Global alternative splicing analysis of mutations in two new spliceosome components KIN17 and PRCC in C. elegans
Ontology highlight
ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
PROVIDER: GSE178335 | GEO | 2021/06/30
REPOSITORIES: GEO
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Project description:Dominant mutant alleles of prp-8 and snrp-27 were identifed in a forward genetic screen for suppressors of cryptic splice site activation
2018-07-17 | GSE113275 | GEO
Project description:Global alternative splicing analysis of mutations in two new spliceosome components KIN17 and PRCC in C. elegans
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Project description:The spliceosome undergoes extensive rearrangements as it assembles in multiple steps onto the precursor messenger RNA. In the earliest assembly step, U1snRNA identifies the 5' splice site through base-pairing interactions. However, U1snRNA leaves the spliceosome relatively early in the assembly process. The 5' splice site identity is subsequently maintained through interactions with U6snRNA, protein factor PRP8, and other components of the spliceosome during the complex assembly and rearrangements that build the catalytic site. Using a forward genetic screen in C. elegans, we have identified splicing suppressors of a locomotion defect caused by a 5'ss mutation. Here we report three new extragenic suppressor alleles from this screen, two in PRP8 and one in SNRNP200/Brr2. mRNASeq studies of these suppressor strains indicates that there are specific native targets with alternative 5' and alternative 3' splicing events affected by these suppressors, especially for the suppressor PRP8 D 1549N (position D1556 in humans). The strong suppressor at the unstructured N-terminus of SNRP200, N18K, indicates a potential regulatory role for this region. By examining distinct changes in the splicing of native genes, and by mapping these conserved suppressor residues onto cryoEM structural models of assembling human spliceosomes, we conclude that there are multiple interactions in the spliceosome that are required to ensure that the initial 5'ss identified by U1snRNA early in spliceosome assembly is the one that gets loaded into the catalytic core.The spliceosome undergoes extensive rearrangements as it assembles in multiple steps onto the precursor messenger RNA. In the earliest assembly step, U1snRNA identifies the 5' splice site through base-pairing interactions. However, U1snRNA leaves the spliceosome relatively early in the assembly process. The 5' splice site identity is subsequently maintained through interactions with U6snRNA, protein factor PRP8, and other components of the spliceosome during the complex assembly and rearrangements that build the catalytic site. Using a forward genetic screen in C. elegans, we have identified splicing suppressors of a locomotion defect caused by a 5'ss mutation. Here we report three new extragenic suppressor alleles from this screen, two in PRP8 and one in SNRNP200/Brr2. mRNASeq studies of these suppressor strains indicates that there are specific native targets with alternative 5' and alternative 3' splicing events affected by these suppressors, especially for the suppressor PRP8 D 1549N (position D1556 in humans). The strong suppressor at the unstructured N-terminus of SNRP200, N18K, indicates a potential regulatory role for this region. By examining distinct changes in the splicing of native genes, and by mapping these conserved suppressor residues onto cryoEM structural models of assembling human spliceosomes, we conclude that there are multiple interactions in the spliceosome that are required to ensure that the initial 5'ss identified by U1snRNA early in spliceosome assembly is the one that gets loaded into the catalytic core.
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Project description:Spatial separation of eukaryotic cells into the nuclear and cytoplasmic compartment permits uncoupling of DNA transcription from translation of mRNAs and allows cells to modify newly transcribed pre mRNAs extensively. Intronic sequences (introns), which interrupt the coding elements (exons), are excised ("spliced") from pre-mRNAs in the nucleus to yield mature mRNAs. This not only enables alternative splicing as an important source of proteome diversity, but splicing is also an essential process in all eukaryotes and knock-out or knock-down of splicing factors frequently results in defective cell proliferation and cell division. However, higher eukaryotes progress through cell division only after breakdown of the nucleus ("open mitosis"). Open mitosis suppresses basic nuclear functions such as transcription and splicing, but allows separate, mitotic functions of nuclear proteins in cell division. Mitotic defects arising after loss-of-function of splicing proteins therefore could be an indirect consequence of compromised splicing in the closed nucleus of the preceding interphase or reflect a direct contribution of splicing proteins to open mitosis. Although experiments to directly distinguish between these two alternatives have not been reported, indirect evidence exists for either hypotheses. In this review, we survey published data supporting an indirect function of splicing in open mitosis or arguing for a direct function of spliceosomal proteins in cell division.
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Project description:Recently, it has been reported that there is a differential subcellular distribution of components of the minor U12-dependent and major U2-dependent spliceosome, and further that the minor spliceosome functions in the cytoplasm. To study the subcellular localization of the snRNA components of both the major and minor spliceosomes, we performed in situ hybridizations with mouse tissues and human cells. In both cases, all spliceosomal snRNAs were nearly exclusively detected in the nucleus, and the minor U11 and U12 snRNAs were further shown to colocalize with U4 and U2, respectively, in human cells. Additionally, we examined the distribution of several spliceosomal snRNAs and proteins in nuclear and cytoplasmic fractions isolated from human cells. These studies revealed an identical subcellular distribution of components of both the U12- and U2-dependent spliceosomes. Thus, our data, combined with several earlier publications, establish that, like the major spliceosome, components of the U12-dependent spliceosome are localized predominantly in the nucleus.
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Project description:HGPS is a rare premature ageing disease, caused by a mutation in the LMNA gene, which activates a cryptic splice site, resulting in the production of a mutant lamin A isoform, called progerin. Sporadic usage of the same cryptic splice site has been observed with normal physiological aging. As it is unknown how HGPS causes premature ageing defects, we set out to determine the gene signature of both young healthy individuals, old healthy individuals, as well as HGPS patients.
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Project description:The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926A>C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5’-splice site (5’SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5’SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.
2024-03-01 | PXD043645 | Pride
Project description:Retinitis pigmentosa is a rare, progressive disease which affects photoreceptors and retinal pigment epithelial (RPE) cells with blindness as a final outcome. Despite high medical and social impact, there is currently no therapeutic options to slow down the progression or cure the disease. The development of effective therapies was largely hindered by high genetic heterogeneity, inaccessible disease tissue and unfaithful model organisms. The fact that components of ubiquitously expressed splicing factors lead to the retina-specific disease is an additional intriguing question. Herein, we sought to correlate the retinal cell type-specific disease phenotype with the splicing profile shown by a patient with autosomal recessive RP, caused by a mutation in pre–mRNA splicing factor 8 (PRPF8). In order to get insight into the role of PRPF8 in homeostasis and disease, we capitalize on the ability to generate patient-specific RPE cells and reveal differentially expressed genes unique to RPE cells. We found that spliceosomal complex and ribosomal functions are crucial in determining cell type specificity through differential expression and alternative splicing (AS), and that PRPF8 mutation causes global changes in splice site selection and exon inclusion that particularly affect genes involved in these cellular functions. This finding corroborates the hypothesis that retinal tissue identity is conferred by specific splicing program, and identifies retinal AS events as a framework towards the design of novel therapeutic opportunities.
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