is no degeneration outside the retina despite global expression of these genes. With the assumption that variable oxygenation plays a role in RP forms related to pre-mRNA splicing and the retina and brain are similar, we searched a data collection of ischemia-hypoxia regulated genes of the brain for oxygen regulated genes of the U4/U6.U5 tri-snRNP complex.A database of ischemia-hypoxia response (IHR) genes in the brain was generated from gene expression profiling studies [n=24]. Public databases (NCBI) were searched for RP genes with global function that are expressed in the brain. From the IHR gene list, we extracted genes that were directly related to retinal degeneration through a listed mutation (OMIM, Retnet, RISN). The database was then examined for indirect links to RP forms affecting the U4/U6.U5 tri-snRNP complex by searching for IHR genes contributing to this complex. Potential expression of matched genes in the retina was ascertained using NEIBank. Immunohistochemistry was used to localize a selected protein of the U4/U6.U5 tri-snRNP complex in cynomolgus monkey and human retina specimens.The approach identified genes that cause retinal degeneration (CNGB1, SEMA4A, RRG4) or developmental changes (SOX2) when mutated. One IHR gene, Pim1, is the immediate binding partner for PAP1 (RP9). Three IHR genes linked the U4/U6.U5 tri-snRNP complex to regulation by oxygenation: PRPF4; SART1, also known as 110 kDa SR-related protein of the U4/U6.U5 tri-snRNP or as hypoxia associated factor (HAF); and LSM8, U6 snRNA-associated Sm-like protein. The 110 kDa SR-related protein was localized in all retinal cells including photoreceptors.Regulation by changes in oxygenation within the U4/U6.U5 tri-snRP complex could be particularly important for photoreceptors where oxygen consumption follows a circadian rhythm. If the U4/U6.U5 tri-snRP complex is already impaired by mutations in any of the four genes causing RP, it may be unable to follow properly the physiological demands of oxygenation which are mediated by the four hypoxia-regulated proteins emerging in this study. Selective vulnerability may involve complex combinations of widely expressed genes, specific cellular functions and local energy availability.
Project description:Imperfect conservation of human pre-mRNA splice sites is necessary to produce alternative isoforms. This flexibility is combined with the precision of the message reading frame. Apart from intron-termini GU_AG and the branchpoint A, the most conserved are the exon-end guanine and +5G of the intron start. Association between these guanines cannot be explained solely by base-pairing with U1 snRNA in the early spliceosome complex. U6 succeeds U1 and pairs +5G in the pre-catalytic spliceosome, while U5 binds the exon end. Current U5 snRNA reconstructions by CryoEM cannot explain the conservation of the exon-end G. Conversely, human mutation analyses show that guanines of both exon termini can suppress splicing mutations. Our U5 hypothesis explains the mechanism of splicing precision and the role of these conserved guanines in the pre-catalytic spliceosome. We propose: (1) optimal binding register for human exons and U5-the exon junction positioned at U5Loop1 C<sub>39</sub>|C<sub>38</sub>; (2) common mechanism for base-pairing of human U5 snRNA with diverse exons and bacterial <i>Ll.</i>LtrB intron with new loci in retrotransposition-guided by base pair geometry; and (3) U5 plays a significant role in specific exon recognition in the pre-catalytic spliceosome. Statistical analyses showed increased U5 Watson-Crick pairs with the 5'exon in the absence of +5G at the intron start. In 5'exon positions -3 and -5, this effect is specific to U5 snRNA rather than U1 snRNA of the early spliceosome. Increased U5 Watson-Crick pairs with 3'exon position +1 coincide with substitutions of the conserved -3C at the intron 3'end. Based on mutation and X-ray evidence, we propose that -3C pairs with U2 G<sub>31</sub> juxtaposing the branchpoint and the 3'intron end. The intron-termini pair, formed in the pre-catalytic spliceosome to be ready for transition after branching, and the early involvement of the 3'intron end ensure that the 3'exon contacts U5 in the pre-catalytic complex. We suggest that splicing precision is safeguarded cooperatively by U5, U6, and U2 snRNAs that stabilize the pre-catalytic complex by Watson-Crick base pairing. In addition, our new U5 model explains the splicing effect of exon-start +1G mutations: U5 Watson-Crick pairs with exon +2C/+3G strongly promote exon inclusion. We discuss potential applications for snRNA therapeutics and gene repair by reverse splicing.
Project description:Cullin-RING E3-Ligases (CRLs), the largest family of E3 ubiquitin-Ligases, regulate diverse cellular processes by promoting ubiquitination of target proteins. The evolutionarily conserved Leucine Rich Repeat protein 1 (LRR-1) is a substrate-recognition subunit of a CRL2LRR-1 E3-ligase. Here we provide genetic evidence supporting a role of this E3-enzyme in the maintenance of DNA replication integrity in Caenorhabditis elegans Through RNAi-based suppressor screens of lrr-1(0) and cul-2(or209ts) mutants, we identified two genes encoding components of the GINS complex, which is part of the Cdc45-MCM-GINS (CMG) replicative helicase, as well as CDC-7 and MUS-101, which drives the assembly of the CMG helicase during DNA replication. In addition, we identified the core components of the ATR/ATL-1 DNA replication checkpoint pathway (MUS-101, ATL-1, CLSP-1, CHK-1). These results suggest that the CRL2LRR-1 E3-ligase acts to modify or degrade factor(s) that would otherwise misregulate the replisome, eventually leading to the activation of the DNA replication checkpoint.
Project description:The spliceosome is formed on pre-mRNA substrates from five small nuclear ribonucleoprotein particles (U1, U2, U4/U6 and U5 snRNPs), and numerous non-snRNP factors. Saccharomyces cerevisiae U4/U6.U5 tri-snRNP comprises U5 snRNA, U4/U6 snRNA duplex and approximately 30 proteins and represents a substantial part of the spliceosome before activation. Schizosaccharomyces pombe U2.U6.U5 spliceosomal complex is a post-catalytic intron lariat spliceosome containing U2 and U5 snRNPs, NTC (nineteen complex), NTC-related proteins (NTR), U6 snRNA, and an RNA intron lariat. Two recent papers describe near-complete atomic structures of these complexes based on cryoEM single-particle analysis. The U4/U6.U5 tri-snRNP structure provides crucial insight into the activation mechanism of the spliceosome. The U2.U6.U5 complex reveals the striking architecture of NTC and NTR and important features of the group II intron-like catalytic RNA core remaining after spliced mRNA is released. These two structures greatly advance our understanding of the mechanism of pre-mRNA splicing.
Project description:Splicing of pre-messenger RNAs into functional messages requires a concerted assembly of proteins and small RNAs that identify the splice junctions and facilitate cleavage of exon-intron boundaries and ligation of exons. One of the key steps in the splicing reaction is the recruitment of a tri-snRNP harboring the U5/U4/U6 snRNPs. The U5 snRNP is also required for both steps of splicing and exon-exon joining. One of the key components of the tri-snRNP is the U5 200kd helicase. The human U5-200kD gene isolated from Hela cells encodes a 200 kDa protein with putative RNA helicase function. Surprisingly, little is known about the functional role of this protein in humans. Therefore, we have investigated the role of the U5-200kD RNA helicase in mammalian cell culture. We created and expressed a dominant negative domain I mutant of the RNA helicase in HEK293 cells and used RNAi to downregulate expression of the endogenous protein. Transient and stable expression of the domain I mutant U5-200kD protein using an ecdysone-inducible system and transient expression of an anti-U5-200kD short hairpin RNA (shRNA) resulted in differential splicing and growth defects in the 293/EcR cells. Cell cycle analysis of the dominant negative clones revealed delayed exit from the G2/M phase of the cell cycle due to a mild splicing defect. In contrast to the domain I dominant negative mutant expressing cells, transient expression of an anti-U5-200kD shRNA resulted in a pronounced S phase arrest and a minute splicing defect. Collectively, this work demonstrates for the first time establishment of differential human cell culture splicing and cell cycle defect models due to perturbed levels of an essential core splicing factor.
Project description:Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication.
Project description:The U2 and U6 snRNAs contribute to the catalysis of intron removal while U5 snRNA loop 1 holds the exons for ligation during pre-mRNA splicing. It is unclear how different exons are positioned precisely with U5 loop 1. Here, we investigate the role of U2 and U6 in positioning the exons with U5 loop 1. Reconstitution in vitro of spliceosomes with mutations in U2 allows U5-pre-mRNA interactions before the first step of splicing. However, insertion in U2 helix Ia disrupts U5-exon interactions with the intron lariat-3' exon splicing intermediate. Conversely, U6 helix Ia insertions prevent U5-pre-mRNA interactions before the first step of splicing. In vivo, synthetic lethal interactions have been identified between U2 insertion and U5 loop 1 insertion mutants. Additionally, analysis of U2 insertion mutants in vivo reveals that they influence the efficiency, but not the accuracy of splicing. Our data suggest that U2 aligns the exons with U5 loop 1 for ligation during the second step of pre-mRNA splicing.