Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Functional analysis of Cwc24 ZF-domain in 5' splice site selection.

ABSTRACT: The essential splicing factor Cwc24 contains a zinc-finger (ZF) domain required for its function in splicing. Cwc24 binds over the 5' splice site after the spliceosome is activated, and its binding prior to Prp2-mediated spliceosome remodeling is important for proper interactions of U5 and U6 with the 5' splice site sequence and selection of the 5' splice site. Here, we show that Cwc24 transiently interacts with the 5' splice site in formation of the functional RNA catalytic core during spliceosome remodeling, and the ZF-motif is required for specific interaction of Cwc24 with the 5' splice site. Deletion of the ZF domain or mutation of the conserved ZF residues greatly weakened the association of Cwc24 with the spliceosome, and lowered the affinity and specificity of its interaction with the 5' splice site, resulting in atypical interactions of U5, U6 and Prp8 with the 5' splice site, and aberrant cleavage at the 5' splice site. Our results reveal a crucial role of the Cwc24 ZF-motif for defining 5' splice site selection in the first splicing step.

SUBMITTER: Wu NY

PROVIDER: S-EPMC6821175 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Functional analysis of Cwc24 ZF-domain in 5' splice site selection.

Wu Nan-Ying NY Cheng Soo-Chen SC

Nucleic acids research 20191101 19

The essential splicing factor Cwc24 contains a zinc-finger (ZF) domain required for its function in splicing. Cwc24 binds over the 5' splice site after the spliceosome is activated, and its binding prior to Prp2-mediated spliceosome remodeling is important for proper interactions of U5 and U6 with the 5' splice site sequence and selection of the 5' splice site. Here, we show that Cwc24 transiently interacts with the 5' splice site in formation of the functional RNA catalytic core during spliceos ...[more]

PMID: 31504764

Similar Datasets

A Novel Role for Nucleolin in Splice Site Selection

Project description:In search for nuclear partners of initiator-tRNA, previously shown to be involved in a quality control of splice site selection, we identified nucleolin (NCL) as specifically and directly bound to it in the nucleus and not in the cytoplasm (using affinity purification of UV crosslinked nuclear initiator-tRNA followed by mass spectrometry). To further explore the role of NCL in splice site selection, we knocked down NCL. We report the activation of 399 latent splice sites upon NCL knockdown, as revealed by RNA deep sequencing of siRNA treated NCL compared to control siRNA. Our study identifies NCL as the first protein component of this quality control mechanism of splice site selection. Our study thus establishes an experimental strategy and computational pipeline that could be used to identify other components of this quality control mechanism.

2022-03-08 | GSE142836 | GEO

POT1 splice site mutant analysis

Project description:We are interested in the contribution mutations in the Shelterin complex protein POT1 may have to the development of melanoma. We have identified a patient who carries a splice site mutation in POT1 and as part of our analysis of this gene we aim to sequence the transcriptome of this patient to see how this mutation influences splicing. RNA has been obtained from lymphocytes collected from the patient.

| EGAS00001000571 | EGA

Principles and correction of 5'-splice site selection.

Project description:In Eukarya, immature mRNA transcripts (pre-mRNA) often contain coding sequences, or exons, interleaved by non-coding sequences, or introns. Introns are removed upon splicing, and further regulation of the retained exons leads to alternatively spliced mRNA. The splicing reaction requires the stepwise assembly of the spliceosome, a macromolecular machine composed of small nuclear ribonucleoproteins (snRNPs). This review focuses on the early stage of spliceosome assembly, when U1 snRNP defines each intron 5'-splice site (5'ss) in the pre-mRNA. We first introduce the splicing reaction and the impact of alternative splicing on gene expression regulation. Thereafter, we extensively discuss splicing descriptors that influence the 5'ss selection by U1 snRNP, such as sequence determinants, and interactions mediated by U1-specific proteins or U1 small nuclear RNA (U1 snRNA). We also include examples of diseases that affect the 5'ss selection by U1 snRNP, and discuss recent therapeutic advances that manipulate U1 snRNP 5'ss selectivity with antisense oligonucleotides and small-molecule splicing switches.

| S-EPMC9311317 | biostudies-literature

A novel role for nucleolin in splice site selection.

Project description:Latent 5' splice sites, not normally used, are highly abundant in human introns, but are activated under stress and in cancer, generating thousands of nonsense mRNAs. A previously proposed mechanism to suppress latent splicing was shown to be independent of NMD, with a pivotal role for initiator-tRNA independent of protein translation. To further elucidate this mechanism, we searched for nuclear proteins directly bound to initiator-tRNA. Starting with UV-crosslinking, we identified nucleolin (NCL) interacting directly and specifically with initiator-tRNA in the nucleus, but not in the cytoplasm. Next, we show the association of ini-tRNA and NCL with pre-mRNA. We further show that recovery of suppression of latent splicing by initiator-tRNA complementation is NCL dependent. Finally, upon nucleolin knockdown we show activation of latent splicing in hundreds of coding transcripts having important cellular functions. We thus propose nucleolin, a component of the endogenous spliceosome, through its direct binding to initiator-tRNA and its effect on latent splicing, as the first protein of a nuclear quality control mechanism regulating splice site selection to protect cells from latent splicing that can generate defective mRNAs.

| S-EPMC8890436 | biostudies-literature

Functional analysis of a novel splice site variant in the ASAH1 gene.

Project description:BackgroundAcid ceramidase (ACDase) deficiency is an ultrarare autosomal recessive lysosomal disorder caused by pathogenic N-acylsphingosine amidohydrolase (ASAH1) variants. It presents with either Farber disease (FD) or spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME).ObjectiveThe study aims to identify a novel splice site variant in a hydrops fetus that causes ASAH1-related disorder, aid genetic counseling, and accurate prenatal diagnosis.MethodsWe report a case of hydrops fetalis with a novel homozygous mutation in ASAH1 inherited from non-consanguineous parents. We performed copy number variation sequencing (CNV-Seq) and whole exome sequencing (WES) on the fetus and family, respectively. Minigene splicing analyses were conducted to confirm the pathogenic variants.ResultsWES data revealed a splice site variant of the ASAH1 (c.458-2A>T), which was predicted to affect RNA splicing. Minigene splicing analyses found that the c.458-2A>T variant abolished the canonical splicing of intron 6, thereby activating two cryptic splicing products (c.456_458ins56bp and c.458_503del).ConclusionsOverall, we identified a novel splice site variant in the mutational spectrum of ASAH1 and its aberrant effect on splicing. These findings highlight the importance of ultrasonic manifestation and family history of fetal hydrops during ASAH1-related disorders and could also aid genetic counseling and accurate prenatal diagnosis. To the best of our knowledge, this is the shortest-lived account of ASAH1-related disorders in utero with severe hydrops fetalis.

| S-EPMC10767590 | biostudies-literature

Postcatalytic spliceosome structure reveals mechanism of 3'-splice site selection.

Project description:Introns are removed from eukaryotic messenger RNA precursors by the spliceosome in two transesterification reactions-branching and exon ligation. The mechanism of 3'-splice site recognition during exon ligation has remained unclear. Here we present the 3.7-angstrom cryo-electron microscopy structure of the yeast P-complex spliceosome immediately after exon ligation. The 3'-splice site AG dinucleotide is recognized through non-Watson-Crick pairing with the 5' splice site and the branch-point adenosine. After the branching reaction, protein factors work together to remodel the spliceosome and stabilize a conformation competent for 3'-splice site docking, thereby promoting exon ligation. The structure accounts for the strict conservation of the GU and AG dinucleotides at the 5' and 3' ends of introns and provides insight into the catalytic mechanism of exon ligation.

| S-EPMC5808836 | biostudies-literature

COSSMO: predicting competitive alternative splice site selection using deep learning.

Project description:MotivationAlternative splice site selection is inherently competitive and the probability of a given splice site to be used also depends on the strength of neighboring sites. Here, we present a new model named the competitive splice site model (COSSMO), which explicitly accounts for these competitive effects and predicts the percent selected index (PSI) distribution over any number of putative splice sites. We model an alternative splicing event as the choice of a 3' acceptor site conditional on a fixed upstream 5' donor site or the choice of a 5' donor site conditional on a fixed 3' acceptor site. We build four different architectures that use convolutional layers, communication layers, long short-term memory and residual networks, respectively, to learn relevant motifs from sequence alone. We also construct a new dataset from genome annotations and RNA-Seq read data that we use to train our model.ResultsCOSSMO is able to predict the most frequently used splice site with an accuracy of 70% on unseen test data, and achieve an R2 of 0.6 in modeling the PSI distribution. We visualize the motifs that COSSMO learns from sequence and show that COSSMO recognizes the consensus splice site sequences and many known splicing factors with high specificity.Availability and implementationModel predictions, our training dataset, and code are available from http://cossmo.genes.toronto.edu.Supplementary informationSupplementary data are available at Bioinformatics online.

| S-EPMC6022534 | biostudies-literature

Inducible nuclear import by TetR aptamer-controlled 3' splice site selection.

Project description:Correct cellular localization is essential for the function of many eukaryotic proteins and hence cell physiology. Here, we present a synthetic genetic device that allows the control of nuclear and cytosolic localization based on controlled alternative splicing in human cells. The device is based on the fact that an alternative 3' splice site is located within a TetR aptamer that in turn is positioned between the branch point and the canonical splice site. The novel splice site is only recognized when the TetR repressor is bound. Addition of doxycycline prevents TetR aptamer binding and leads to recognition of the canonical 3' splice site. It is thus possible to produce two independent splice isoforms. Since the terminal loop of the aptamer may be replaced with any sequence of choice, one of the two isoforms may be extended by the respective sequence of choice depending on the presence of doxycycline. In a proof-of-concept study, we fused a nuclear localization sequence to a cytosolic target protein, thus directing the protein into the nucleus. However, the system is not limited to the control of nuclear localization. In principle, any target sequence can be integrated into the aptamer, allowing not only the production of a variety of different isoforms on demand, but also to study the function of mislocalized proteins. Moreover, it also provides a valuable tool for investigating the mechanism of alternative splicing in human cells.

| S-EPMC7812871 | biostudies-literature

Stop codons affect 5' splice site selection by surveillance of splicing.

Project description:Pre-mRNA splicing involves recognition of a consensus sequence at the 5' splice site (SS). However, only some of the many potential sites that conform to the consensus are true ones, whereas the majority remain silent and are not normally used for splicing. We noticed that in most cases the utilization of such a latent intronic 5' SS for splicing would introduce an in-frame stop codon into the resultant mRNA. This finding suggested a link between SS selection and maintenance of an ORF within the mRNA. Here we tested this idea by analyzing the splicing of pre-mRNAs in which in-frame stop codons upstream of a latent 5' SS were mutated. We found that splicing with the latent site is indeed activated by such mutations. Our findings predict the existence of a checking mechanism, as a component of the nuclear pre-mRNA splicing machine, to ensure the maintenance of an ORF. This notion is highly important for accurate gene expression, as perturbations that would lead to splicing at these latent sites are expected to introduce in-frame stop codons into the majority of mRNAs.

| S-EPMC122760 | biostudies-literature

Noncanonical registers and base pairs in human 5' splice-site selection.

Project description:Accurate recognition of splice sites is essential for pre-messenger RNA splicing. Mammalian 5' splice sites are mainly recognized by canonical base-pairing to the 5' end of U1 small nuclear RNA, yet we described multiple noncanonical base-pairing registers by shifting base-pair positions or allowing one-nucleotide bulges. By systematic mutational and suppressor U1 analyses, we prove three registers involving asymmetric loops and show that two-nucleotide bulges but not longer can form in this context. Importantly, we established that a noncanonical uridine-pseudouridine interaction in the 5' splice site/U1 helix contributes to the recognition of certain 5' splice sites. Thermal melting experiments support the formation of noncanonical registers and uridine-pseudouridine interactions. Overall, we experimentally validated or discarded the majority of predicted noncanonical registers, to derive a list of 5' splice sites using such alternative mechanisms that is much different from the original. This study allows not only the mechanistic understanding of the recognition of a wide diversity of mammalian 5' splice sites, but also the future development of better splice-site scoring methods that reliably predict the effects of disease-causing mutations at these sequences.

| S-EPMC4856993 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data