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At least 19 recordsLinked to original sources
PubMed1991

An RNA splice site mutation in the C1-inhibitor gene causes type I hereditary angio-oedema.

Restriction fragment length polymorphism analysis, the polymerase chain reaction and nucleotide sequencing have been used to characterise a single base substitution (G----T) at nucleotide 8863 in the C1-inhibitor gene. This destroys the 5' donor splice site recognition motif of the sixth intron. Family studies suggest that the mutation is responsible for type I hereditary angio-oedema in a studied kindred.

Angioedema↗
PubMed2026

Pervasive noise in human pre-mRNA splice site selection.

RNA splicing has historically been thought to be highly efficient and accurate, with little opportunity for deviation from regulated alternative splicing. This dogma has been challenged by recent observations that biological noise may contribute substantially to transcriptome diversity. However, quantitative understanding of stochastic splicing variation is challenging because these transcripts are likely subject to rapid degradation. Here, we use deep sequencing across RNA compartments to track splicing intermediates in human cells and see abundant cryptic splicing associated with genomic features that promote splicing noise. We observe pervasive usage of low-fidelity splice sites, likely due to stochasticity in recruitment or binding of the spliceosome. These sites are turned over quickly and show evidence for nuclear and cytoplasmic degradation, suggesting widespread surveillance and rapid quality control of non-productive transcripts. Our findings provide insights into the propensity for error in RNA processing mechanisms and regulation of alternative splice sites across a gene.

Humans↗
PubMed1992

The 5' splice site consensus RNA oligonucleotide induces assembly of U2/U4/U5/U6 small nuclear ribonucleoprotein complexes.

A short RNA oligonucleotide comprising the 5' splice site consensus sequence (5'SS RNA oligo) efficiently inhibits splicing of mRNA precursors in HeLa cell nuclear extracts. Addition of 5'SS RNA oligo inhibits early, but not late, steps in the splicing reaction, affecting the process of spliceosome assembly. In the presence of 5'SS RNA oligo a majority of U4/U5/U6 triple small nuclear ribonucleoprotein (snRNP) complex present in HeLa nuclear extracts associates with U2 snRNP to form a multi-snRNP complex, which could account for the observed inhibition of splicing by the oligo. This same set of snRNPs has been shown to assemble on pre-mRNAs during in vitro splicing to form splicing complex B. Removal of the 5' end of U1 snRNA, which is complementary to the 5' splice site, does not prevent association of snRNPs into U2/U4/U5/U6 complex in the presence of 5'SS RNA oligo. This suggests that interactions other than U1 snRNA.5'SS RNA oligo base pairing are used in recognition of the oligo sequence. 5'SS RNA oligo-induced assembly of the multi-snRNP complex may thus serve as a model to study the mechanism of 5' splice site recognition during splicing.

Base Sequence↗
PubMed2025

Pervasive noise in human splice site selection.

RNA splicing has historically been thought to be highly efficient and accurate, with little opportunity for deviation from regulated alternative splicing decisions. This dogma has been challenged by recent observations that suggest that biological noise may contribute substantially to transcriptome diversity. However, quantitative understanding of stochastic variations in splicing is challenging because these transcripts are likely subject to rapid degradation. Here, we use ultra-deep sequencing across RNA compartments to track splicing intermediates in human cells and see abundant cryptic splicing associated with genomic features that promote splicing noise. We observe pervasive usage of low-fidelity splice sites, likely due to stochasticity in recruitment or binding of the spliceosome. These sites are most likely degraded in the nucleus rather than targeted by translation-dependent degradation processes, suggesting widespread surveillance and rapid quality control of non-productive RNA transcripts. Our findings provide unprecedented insights into the propensity for error in RNA processing mechanisms and the regulation of alternative splice sites across a gene.

Journal Article↗
PubMed1992

Molecular diversity of L-type Ca2+ channel transcripts in human fibroblasts.

The nucleotide sequence of cDNA encoding the human fibroblast Ca2+ channel of L type (HFCC) has been determined. It is highly homologous to L-type channels previously cloned from rabbit lung and heart as well as from rat brain. At least four sites of molecular diversity were identified in the nucleotide sequence of HFCC. Three of these include regions encoding the transmembrane segments IIS6, IIIS2, and IVS3, which are known to be important for channel gating properties. The positions of these sites correlate with RNA splice sites, indicating that the molecular diversity of the transcripts is a result of alternative splicing. The fourth diversity region is located at the C-terminal region and comprises insertions and deletions. It is suggested that these variations may give rise to multiple subforms of HFCC with altered electrophysiological properties.

Amino Acid Sequence↗
PubMed1992

Intronic sequences and 3' splice sites control Rous sarcoma virus RNA splicing.

cis-acting sequences of Rous sarcoma virus (RSV) RNA involved in control of the incomplete splicing that is part of the retroviral life cycle have been studied. The 5' and two alternative 3' splice sites, as well as negative regulator of splicing element in the intron, have been introduced into chimeric constructs, and their responsive roles in splicing inhibition have been evaluated by transient transfection experiments. Although the RSV 5' splice site was used efficiently in these assays, substrates containing either the RSV env or the RSV src 3' splice site were not spliced completely, resulting in 40 to 50% unspliced RNA. Addition of the negative regulator of splicing element to substrates containing RSV 3' splice sites resulted in greater inhibition of splicing (70 to 80% unspliced RNA), suggesting that the two elements function independently and additively. Deletion of sequences more than 70 nucleotides upstream of the src 3' splice site resulted in efficient splicing at this site, suggesting that inefficient usage is not inherent in this splice site but is instead due to to sequences upstream of it. Insertion of these upstream sequences into the intron of a heterologous pre-mRNA resulted in partial inhibition of its splicing. In addition, secondary structure interactions were predicted to occur between the src 3' splice site and the inhibitory sequences upstream of it. Thus, RSV splicing control involves both intronic sequences and 3' splice sites, with different mechanisms involved in the underutilization of the env and src splice acceptor sites.

Avian Sarcoma Viruses↗
PubMed2025

RNase III cleavage sites spread across splice junctions enforce sequential snoRNA processing.

Small nucleolar RNAs (snoRNAs) are a class of eukaryotic non-coding RNA molecules whose precursor transcripts are capped and polyadenylated. However, these end modifications are detrimental to snoRNA function and must be removed, a process typically involving excision from introns and/or endonucleolytic cleavage. For RNA precursors that host multiple snoRNAs, the sequence of maturation events is potentially important, but not well understood. Here, we report a new mode of maturation concerning snoRNA pairs that are co-hosted in the intron and the adjacent 3' exon of a precursor transcript. For a snoRNA pair with this arrangement in Schizosaccharomyces pombe, we found that the sequence surrounding an exon-exon junction within their precursor transcript folds into a hairpin after splicing of the intron. This hairpin recruits the RNase III ortholog Pac1, which participates in the maturation of the downstream snoRNA by cleaving the precursor. Our findings suggest that conditional RNase III cleavage signals hidden in an exon-exon junction evolved to enforce sequential snoRNA processing. Sequence analysis suggests that this mechanism is conserved in animals and plants.

RNA, Small Nucleolar↗
PubMed2026

The structured mRNA element 45ABC mediates auto- and cross-regulation of RBP45 genes via alternative splicing.

Alternative splicing (AS) is a common gene regulatory mechanism involving distinct interactions between trans-acting factors and cis-regulatory elements on the precursor messenger RNA (pre-mRNA). In this study, we have functionally characterized the structured motif 45ABC, which is located in the pre-mRNAs of RNA-binding protein (RBP) 45 genes in many plant species. Our data revealed that this element mediates a negative auto- and cross-regulatory feedback loop via AS of the three 45ABC-containing RBP45 genes in Arabidopsis thaliana. We identified a G-rich stretch within the first stem as a potential RBP45 binding site and observed increased RBP45-dependent AS upon structural weakening of this pairing element. The second stem includes the alternative 5' splice site being activated in the presence of RBP45. Based on the known interaction between RBP45 homologs and U1 snRNP components required for 5' splice site recognition, we propose that RBP45 recruitment to stem I of 45ABC may induce usage of the alternative 5' splice site in stem II. The resulting splicing variant is unproductive, thereby diminishing RBP45 expression. Analysing the splicing-regulatory impact of the three At-RBP45 genes in auto- and cross-regulation and a transcriptome-wide manner revealed unequal genetic redundancy with a major role of RBP45B. Furthermore, phenotypical analysis of single- and higher-order rbp45 mutants pointed at these genes' functions in controlling primary root growth and flowering time. Taken together, we demonstrated that both sequence and structural features of 45ABC are critical for proper splicing control, balancing RBP45 expression and functions in plants via a conserved mRNA motif.

Alternative Splicing↗
PubMed1992

Evidence for a base-pairing interaction between U6 small nuclear RNA and 5' splice site during the splicing reaction in yeast.

U6 small nuclear RNA (snRNA) is an essential factor in mRNA splicing. On the basis of the high conservation of its sequence, it has been proposed that U6 snRNA may function catalytically during the splicing reaction. If this is the case, it is likely that U6 snRNA interacts with the splice sites in the spliceosome to catalyze the reaction. We have used UV crosslinking to analyze the interactions of U6 snRNA with the splicing substrates during the yeast splicing reaction. Crosslinked products in which the central region of U6 snRNA was joined to the 5' splice site region of mRNA precursor and lariat intermediate were identified. The crosslinking sites were precisely located in one of these products. The results suggest a possible base-pairing interaction between U6 snRNA and the 5' splice site of the mRNA precursor.

Base Sequence↗
PubMedDate not supplied

Generation of DM-20 splice site in myelin proteolipid protein gene: a hypothesis based on analysis of the amphibian protein.

The DM-20 isoform of proteolipid protein (PLP) of central nervous system myelin is undetectable in amphibia, but readily detectable in reptiles, birds and mammals. To explain the phylogenetic origin of DM-20, it has been proposed that a donor-acceptor RNA splice site was generated by the introduction of a single base change in the codon encoding amino acid 116 of PLP. We tested this hypothesis by isolating and sequencing peptides from bullfrog PLP. One of the peptides corresponded to mammalian residues 110-122, which contain the N-terminal boundary region for the domain excluded from DM-20 in higher vertebrates. We found that the Thr115-Val116 sequence is conserved between frog and mammal. Therefore, we propose that a single base change in the third position of the codon for Thr115 would account for the appearance of the new donor-acceptor splice site. Three amino acids elsewhere in this thirteen-residue peptide were found to differ between bullfrog and mammal, which could account for the previously reported weak recognition of amphibian PLP by an antiserum specific for peptide 109-128. A second peptide from bullfrog PLP had a sequence identical to that of residues 45-52 in mammalian PLP. Our findings explain how a specific change in the myelin PLP gene could generate a new form of the PLP protein.

Amino Acid Sequence↗
PubMed1992

Reduced secreted mu mRNA synthesis in selective IgM deficiency of Bloom's syndrome.

Serum IgM concentrations were low although serum IgG and IgA concentrations were normal in both our patients with Bloom's syndrome. Although the percentages of surface IgM-bearing cells were not reduced, the numbers of IgM-secreting cells were markedly reduced. The membrane-bound mu (microns) and secreted mu (microseconds) mRNAs are produced from transcripts of a single immunoglobulin mu gene by alternative RNA processing pathways. The control of microseconds mRNA synthesis depends on the addition of poly(A) to microseconds C-terminal segment. In both patients, mu mRNA was well detected but microseconds C-terminal mRNA was scarcely detected, suggesting that microns mRNA was well transcribed but microseconds mRNA was not. There was, at least, no mutation or deletion in the microseconds C-terminal coding sequence, the RNA splice site (GG/TAAAC) at the 5' end of microseconds C-terminal segment and the AATAAA poly(A) signal sequence in both patients. Our results suggest that selective IgM deficiency in Bloom's syndrome is due to an abnormality in the maturation of surface IgM-bearing B cells into IgM-secreting cells and a failure of microseconds mRNA synthesis. Moreover, reduced microseconds mRNA synthesis may be due to the defect on developmental regulation of the site at which poly(A) is added to transcripts of the mu gene.

Adult↗
PubMed1992

Linkage of two pseudogenes from V kappa 1 and V kappa 9 murine immunoglobulin families.

As an initial step towards the molecular analysis of the murine V kappa locus, a cosmid library from BALB/cJ mouse liver DNA was screened with probes representing 10 V kappa families. Of eight cosmids that were isolated from the initial screen, five contained a single restriction fragment that hybridized to the probes. Two cosmids contained two fragments that hybridized to the same probe, V kappa 4, indicating that some V kappa 4 gene segments are linked. One cosmid had two genes that belonged to different families, V kappa 1 and V kappa 9. The two gene segments were located within 12 kb of each other and lay in the same transcriptional orientation. Linkage of gene segments from the V kappa 1 and V kappa 9 families is consistent with a genetic map of the locus, and provides physical evidence for the first time that two genes from different families are closely linked in the murine kappa locus. Sequence analysis revealed that both genes are pseudogenes: the V kappa psi 1.7 gene segment has eight mutations, including termination codons, insertions, and deletions, and the V kappa psi 9B.8 gene segment has two mutations of an insertion and altered RNA splice site. Both genes have the potential to rearrange based on the sequence of their heptamer-nonamer motifs. The identification of pseudogenes raises the question of how many nonfunctional genes are present in the murine germline repertoire.

Amino Acid Sequence↗
PubMed1992

Characterization of the allelic differences in the mouse cardiac alpha-myosin heavy chain coding sequence.

We have obtained and sequenced the coding sequence of the mouse cardiac alpha-myosin heavy chain (Myhc alpha) from the A/J, BALB/cByJ, C57BL/6J, and DBA/2J inbred mouse strains. Overlapping cDNA sequences were obtained using RNA-PCR and anchor-PCR techniques for these studies. In the A/J mouse strain, the full-length message is 5989 bp long and encodes for a protein consisting of 1938 amino acids (Mr 223,689). The protein deduced sequence of the A/J Myhc alpha was compared with corresponding sequences of human and rat Myhc alpha and beta. These results demonstrated that the mouse Myhc alpha is highly conserved and has maintained the alpha-isoform-specific divergent cluster observed in other Myhc alpha proteins. One difference was the loss of a glutamine at residue 1932, which is due to a change in an RNA splicing site sequence. Allelic variability was observed in both nucleotide and amino acid sequences among the four different inbred mouse strains and generally appears to be random in nature. Three of the nucleotide changes resulted in a different amino acid, while the remaining 46 were silent substitutions.

Alleles↗
PubMed2005

Tissue specificity and regulation of the N-terminal diversity of reticulon 3.

Over the last few years, the widely distributed family of reticulons (RTNs) is receiving renewed interest because of the implication of RTN4/Nogo in neurite regeneration. Four genes were identified in mammals and are referred to as RTN1, 2, 3 and the neurite outgrowth inhibitor RTN4/Nogo. In the present paper, we describe the existence of five new isoforms of RTN3 that differ in their N-termini, and analysed their tissue distribution and expression in neurons. We redefined the structure of human and murine rtn3 genes, and identified two supplementary exons that may generate up to seven putative isoforms arising by alternative splicing or differential promoter usage. We confirmed the presence of five of these isoforms at the mRNA and protein levels, and showed their preferential expression in the central nervous system. We analysed rtn3 expression in the cerebellum further, and observed increased levels of several of the RTN3 isoforms during cerebellum development and during in vitro maturation of cerebellar granule cells. This pattern of expression paralleled that shown by RTN4/Nogo isoforms. Specifically, RTN3A1 expression was down-regulated upon cell death of cerebellar granule neurons triggered by potassium deprivation. Altogether, our results demonstrate that the rtn3 gene generates multiple isoforms varying in their N-termini, and that their expression is tightly regulated in neurons. These findings suggest that RTN3 isoforms may contribute, by as yet unknown mechanisms, to neuronal survival and plasticity.

Alternative Splicing↗
PubMed2026

Promoter identity shapes splicing outcomes and fidelity.

Gene expression is a complex process subject to regulation at multiple functionally interconnected levels. One prominent example is the crosstalk between transcription and splicing regulation. Past work has shown that transcription can influence splicing in multiple ways, but a systematic investigation of this complex interplay is lacking. Here we employ massively parallel reporter assays of large combinatorial promoter-splice site libraries to dissect how promoter identity and transcription dynamics affect alternative splicing in human cells. We find that promoter identity, rather than expression level, exerts strong and highly context-specific effects on cassette exon inclusion, exceeding the effect of pharmacological inhibitors of transcription initiation or elongation. Groups of exons display coordinated promoter-dependent splicing behavior, and we identified predictive sequence and structural features underlying this sensitivity. Promoter and gene architecture also shape isoform diversity by modulating cryptic splice site usage. These findings present promoters as central regulators of splicing outcomes and fidelity.

Humans↗
PubMed1992

U1-U2 snRNPs interaction induced by an RNA complementary to the 5' end sequence of U1 snRNA.

Several lines of evidences indicate that U1 and U2 snRNPs become interacting during pre-mRNA splicing. Here we present data showing that an U1-U2 snRNPs interaction can be mediated by an RNA only containing the consensus 5' splice site of all of the sequences characteristic of pre-mRNAs. Using monospecific antibodies (anti-(U1) RNP and anti-(U2) RNP), we have found that a tripartite complex comprising U1 and U2 snRNPs is immunoprecipitated in the presence of a consensus 5' splice site containing RNA, either from a crude extract or from an artificial mixture enriched in U1 and U2 snRNPs. This complex does not appear in the presence of an RNA lacking the sequence complementary to the 5' terminus of U1 snRNA. Moreover, RNAse T1 protection coupled to immunoprecipitation experiments have demonstrated that only the 5' end sequence of U1 snRNA contacts the consensus 5' splice site containing RNA, arguing that U2 snRNP binding in the tripartite complex is mediated by U1 snRNP.

Base Sequence↗
PubMed2025

Utility of genome sequencing and group-enrichment to support splice variant interpretation in Marfan syndrome.

PURPOSE: To quantify the impact of noncanonical FBN1 splice site variants in undiagnosed Marfan syndrome (MFS), a connective tissue disorder associated with skeletal abnormalities and familial thoracic aortic aneurysm disease (FTAAD). METHODS: A systematic analysis of ultrarare FBN1 variants was performed using genome sequencing data from the 100,000 Genomes Project. Variants were annotated with SpliceAI and the significance of enrichment among individuals with FTAAD was assessed using Fisher's exact test. Experimental validation used RNA sequencing, reverse transcriptase polymerase chain reaction, minigene constructs, and replication analysis was with data from UK Biobank. RESULTS: Using aggregate data for 78,195 individuals, we identified 13,864 singleton single-nucleotide variants in FBN1 of which 21 were predicted to affect splicing (SpliceAI > 0.5). Incidence of candidate splice variants in individuals recruited with FTAAD (9/703) was significantly elevated compared with that seen in non-FTAAD participants (12/77,492; odds ratio = 84, P = 9.7 × 10-14). Additional analysis uncovered a further 14 families harboring 11 different FBN1 splice variants. A total of 20 candidate splice variants in 23 families were identified, of which 70% lay beyond the ±8 splice regions. RNA testing confirmed the predicted splice aberration in 16 of 20 and for 9 of 20, pseudoexonization was the likely splicing anomaly. CONCLUSION: Our findings indicate that noncanonical splice variants may account for approximately 3% of families with undiagnosed FTAAD, highlighting the importance of incorporating analysis of introns and confirmatory RNA testing into genetic testing for Marfan syndrome.

Humans↗