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Results for “RNA Splicing”

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At least 145 records · Page 8Linked to original sources

PubMed1993

Tissue-specific RNA splicing generates an ankyrin-like domain that affects the dimerization and DNA-binding properties of a bHLH protein.

mRNAs encoding two rat bHLH proteins, referred to as REB alpha and REB beta, have been identified as alternatively spliced transcripts derived from a single genomic locus. Alternative RNA processing events results in tissue-specific differences in the ratios of these two mRNAs. Although it exhibits a highly enriched level of expression in the developing neural tube, the REB gene is expressed at variable levels in many organs of the mature animal. The REB alpha sequence contains a region characterized by a leucine heptad repeat that is situated amino-terminal of the carboxy-terminally located bHLH domain. REB beta is identical to REB alpha except for a 24-amino-acid insertion in the leucine heptad repeat that results from the inclusion of an additional 72-bp exon in the REB beta transcript. As a consequence of this insertion, REB beta exhibits a markedly diminished capacity to bind to cognate E-box-binding sites and to form homodimers and heterodimers with other members of the bHLH gene family. Analysis of the 24-amino-acid REB beta-specific insert revealed that it mediates an inhibitory function and exhibits a significant degree of sequence similarity to ankyrin-like repeats. It is proposed that this tissue-specific pattern of REB RNA splicing is involved in the determination of corresponding tissue-specific combinations of heterodimeric complexes of ubiquitous and tissue-restricted bHLH proteins. Thus, REB alpha and REB beta represent a novel example of a regulated formation of an ankyrin-like domain within a bHLH protein, thereby mediating control of protein-protein interactions.

Amino Acid Sequence↗

PubMed2005

Role of CDK/cyclin complexes in transcription and RNA splicing.

The production of mRNAs in all living organisms is an extremely complex process that includes multiple catalytic activities such as transcription, capping, splicing, polyadenylation, cleavage and export. All of these processes are controlled by a large group of proteins which form very dynamic complexes interacting with DNA and pre-mRNAs to coordinate these activities. Phosphorylations play a central role in regulating formation, activation and inactivation of these complexes. A growing number of protein kinases have been identified that are capable of phosphorylating proteins involved in mRNA production. Among them, Cyclin-dependent Kinases (CDKs) represent a family of serine/threonine protein kinases that become active upon binding to a cyclin regulatory partner. CDK/cyclin complexes were first identified as crucial regulators of cell cycle progression. More recently, CDK/cyclin complexes have also been implicated in transcription and mRNA processing leading to the concept of an intricate network of CDK/cyclin complexes regulating cell cycle, transcription and mRNA processing via cross-talk between multiple CDKs. In this review, we discuss the role of CDK/cyclin-dependent phosphorylation in the regulation of transcription and RNA splicing and highlight recent findings that indicate the involvement of CDK/cyclin complexes in connecting transcription and RNA splicing.

Amino Acid Sequence↗

PubMed2026

RNA splicing and cardiovascular disease: a guide for cardiologists.

Alternative splicing (AS) is a fundamental RNA processing mechanism, which generates different RNA transcripts and consequently different protein isoforms from a single gene. This increases the diversity of proteins within an organism and can fine-tune biological processes. This review examines how cardiac-enriched RNA-binding proteins establish heart-specific splicing programs governing aspects of cardiac development, function, and disease. Developmentally, coordinated sarcomeric isoform switches underpin the foetal-to-adult transition and further isoform rewiring in ion channel and kinase genes determine electrophysiology and excitation-contraction coupling. AS contributes to the pathogenesis of several cardiomyopathies and emerging datasets suggest that pathological hypertrophy engages distinct splicing signatures compared with physiological hypertrophy. This review summarizes diagnostic and prognostic opportunities arising from bulk, long-read, and single-cell/nucleus transcriptomics, which resolve cell type-specific isoforms and disease-associated switches. Circulating RNA biomarkers (including splice ratios and circularRNAs) may signify myocardial remodelling and arrhythmic risk. Integrative approaches that link AS with proteomics and genomics improve variant interpretation, reveal previously unannotated protein isoforms, and enable tracking of disease progression and therapy response. Finally, an outline of therapeutic strategies to modulate AS in cardiovascular disease (CVD), including antisense oligonucleotides, small molecules, and genome-editing modalities (CRISPR, base, and prime editing), is provided. The major challenges that remain before splice-targeting therapeutics can be targeted to treat cardiovascular disease are highlighted. Lessons from neuromuscular indications establish clinical feasibility of splicing correction and motivate translation to cardiology. Together, mechanistic insight, biomarker development, and therapeutic innovation position RNA splicing as a tractable axis for precision cardiovascular medicine.

Humans↗

PubMed1994

RNA splicing regulates the activity of a SH2 domain-containing protein tyrosine phosphatase.

A cDNA which encodes a protein tyrosine phosphatase with two src homology 2 (SH2) domains was isolated from a rat brain cDNA library. This phosphatase appears to be a rat homologue of PTP1D based on its amino acid sequence. The gene is expressed in a variety of tissues, and its mRNA is enriched in the brain, skeletal muscle, and lung. An RNA splice variant (PTP1Di) was also isolated which has four additional amino acid residues (Ala-Leu-Leu-Gln) in the catalytic domain. The catalytic domains of PTP1D and PTP1Di were expressed in Escherichia coli as glutathione S-transferase fusion proteins and purified to near homogeneity. Whereas both PTP1D and PTP1Di had catalytic activity, the Vmax of PTP1Di relative to that of PTP1D was 8-fold lower for para-nitrophenylphosphate, 20-fold lower for nicotinic acetylcholine receptor, and 14-fold lower for myelin basic protein. The Km values of PTP1Di were lower than those of PTP1D for both nicotinic acetylcholine receptor and myelin basic protein, suggesting a higher affinity of PTP1Di for a protein substrate. These two forms also differed in optimum pH for para-nitrophenylphosphate and sensitivity to the inhibitory effects of vanadate, molybdate, and spermidine. In order to see if this insert would affect the catalytic activity of other related phosphatases, the 4-amino acids were inserted in the corresponding region of the catalytic domain of PTP1C. Whereas both the wild type and PTP1Ci which contained the 4-amino acid insert dephosphorylated para-nitrophenylphosphate, nicotinic receptor, and myelin basic protein, the enzyme activity of PTP1Ci was only 11-24% of that of PTP1C wild type. These results demonstrate that the 4-amino acid insert in the catalytic domains of PTP1D down-regulates its phosphatase activity and suggests that RNA splicing may serve as a regulatory mechanism of protein tyrosine phosphatase activity.

Amino Acid Sequence↗

PubMed1992

Connections between RNA splicing and DNA intron mobility in yeast mitochondria: RNA maturase and DNA endonuclease switching experiments.

The intron-encoded proteins bI4 RNA maturase and aI4 DNA endonuclease can be faithfully expressed in yeast cytoplasm from engineered forms of their mitochondrial coding sequences. In this work we studied the relationships between these two activities associated with two homologous intron-encoded proteins: the bI4 RNA maturase encoded in the fourth intron of the cytochrome b gene and the aI4 DNA endonuclease (I-SceII) encoded in the fourth intron of the gene coding for the subunit I of cytochrome oxidase. Taking advantage of both the high recombinogenic properties of yeast and the similarities between the two genes, we constructed in vivo a family of hybrid genes carrying parts of both RNA maturase and DNA endonuclease coding sequences. The presence of a sequence coding for a mitochondrial targeting peptide upstream from these hybrid genes allowed us to study the properties of their translation products within the mitochondria in vivo. We thus could analyze the ability of the recombinant proteins to complement RNA maturase deficiencies in different strains. Many combinations of the two parental intronic sequences were found in the recombinants. Their structural and functional analysis revealed the following features. (i) The N-terminal half of the bI4 RNA maturase could be replaced in total by its equivalent from the aI4 DNA endonuclease without affecting the RNA maturase activity. In contrast, replacing the C-terminal half of the bI4 RNA maturase with its equivalent from the aI4 DNA endonuclease led to a very weak RNA maturase activity, indicating that this region is more differentiated and linked to the maturase activity. (ii) None of the hybrid proteins carrying an RNA maturase activity kept the DNA endonuclease activity, suggesting that the latter requires the integrity of the aI4 protein. These observations are interesting because the aI4 DNA endonuclease is known to promote the propagation, at the DNA level, of the aI4 intron, whereas the bI4 RNA maturase, which is required for the splicing of its coding intron, also controls the splicing process of the aI4 intron. We propose a scenario for the evolution of these intronic proteins that relies on a switch from DNA endonuclease to RNA maturase activity.

Amino Acid Sequence↗

PubMed1994

RNA splicing in Borna disease virus, a nonsegmented, negative-strand RNA virus.

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus related to rhabdoviruses and paramyxoviruses. Unlike animal viruses of these two families, BDV transcribes RNAs in the nuclei of infected cells and produces high levels of transcripts containing multiple open reading frames. Previous Northern blot analysis of RNA from BDV-infected rat brain tissue has shown that two viral transcripts, a 6.1-kb RNA and a 1.5-kb RNA, lack regions that are internal to two otherwise identical transcripts, the 7.1-kb RNA and the 2.8-kb RNA, respectively (T. Briese, A. Schneemann, A. Lewis, Y. Park, S. Kim, H. Ludwig, and W. I. Lipkin, Proc. Natl. Acad. Sci. USA 91:4362-4366, 1994). To determine the precise location of this deletion, we performed reverse transcription PCR analysis using total RNA from BDV-infected rat brain tissue. This investigation resulted in the identification of two introns in the 7.1- and 2.8-kb RNAs, which can be alternatively spliced to yield additional RNA species, including the 6.1- and 1.5-kb RNAs. Transient transfection of COS-7 cells with a cDNA clone of the 2.8-kb RNA resulted in the production of both the 2.8-kb RNA and the 1.5-kb RNA, confirming the theory that the 2.8-kb RNA is a sufficient substrate for splicing in mammalian cells. Splicing has not previously been observed in nonsegmented, negative-strand RNA viruses and presumably serves as a mechanism by which expression of BDV proteins is regulated in infected cells.

Animals↗

PubMed1999

Colocalization of intranuclear lamin foci with RNA splicing factors.

The lamins form a fibrous network underlying the inner nuclear membrane termed the nuclear lamina. In order to gain insights into the role of lamins in nuclear organization, we have characterized a monoclonal antibody (LA-2H10) raised against recombinant rat lamin A that labels nuclei in a speckled pattern in all cells of unsynchronized populations of HeLa and rat F-111 fibroblast cells, unlike the typical nuclear periphery staining by another monoclonal antibody to lamin A, LA-2B3. In immunolocalization studies the lamin A speckles or foci were found to colocalize with the RNA splicing factors SC-35 and U5-116 kD, but not with p80 coilin found in coiled bodies. Lamin B1 was also associated with these foci. These foci dispersed when cells entered mitosis and reformed during anaphase. The differential reactivity of LA-2H10 and LA-2B3 was retained after nuclei were extracted with detergents, nucleases and salt to disrupt interactions of lamins with chromatin and other nuclear proteins. Using deletion fragments of recombinant lamin A, the epitope recognized by LA-2H10 was located between amino acids 171 and 246. Our findings are consistent with a structural role for lamins in supporting nuclear compartments containing proteins involved in RNA splicing.

Animals↗

PubMed1982

RNA splicing in Neurospora mitochondria. Characterization of new nuclear mutants with defects in splicing the mitochondrial large rRNA.

In Neurospora, the gene encoding the mitochondrial large (25S) ribosomal RNA contains an intervening sequence of 2.3 kb. We have identified eight nuclear mutants that are defective in splicing the mitochondrial large ribosomal RNA and that accumulate unspliced precursor RNA. These mutants identify three different nuclear genes required for the same mitochondrial RNA splicing reaction. Some of the mutants have unique phenotypic characteristics (for example, accumulation of an unusual intron RNA) that may provide insight into specific aspects of mitochondrial RNA splicing. Mutations at one locus, cyt4, are subject to partial phenotypic suppression by the electron-transport inhibitor antimycin. This phenomenon suggests that at least one component required for mitochondrial RNA splicing is regulated such that its synthesis or activity is increased in response to impairment of electron transport.

Antimycin A↗

PubMed2001

Multiple forms of mouse vascular endothelial growth factor-D are generated by RNA splicing and proteolysis.

The secreted glycoprotein vascular endothelial growth factor-D (VEGF-D) is angiogenic, lymphangiogenic, and promotes metastatic spread of tumor cells via lymphatic vessels. VEGF-D consists of a receptor-binding domain (VEGF homology domain) and N- and C-terminal propeptides. Proteolytic processing produces numerous forms of human VEGF-D, including fully processed derivatives (containing only the VEGF homology domain), partially processed, and unprocessed derivatives. Proteolysis is essential to generate human VEGF-D that binds the angiogenic receptor VEGF receptor-2 (VEGFR-2) and the lymphangiogenic receptor VEGFR-3 with high affinity. Here, we report that alternative use of an RNA splice donor site in exon 6 of the mouse VEGF-D gene produces two different protein isoforms, VEGF-D(358) and VEGF-D(326), with distinct C termini. The two isoforms were both expressed in all adult mouse tissues and embryonic stages of development analyzed. Both isoforms are proteolytically processed in a similar fashion to human VEGF-D to generate a range of secreted derivatives and bind and cross-link VEGFR-3 with similar potency. The isoforms are differently glycosylated when expressed in vitro. This study demonstrates that RNA splicing, protein glycosylation, and proteolysis are mechanisms for generating structural diversity of mouse VEGF-D.

Amino Acid Sequence↗

PubMed1994

RNA splicing contributes to the generation of mature mRNAs of Borna disease virus, a non-segmented negative strand RNA virus.

We recently demonstrated that Borna disease virus (BDV) has a negative non-segmented single stranded (NNS) RNA genome, whose organization is similar to that of other members of the Mononegavirales order. However, we have also documented that in contrast to the rest of the NNS-RNA animal viruses, BDV replication and transcription occur in the nucleus of infected cells. Here, we provide evidence that BDV uses the host nuclear splicing machinery to generate some of the viral mRNAs, representing the first documentation of RNA splicing in NNS-RNA animal viruses. Possible implications of RNA splicing for the regulation of BDV gene expression are discussed.

Base Sequence↗

PubMed1982

Making ends meet: a model for RNA splicing in fungal mitochondria.

On the basis of available nucleotide sequence and genetic data; we present a model for RNA splicing in fungal mitochondria. Seven intron RNAs of two fungal species can form identical secondary structures, involving four conserved sequences, which bring the ends of each intron together and allow an internal guide RNA sequence to pair with exon bases adjacent to the splice junctions. The splicing sites are thus aligned precisely within a conserved structure, which we suggest could present specific recognition signals to the proteins that catalyse the splicing reaction.

Aspergillus nidulans↗

PubMed2003

Unusual misregulation of RNA splicing caused by insertion of a transposable element into the T (Brachyury) locus.

BACKGROUND: The TWis mutant allele of the Brachyury, or T, gene was created by insertion of an endogenous retrovirus-like early transposon (ETn) element into the exon 7 splice donor consensus sequence of the 8 exon T locus. While the developmental consequences of this disruption have been well characterized, the molecular consequences have not been previously investigated, and it has been assumed that the insertion results in a truncated protein. This study sought to further characterize the mutant TWis allele by investigating the nature of the transcripts produced by insertion of this transposable element. RESULTS: Using an RT-PCR based approach, we have shown that at least 8 different mutant transcripts are produced from the TWis allele. All TWis transcripts bypass the mutated exon 7 splice donor site, such that wild type T transcripts are not produced from the TWis allele. CONCLUSIONS: This result shows an unsuspected misregulation of RNA splicing caused by insertion of a transposable element, that could have more widespread consequences in the genome.

Alleles↗

PubMed1990

Simian immunodeficiency virus displays complex patterns of RNA splicing.

The human and simian immunodeficiency viruses encode at least six gene products that apparently serve regulatory functions. To evaluate the regulation of simian immunodeficiency virus gene expression at the level of RNA splicing, we used the polymerase chain reaction to amplify and clone cDNAs corresponding to a large array of mRNAs from infected cells. We identified mRNAs that used splice acceptor sites upstream of the initiator codons for tat, rev, vpr, nef, vif, and vpx, suggesting that these proteins may be expressed from different mRNAs. We also provide hybridization data suggesting that the same splice acceptor site may be used for both rev and env mRNAs. Furthermore, we isolated both tat and rev cDNAs that utilized three alternative splice acceptor sites at the start of coding exon 2, indicating that different versions of these proteins may be encoded. Finally, approximately 10 to 20% of simian immunodeficiency virus mRNAs spliced an intron from their untranslated 5' ends, and sequences contained within this intron constituted a portion of the tat-responsive TAR element. Thus, alternative pre-mRNA splicing adds a level of complexity to simian immunodeficiency virus expression, which may affect several levels of gene regulation.

Animals↗

PubMed1985

Site-specific deletion in cauliflower mosaic virus DNA: possible involvement of RNA splicing and reverse transcription.

A frequent site-specific deletion was observed in the life cycle of cauliflower mosaic virus (S strain). Analysis of the sequence around the deletion site and the parental sequence implied that the deletion was promoted at sequences similar to the donor and acceptor consensus sequences of RNA splicing, designated as the deletion donor and acceptor sequences, respectively. To elucidate the mechanism of this site-specific deletion, point mutations were introduced into the deletion donor sequence (GT to GG or GA transversion). Deletion at the original deletion donor site did not occur in these mutants, instead, new (cryptic) donor sites were activated. All of these activated cryptic sites had sequences similar to the splicing consensus sequence. In all cases except one, the original deletion acceptor site was used. These results can be most readily explained by postulating that the site-specific deletion occurs by reverse transcription of spliced viral RNA. This frequent site-specific deletion was not observed in other strains. For a virus which replicates by reverse transcription, a mechanism to regulate the rate of splicing is required to ensure the intactness of the viral genome. We discuss the possibility that the S strain has a mutation in this regulatory mechanism.

Journal Article↗

PubMed1987

The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability.

Polyoma virus late RNA processing provides a convenient model system in which to study the mechanics of splicing in vivo. In order to understand further the role of the untranslated "late leader" unit in late RNA processing we have constructed a group of polyoma viruses with deletions and substitutions in the leader exon. This has allowed us to determine that there is a minimum exon size required for both pre-mRNA splicing and stability in this system. We show here that the non-viability of a mutant (ALM) with a 9 base late leader unit is due to a general defect in late RNA splicing. In addition, ALM-infected cells show at least 40-fold depression in the accumulation of late nuclear RNA (spliced or unspliced). The ALM late promoter, however, functions nearly normally. Substituted leader variants with 51- to 96-base long exons of unrelated sequence are viable (G. Adami and G. Carmichael, J. Virol. 58, 417-425, 1986). We show here that late RNA from one of these substituted leader mutants (containing a 51-base leader exon) is spliced at wild type levels, with virtually no defect in accumulation. Thus, in the polyoma system, splice sites separated by only 9 bases can inhibit each others usage, presumably by steric interference. We suggest that this type of inhibition leads to extreme RNA instability.

Animals↗

PubMed1997

HIV Rev-dependent binding of SF2/ASF to the Rev response element: possible role in Rev-mediated inhibition of HIV RNA splicing.

Production of the structural and enzymatic proteins of type 1 human immunodeficiency virus (HIV-1) is controlled by the rev regulatory gene product. The 116-amino acid Rev protein acts by binding to the Rev response element (RRE), a complex RNA stem-loop structure located within the env gene of HIV. Rev exerts a series of posttranscriptional effects, including the inhibition of viral RNA splicing, the activation of nuclear export of incompletely spliced viral RNAs, and the enhancement of translation of RRE-containing RNAs. Our studies now demonstrate that at least one member of the SR family of splicing factors, SF2/ASF, specifically binds to a subregion of the RRE in vitro in a Rev-dependent manner. Furthermore, expression of high levels of SF2/ASF inhibits Rev function and impairs HIV replication in vivo. Both the in vitro binding of SF2/ASF to the Rev/RRE complex and the in vivo inhibition of Rev action by SF2/ASF are abrogated by mutation of the N-terminal RNA recognition motif but are not affected by mutation of the C-terminal arginine-serine-rich domain. These findings suggest that Rev inhibition of HIV splicing likely involves recruitment of the essential splicing factor SF2/ASF to the Rev/RRE complex. However, these inhibitory effects of Rev on viral RNA splicing are apparently overcome by augmenting the intracellular levels of SF2/ASF expression.

Animals↗

PubMed1997

Naegleria nucleolar introns contain two group I ribozymes with different functions in RNA splicing and processing.

We have characterized the structural organization and catalytic properties of the large nucleolar group I introns (NaSSU1) of the different Naegleria species N. jamiesoni, N. andersoni, N. italica, and N. gruberi. NaSSU1 consists of three distinct RNA domains: an open reading frame encoding a homing-type endonuclease, and a small group I ribozyme (NaGIR1) inserted into the P6 loop of a second group I ribozyme (NaGIR2). The two ribozymes have different functions in RNA splicing and processing. NaGIR1 is an unusual self-cleaving group I ribozyme responsible for intron processing at two internal sites (IPS1 and IPS2), both close to the 5' end of the open reading frame. This processing is hypothesized to lead to formation of a messenger RNA for the endonuclease. Structurally, NaGIR2 is a typical group IC1 ribozyme, catalyzing intron excision and exon ligation reactions. NaGIR2 is responsible for circularization of the excised intron, a reaction that generates full-length RNA circles of wild-type intron. Although it is only distantly related in primary sequence, NaSSU1 RNA has a predicted organization and function very similar to that of the mobile group I intron DiSSU1 of Didymium, the only other group I intron known to encode two ribozymes. We propose that these twin-ribozyme introns define a distinct category of group I introns with a conserved structural organization and function.

Animals↗

PubMed2006

L4-33K, an adenovirus-encoded alternative RNA splicing factor.

Splicing of the adenovirus IIIa mRNA is subjected to a strict temporal regulation during virus infection such that efficient IIIa 3' splice site usage is confined to the late phase of the infectious cycle. Here we show that the adenovirus L4-33K protein functions as a virus-encoded RNA splicing factor that preferentially activates splicing of transcripts with a weak 3' splice site sequence context, a sequence configuration that is shared by many of the late adenovirus 3' splice sites. Furthermore, we show that L4-33K activates IIIa splicing through the IIIa virus infection-dependent splicing enhancer element (3VDE). This element was previously shown to be the minimal element, both necessary and sufficient, for activation of IIIa splicing in the context of an adenovirus-infected cell. L4-33K stimulates an early step in spliceosome assembly and appears to be the only viral protein necessary to convert a nuclear extract prepared from uninfected HeLa cells to an extract with splicing properties very similar to a nuclear extract prepared from adenovirus late-infected cells. Collectively, our results suggest that L4-33K is the key viral protein required to activate the early to late switch in adenovirus major late L1 alternative splicing.

Adenoviridae↗