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At least 253 records · Page 14Linked to original sources
PubMed2004

Spliced-leader RNA trans splicing in a chordate, Oikopleura dioica, with a compact genome.

trans splicing of a spliced-leader RNA (SL RNA) to the 5' ends of mRNAs has been shown to have a limited and sporadic distribution among eukaryotes. Within metazoans, only nematodes are known to process polycistronic pre-mRNAs, produced from operon units of transcription, into mature monocistronic mRNAs via an SL RNA trans-splicing mechanism. Here we demonstrate that a chordate with a highly compact genome, Oikopleura dioica, now joins Caenorhabditis elegans in coupling trans splicing with processing of polycistronic transcipts. We identified a single SL RNA which associates with Sm proteins and has a trimethyl guanosine cap structure reminiscent of spliceosomal snRNPs. The same SL RNA, estimated to be trans-spliced to at least 25% of O. dioica mRNAs, is used for the processing of both isolated or first cistrons and downstream cistrons in a polycistronic precursor. Remarkably, intercistronic regions in O. dioica are far more reduced than those in either nematodes or kinetoplastids, implying minimal cis-regulatory elements for coupling of 3'-end formation and trans splicing.

Animals↗
PubMed1991

Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein.

The removal of introns from eukaryotic messenger RNA precursors shares mechanistic characteristics with the self-splicing of certain introns, prompting speculation that the catalytic reactions of nuclear pre-messenger RNA splicing are fundamentally RNA-based. The participation of five small nuclear RNAs (snRNAs) in splicing is now well documented. Genetic analysis in yeast has revealed the requirement, in addition, for several dozen proteins. Some of these are tightly bound to snRNAs to form small nuclear ribonucleoproteins (snRNPs); such proteins may promote interactions between snRNAs or between an snRNA and the intron. Other, non-snRNP proteins appear to associate transiently with the spliceosome. Some of these factors, which include RNA-dependent adenosine triphosphatases, may promote the accurate recognition of introns.

Adenosine Triphosphate↗
PubMed1983

Specific transcription and RNA splicing defects in five cloned beta-thalassaemia genes.

Transcriptional analysis of five different cloned beta-thalassaemia genes introduced into cultured mammalian cells revealed specific defects in transcription and RNA splicing. A single base change 87 base pairs to the 5' side of the mRNA cap site significantly lowers the level of transcription and therefore appears to represent a promoter mutation. Three genes contain different single base changes in the first intervening sequence (IVS) 5' splice site. One mutation, at IVS1 position 1, inactivates the splice site completely; the other two, at IVS1 positions 5 and 6, reduce its activity. Each mutation activates the same three cryptic splice sites. The fifth gene contains a single base change within IVS2 at position 745, which results in the formation of abnormal beta-globin RNA that contains an extra exon.

Alleles↗
PubMed1994

Presence of negative and positive cis-acting RNA splicing elements within and flanking the first tat coding exon of human immunodeficiency virus type 1.

The human immunodeficiency virus type 1 (HIV-1) RNA follows a complex splicing pathway in which a single primary transcript either remains unspliced or is alternatively spliced to more than 30 different singly and multiply spliced mRNAs. We have used an in vitro splicing assay to identify cis elements within the viral genome that regulate HIV-1 RNA splicing. A novel splicing regulatory element (SRE) within the first tat coding exon has been detected. This element specifically inhibits splicing at the upstream 3' splice site flanking this tat exon. The element only functions when in the sense orientation and is position dependent when inserted downstream of a heterologous 3' splice site. In vivo, an HIV-1 SRE mutant demonstrated a decrease in unspliced viral RNA, increased levels of single- and double-spliced tat mRNA, and reduced levels of env and rev mRNAs. In addition to the negative cis-acting SRE, the flanking 5' splice site downstream of the first tat coding exon acts positively to increase splicing at the upstream 3' splice sites. These results are consistent with hypotheses of bridging interactions between cellular factors that bind to the 5' splice site and those that bind at the upstream 3' splice site.

Alternative Splicing↗
PubMed1981

RNA splicing mutation in an aberrantly rearranged immunoglobulin lambda I gene.

The mouse cell line MOPC 315 is an IgA (lambda II)-producing myeloma. We have studied a derivative of MOPC 315 that secretes normal lambda II chains but no heavy chain. This derivative, MOPC 315-26, was found to contain a rearranged lambda I gene in addition to a rearranged lambda II gene. The rearranged lambda I gene was cloned into bacteriophage lambda DNA and its structure was studied. The lambda I gene was found to have arisen by an aberrant recombination event that resulted in a single base insertion at the site of V-J region joining. In addition, the gene contained numerous point mutations in the vicinity of the junction of the V and J regions. Two point mutations occurred in the donor splice sequence normally used for the removal of the intron between the J and C regions, suggesting that the RNA synthesized from the aberrantly rearranged lambda I gene would be unable to undergo proper RNA splicing.

Amino Acid Sequence↗
PubMed1990

Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing.

U6 is one of the five small nuclear RNA's (snRNA's) that are required for splicing of nuclear precursor messenger RNA (pre-mRNA). The size and sequence of U6 RNA are conserved among organisms as diverse as yeast and man, and so it has been proposed that U6 RNA functions as a catalytic element in splicing. A procedure for in vitro reconstitution of functional yeast U6 small nuclear ribonucleoproteins (snRNP's) with synthetic U6 RNA was applied in an attempt to elucidate the function of yeast U6 RNA. Two domains in U6 RNA were identified, each of which is required for in vitro splicing. Single nucleotide substitutions in these two domains block splicing either at the first or the second step. Invariably, U6 RNA mutants that block the first step of splicing do not enter the spliceosome. On the other hand, those that block the second step of splicing form a spliceosome but block cleavage at the 3' splice site of the intron. In both domains, the positions of base changes that block the second step of splicing correspond exactly to the site of insertion of pre-mRNA-type introns into the U6 gene of two yeast species, providing a possible explanation for the mechanism of how these introns originated and adding further evidence for the proposed catalytic role of U6 RNA.

Base Sequence↗
PubMed1984

Mapping of Early and Late Transcripts Encoded by the Autographa californica Nuclear Polyhedrosis Virus Genome: Is Viral RNA Spliced?

Early and late transcripts were mapped on the Autographa californica nuclear polyhedrosis virus genome by Northern blotting and hybridization with the cloned viral EcoRI fragments. At least 11 early and about 90 late RNAs were compared with over 32 polypeptides synthesized by in vitro translation of hybrid-selected RNA. The latter method, of course, had its limitations also and did not guarantee that all viral RNAs could be detected in this way. A comparison of cytoplasmic and total cellular RNAs showed no clear-cut differences in their size distributions. We found that there were more RNA classes than corresponding proteins encoded by them and mapped by in vitro translation. By using the Berk-Sharp method and analyses of DNA-RNA hybrids by one-dimensional or two-dimensional neutral and alkaline gel electrophoreses, we were unable to adduce evidence for RNA splicing in this viral system. Minor splices, particularly at sites close to the termini of RNA molecules, could not be excluded.

Journal Article↗
PubMed1988

cis-acting intron mutations that affect the efficiency of avian retroviral RNA splicing: implication for mechanisms of control.

The full-length retroviral RNA transcript serves as (i) mRNA for the gag and pol gene products, (ii) genomic RNA that is assembled into progeny virions, and (iii) a pre-mRNA for spliced subgenomic mRNAs. Therefore, a balance of spliced and unspliced RNA is required to generate the appropriate levels of protein and RNA products for virion production. We have introduced an insertion mutation near the avian sarcoma virus env splice acceptor site that results in a significant increase in splicing to form functional env mRNA. The mutant virus is replication defective, but phenotypic revertant viruses that have acquired second-site mutations near the splice acceptor site can be isolated readily. Detailed analysis of one of these viruses revealed that a single nucleotide change at -20 from the splice acceptor site, within the original mutagenic insert, was sufficient to restore viral growth and significantly decrease splicing efficiency compared with the original mutant and wild-type viruses. Thus, minor sequence alterations near the env splice acceptor site can produce major changes in the balance of spliced and unspliced RNAs. Our results suggest a mechanism of control in which splicing is modulated by cis-acting sequences at the env splice acceptor site. Furthermore, this retroviral system provides a powerful genetic method for selection and analysis of mutations that affect splicing control.

Alpharetrovirus↗
PubMed2001

A recurrent RNA-splicing mutation in the SEDL gene causes X-linked spondyloepiphyseal dysplasia tarda.

Spondyloepiphyseal dysplasia tarda (SEDL) is a genetically heterogeneous disorder characterized by mild-to-moderate short stature and early-onset osteoarthritis. Both autosomal and X-linked forms have been described. Elsewhere, we have reported the identification of the gene for the X-linked recessive form, which maps to Xp22.2. We now report characterization of an exon-skipping mutation (IVS3+5G-->A at the intron 3 splice-donor site) in two unrelated families with SEDL. Using reverse transcriptase (RT)-PCR, we demonstrated that the mutation resulted in elimination of the first 31 codons of the open reading frame. The mutation was not detected in 120 control X chromosomes. Articular cartilage from an adult who had SEDL and carried this mutation contained chondrocytes with abundant Golgi complexes and dilated rough endoplasmic reticulum (ER). RT-PCR experiments using mouse/human cell hybrids revealed that the SEDL gene escapes X inactivation. Homologues of the SEDL gene include a transcribed retropseudogene on chromosome 19, as well as expressed genes in mouse, rat, Drosophila melanogaster Caenorhabditis elegans, and Saccharomyces cerevisiae. The latter homologue, p20, has a putative role in vesicular transport from ER to Golgi complex. These data suggest that SEDL mutations may perturb an intracellular pathway that is important for cartilage homeostasis.

Adult↗
PubMed1994

Scallop striated and smooth muscle myosin heavy-chain isoforms are produced by alternative RNA splicing from a single gene.

We report here that the catch and striated adductor muscle myosin heavy-chain (MHC) isoforms of scallop (Argopecten irradians, previously Aequipecten irradians) are generated by alternative RNA splicing from a single gene. Scallop catch muscle cDNA and genomic DNA were amplified by PCR using primers based on the previously sequenced scallop striated muscle MHC cDNA. Mapping of the exon/intron borders and sequencing of a full-length catch muscle MHC in overlapping fragments revealed that the 24-kb gene encodes the MHC polypeptide in 27 exons and that four sets of tandem exon pairs are alternatively spliced into a striated and a catch MHC isoform. An additional alternative exon was identified in catch cDNA and is apparently spliced into a minor MHC isoform. The striated muscle-specific isoform is not expressed in other tissues, whereas the catch-type isoforms were also detected in various smooth muscles, but not in the striated one. Of the alternative exons, exons 5 and 6 encode part of the ATP-binding region and the 25-kDa/50-kDa proteolytic junction; exon 13 encodes part of one of the actin-binding regions and extends to the active site; exon 20 encodes the middle of the rod hinge region; exon 26 in the striated-specific sequence starts with the stop codon, whereas the catch-specific exon codes for an additional 10 residues. Differences between the alternative exons presumably determine the lower ATPase activity of smooth muscle myosin, contribute to the different structure of the striated and smooth muscle thick filaments, and may also be important for the molecular mechanism of the catch phenomenon.

Alternative Splicing↗
PubMed2005

Steroid hormone receptor coactivation and alternative RNA splicing by U2AF65-related proteins CAPERalpha and CAPERbeta.

Increasing evidence indicates that transcription and pre-mRNA processing are functionally coupled to modulate gene expression. Here, we report that two members of the U2AF65 family of proteins, hCC1.3, which we call CAPERalpha, and a related protein, CAPERbeta, regulate both steroid hormone receptor-mediated transcription and alternative splicing. The CAPER proteins coactivate the progesterone receptor in luciferase transcription reporter assays and alter alternative splicing of a calcitonin/calcitonin gene-related peptide minigene in a hormone-dependent manner. The importance of CAPER coactivators in the regulation of alternative RNA splicing of an endogenous cellular gene (VEGF) was substantiated by siRNA knockdown of CAPERalpha. Mutational analysis of CAPERbeta indicates that the transcriptional and splicing functions are located in distinct and separable domains of the protein. These results indicate that steroid hormone receptor-regulated transcription and pre-mRNA splicing can be directly linked through dual function coactivator molecules such as CAPERalpha and CAPERbeta.

Alternative Splicing↗
PubMed1985

Transfer RNA splicing in Saccharomyces cerevisiae. Secondary and tertiary structures of the substrates.

Secondary and tertiary structures of four yeast tRNA precursors that contain introns have been elucidated using limited digestion with a variety of single-strand- and double-strand-specific nucleases. The pre-tRNAs, representing the variety of intron sizes and potential structures, were: pre-tRNALeuCAA, pre-tRNALeuUAG, pre-tRNAIleUAU, and pre-tRNAPro-4UGG. Conventional tRNA cloverleaf structure is maintained in these precursors except that the anticodon loop is interrupted by the intron. The intron contains a sequence which is complementary to a portion of the anticodon loop and allows the formation of a double helix often extending the anticodon stem. The 5' and 3' splicing cleavage sites are located at either end of this helix and are single-stranded. The intron is the most sensitive region to nuclease cleavage, suggesting that it is on the surface of the molecule and available for interaction with the splicing endonuclease. Absence of Mg2+ or spermidine renders the dihydrouridine and T psi C loops of these precursors highly sensitive to nuclease digestion. These ionic effects mimic those observed for tRNAPhe and suggest that the tRNA portion of these precursors has native tRNA structure. We propose consensus secondary and tertiary structures which may be of significance to eventual understanding of the mechanism of yeast tRNA splicing.

Macromolecular Substances↗
PubMed1990

Function of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing requires an idiosyncratic domain not found in other synthetases.

Neurospora mitochondrial tyrosyl-tRNA synthetase (mt TyrRS), which is encoded by nuclear gene cyt-18, functions in splicing group I introns. Analysis of intragenic partial revertants of the cyt-18-2 mutant and in vitro mutants of the cyt-18 protein expressed in E. coli showed that splicing activity of the cyt-18 protein is dependent on a small N-terminal domain that has no homolog in bacterial or yeast mt TyrRSs. This N-terminal splicing domain apparently acts together with other regions of the protein to promote splicing. Our findings support the hypothesis that idiosyncratic sequences in aminoacyl-tRNA synthetase may function in processes other than aminoacylation. Furthermore, they suggest that splicing activity of the Neurospora mt TyrRs was acquired after the divergence of Neurospora and yeast, and they demonstrate one mechanism whereby splicing factors may evolve from cellular RNA binding proteins.

Amino Acid Sequence↗