Human PRPF39 is an alternative splicing factor recruiting U1 snRNP to weak 5' splice sites
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ABSTRACT:
PROVIDER: PRJNA866129 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR20828725_1.fastq.gz | Fastqsanger.gz | |||
| SRR20828725_2.fastq.gz | Fastqsanger.gz | |||
| SRR20828726_1.fastq.gz | Fastqsanger.gz | |||
| SRR20828726_2.fastq.gz | Fastqsanger.gz | |||
| SRR20828727_1.fastq.gz | Fastqsanger.gz |
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Project description:Removal of introns during pre-mRNA splicing, which is central to gene expression, initiates by base pairing of U1 snRNA with a 5' splice site (5'SS). In mammals, many introns contain weak 5'SSs that are not efficiently recognized by the canonical U1 snRNP, suggesting alternative mechanisms exist. Here, we develop a cross-linking immunoprecipitation coupled to a high-throughput sequencing method, BCLIP-seq, to identify NRDE2 (Nuclear RNAi defective-2) and CCDC174 (Coiled-Coil Domain-Containing 174) as novel RNA-binding proteins in mouse ES cells that associate with U1 snRNA and unspliced 5'SSs. Both proteins bind directly to U1 snRNA independently of canonical U1 snRNP specific proteins, and they are required for the selection and effective processing of weak 5'SSs. Our results reveal that mammalian cells use non-canonical splicing factors bound directly to U1 snRNA to effectively select suboptimal 5'SS sequences in hundreds of genes, promoting proper splice site choice and accurate pre-mRNA splicing.
2023-09-07 | PXD029392 | Pride
Project description:Removal of introns during pre-mRNA splicing, which is central to gene expression, initiates by base pairing of U1 snRNA with a 5' splice site (5'SS). In mammals, many introns contain weak 5'SSs that are not efficiently recognized by the canonical U1 snRNP, suggesting alternative mechanisms exist. Here, we develop a cross-linking immunoprecipitation coupled to a high-throughput sequencing method, BCLIP-seq, to identify NRDE2 (Nuclear RNAi defective-2) and CCDC174 (Coiled-Coil Domain-Containing 174) as novel RNA-binding proteins in mouse ES cells that associate with U1 snRNA and unspliced 5'SSs. Both proteins bind directly to U1 snRNA independently of canonical U1 snRNP specific proteins, and they are required for the selection and effective processing of weak 5'SSs. Our results reveal that mammalian cells use non-canonical splicing factors bound directly to U1 snRNA to effectively select suboptimal 5'SS sequences in hundreds of genes, promoting proper splice site choice and accurate pre-mRNA splicing.
2023-05-04 | GSE179744 | GEO
Project description:Synaptic activity induces well-known changes in enhancer-promoter driven gene expression but also induces changes in splicing and polyadenylation that are understudied. Here, we investigate the mechanism of expression for alternative polyadenylation isoform Homer1a, an immediate early gene essential to synaptic plasticity. We report that neuronal activation, in neuronal cultures and in adult mouse brain, depletes the splice factor U1 snRNP from Homer1 pre-mRNA and that this causes shifted utilization of a cryptic polyadenylation signal within intron 5 resulting in Homer1a expression. Because U1 snRNP is a ubiquitous splice factor, we tested the generality of activity-driven U1 snRNP depletion as a mechanism for gene expression using RNA immunoprecipitation sequencing. Analysis reveals that neuronal activity changes U1 snRNP binding to ~2000 transcripts and for a subset of transcripts, a reduction in U1 snRNP binding was accompanied by utilization of a cryptic intronic polyadenylation site. This subset is enriched for transcripts encoding synaptic proteins involved in excitability control. Genes demonstrating activity-dependent reduced U1 snRNP binding often encode a binding motif for Sam68, a neuronal alternative polyadenylation factor. Findings reveal that activity-driven changes in intron utilization for transcript termination serves an important role in synaptic plasticity.
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Project description:Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction. Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5' end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1-PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1-PAS axis limits pervasive transcription throughout the genome. 3' end sequencing of poly (A) + RNAs in mouse ES cells with and without U1 snRNP inhibition using antisense morpholino oligonucleotides (AMO). Each with two biological replicates.
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Project description:Pervasive transcription in the mammalian genome produces thousands of long noncoding RNAs (lncRNAs) and promoter- or enhancer-associated unstable transcripts. They preferentially locate to chromatin, at which some regulate chromatin structure, transcription and RNA processing. While several RNA sequences responsible for nuclear localization have been identified, such as repeats in the lncRNA Xist and Alu-like elements for long RNAs, how lncRNAs as a class are enriched on chromatin remains elusive. To screen for cis-elements that contribute to RNA-chromatin localization, we developed a high-throughput method named RNA elements for subcellular localization by sequencing (REL-seq), and discovered a U1 small nuclear ribonucleoprotein (snRNP)-recognition motif being critical for chromatin localization of reporter RNAs. Across the genome, chromatin-bound lncRNAs, which are enriched with 5’ splice sites and depleted of 3’ splice sites, exhibit high levels of U1 snRNA binding compared to cytoplasm-localized protein-coding mRNAs. Acute depletion of U1 snRNA, or U1 snRNP protein component SNRNP70, drastically reduces the chromatin association of hundreds of lncRNAs and unstable transcripts without altering the overall transcription rate in cells. In addition, rapid degradation of SNRNP70 reduces the localization of both nascent and polyadenylated lncRNA transcripts to chromatin, and disrupts the nuclear-speckles and genome-wide localization of Malat1, a highly conserved and abundant lncRNA. Moreover, chromatin-bound U1 snRNP interacts with transcriptionally engaged RNA polymerase (Pol) II. Together, these results demonstrate that U1 snRNP acts widely to tether and mobilize lncRNAs to chromatin in a Pol II transcription-dependent manner. Our findings uncover a novel role of U1 snRNP beyond pre-mRNA processing and provide molecular insights into how lncRNAs are recruited to Pol II-transcribed genes and have a propensity for chromatin-associated functions.
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Project description:In the earliest step of spliceosome assembly, the two splice sites flanking an intron are brought into proximity by U1 snRNP and U2AF. The mechanism that facilitates this intron looping is poorly understood. Using a CRISPR interference-based approach to halt RNA polymerase II transcription in the middle of introns, we discovered that the 5 splice site base pairs with a U1 snRNA that is tethered to RNA polymerase II during intron synthesis. Correlation with splicing outcomes demonstrate that these associations are functional. The interactions between 5 splice sites, U1 snRNP, and elongating RNA polymerase II occurs genome-wide. Our findings reveal that during intron synthesis the upstream 5 splice site remains attached to the transcriptional machinery and is thus brought into proximity of the 3 splice site to enable rapid splicing.
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Project description:In the earliest step of spliceosome assembly, the two splice sites flanking an intron are brought into proximity by U1 snRNP and U2AF. The mechanism that facilitates this intron looping is poorly understood. Using a CRISPR interference-based approach to halt RNA polymerase II transcription in the middle of introns, we discovered that the 5 splice site base pairs with a U1 snRNA that is tethered to RNA polymerase II during intron synthesis. Correlation with splicing outcomes demonstrate that these associations are functional. The interactions between 5 splice sites, U1 snRNP, and elongating RNA polymerase II occurs genome-wide. Our findings reveal that during intron synthesis the upstream 5 splice site remains attached to the transcriptional machinery and is thus brought into proximity of the 3 splice site to enable rapid splicing.
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Project description:In the earliest step of spliceosome assembly, the two splice sites flanking an intron are brought into proximity by U1 snRNP and U2AF. The mechanism that facilitates this intron looping is poorly understood. Using a CRISPR interference-based approach to halt RNA polymerase II transcription in the middle of introns, we discovered that the 5 splice site base pairs with a U1 snRNA that is tethered to RNA polymerase II during intron synthesis. Correlation with splicing outcomes demonstrate that these associations are functional. The interactions between 5 splice sites, U1 snRNP, and elongating RNA polymerase II occurs genome-wide. Our findings reveal that during intron synthesis the upstream 5 splice site remains attached to the transcriptional machinery and is thus brought into proximity of the 3 splice site to enable rapid splicing.
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Project description:Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was performed to generate genome-wide binding maps of two U1-snRNP proteins: U1C and U1-70K in Trypanosoma brucei. 3 (2) biological replicates of U1C (U1-70K) -specific co-immunoprecipitated RNA after UV-crosslinking
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Project description:The RNA-binding protein FUS/TLS, mutation in which is causative of the fatal motor neuron disease ALS, is demonstrated to directly bind to the U1-snRNP and SMN complexes. ALS-causative mutations in FUS/TLS are shown to abnormally enhance their interaction with SMN and reduce interaction with U1-snRNP. Correspondingly, global RNA analysis reveals a mutant-dependent loss of splicing activity, with ALS-linked mutants failing to reverse changes caused by loss of wild-type FUS/TLS. Furthermore, a common FUS/TLS mutant-associated RNA splicing signature is identified in ALS patient fibroblasts. Taken together, our studies establish potentially converging disease mechanisms in ALS and spinal muscular atrophy, with ALS-causative mutants acquiring properties representing both gain (dysregulation of SMN) and loss (reduced RNA processing mediated by U1-snRNP) of function. RNA-mediated oligonucleotide Annealing, Selection, and Ligation with Next-Generation sequencing (RASL-seq) method was used for analyzing alternative splicing changes. Oligonucleotide probes are designed to anneal to the exon-exon junctions. The probe library was assembled to assess 5530 unique alternative splicing events, most of which were exon inclusion or skipping, with a minority for alternative 5’- or 3’- splice sites. The splicing changes were compared among groups of reducing FUS/TLS or SMN levels, or expressing various FUS mutations to determine the loss versus gain of FUS/TLS function on splicing regulation.
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