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At least 271 records · Page 15Linked to original sources
PubMed1990

Control of retroviral RNA splicing through maintenance of suboptimal processing signals.

The full-length retroviral transcript serves as genomic RNA for progeny virions, as an mRNA for structural proteins and enzymes, and as a pre-mRNA substrate for splicing that yields subgenomic mRNAs that encode other essential proteins. Thus, RNA splicing to form subgenomic mRNAs must be incomplete or regulated in order to preserve some of the full-length transcripts. We have used the avian sarcoma virus system to delineate the viral functions that are required in the regulation of the splicing event that forms the envelope glycoprotein (env) subgenomic mRNA. We observed previously that a specific insertion mutation just 5' of the env splice acceptor site resulted in nearly complete splicing to form env mRNA and a concomitant replication defect which is presumably due to a deficit of the full-length transcript. Replication-competent pseudorevertants contained second-site mutations that restored splicing control, and these mapped either just upstream or downstream of the env splice acceptor site. In this report, we show that splicing control at this site does not require expression of any known viral replication protein(s), nor does it appear to require the viral splice donor site. From these results and analysis of additional splicing mutations obtained by in vivo selection, we conclude that splicing is controlled through the maintenance of suboptimal cis-acting signals in the viral RNA that alter the efficiency of recognition by the cellular splicing machinery.

Animals↗
PubMed2004

cis-Acting and trans-acting modulation of equine infectious anemia virus alternative RNA splicing.

Equine infectious anemia virus (EIAV), a lentivirus distantly related to HIV-1, encodes regulatory proteins, EIAV Tat (ETat) and Rev (ERev), from a four-exon mRNA. Exon 3 of the tat/rev mRNA contains a 30-nucleotide purine-rich element (PRE) which binds both ERev and SF2/ASF, a member of the SR family of RNA splicing factors. To better understand the role of this element in the regulation of EIAV pre-mRNA splicing, we quantified the effects of mutation or deletion of the PRE on exon 3 splicing in vitro and on alternative splicing in vivo. We also determined the branch point elements upstream of exons 3 and 4. In vitro splicing of exon 3 to exon 4 was not affected by mutation of the PRE, and addition of purified SR proteins enhanced splicing independently of the PRE. In vitro splicing of exon 2 to exon 3 was dependent on the PRE; under conditions of excess SR proteins, either the PRE or the 5' splice site of exon 3 was sufficient to activate splicing. We applied isoform-specific primers in real-time RT-PCR reactions to quantitatively analyze alternative splicing in cells transfected with rev-minus EIAV provirus constructs. In the context of provirus with wild-type exon 3, greater than 80% of the viral mRNAs were multiply spliced, and of these, less than 1% excluded exon 3. Deletion of the PRE resulted in a decrease in the relative amount of multiply spliced mRNA to about 40% of the total and approximately 39% of the viral mRNA excluded exon 3. Ectopic expression of ERev caused a decrease in the relative amount of multiply spliced mRNA to approximately 50% of the total and increased mRNAs that excluded exon 3 to about 4%. Over-expression of SF2/ASF in cells transfected with wild-type provirus constructs inhibited splicing but did not significantly alter exon 3 skipping.

Alternative Splicing↗
PubMed1999

RNA splicing: more clues from spinal muscular atrophy.

Spinal muscular atrophy is caused by mutations in the SMN1 gene, the product of which is part of a multi-component complex involved in the assembly of small nuclear ribonucleoproteins. A recent study indicates that SMN may also play a role in pre-mRNA splicing.

Cyclic AMP Response Element-Binding Protein↗
PubMed1986

Heterogeneous nuclear ribonucleoproteins: role in RNA splicing.

Splicing in vitro of a messenger RNA (mRNA) precursor (pre-mRNA) is inhibited by a monoclonal antibody to the C proteins (anti-C) of the heterogeneous nuclear RNA (hnRNA)-ribonucleoprotein (hnRNP) particles. This antibody, 4F4, inhibits an early step of the reaction: cleavage at the 3' end of the upstream exon and the formation of the intron lariat. In contrast, boiled 4F4, or a different monoclonal antibody (designated 2B12) to the C proteins, or antibodies to other hnRNP proteins (120 and 68 kilodaltons) and nonimmune mouse antibodies have no inhibitory effect. The 4F4 antibody does not prevent the adenosine triphosphate-dependent formation of a 60S splicing complex (spliceosome). Furthermore, the 60S splicing complex contains C proteins, and it can be immunoprecipitated with 4F4. Depletion of C proteins from the splicing extract by immunoadsorption with either of the two monoclonal antibodies to the C proteins (4F4 or 2B12) results in the loss of splicing activity, whereas mock-depletion with nonimmune mouse antibodies bodies has no effect. A 60S splicing complex does not form in a C protein-depleted nuclear extract. These results indicate an essential role for proteins of the hnRNP complex in the splicing of mRNA precursors.

Adenosine Triphosphate↗
PubMed1989

Tissue-dependent plastid RNA splicing in maize: transcripts from four plastid genes are predominantly unspliced in leaf meristems and roots.

Most plastid gene products do not accumulate to high levels in meristem proplastids or in the specialized plastids of roots. To assess whether a modulation of plastid splicing activities might play a role in this tissue-dependent expression of the plastid genome, the ratio of spliced to unspliced transcripts from the atpF, petB, petD, and rpl16 genes was compared between several tissues of maize. Although these transcripts are predominantly spliced in green leaf tissue (both bundle sheath and mesophyll cells), spliced atpF, petB, and petD transcripts are underrepresented relative to their unspliced precursors in roots and leaf meristems. The ratio of spliced to unspliced rpl16 transcripts varies in a similar fashion, but the magnitude of the differences between tissues is not as great. The proportion of RNA that is spliced reflects the tissue of origin and not photosynthetic competency, chlorophyll content, or exposure to light since the leaves of photosynthetic mutants and of seedlings grown in the absence of light contain spliced and unspliced transcripts in normal ratios. These results raise the possibility that low RNA splicing activities are in part responsible for the limited expression of the plastid genome in meristematic and root tissue.

Gene Expression Regulation↗
PubMed1986

RNA splicing and intron turnover are greatly diminished by a mutant yeast branch point.

Two mutant genes, both of which contain an A----C transversion at the absolutely conserved branch point of the yeast "TACTAAC box" (TACTAAC----TACTACC), were constructed and introduced into yeast cells. Splicing and gene expression are almost completely eliminated by this mutation, but a low level (approximately equal to 0.1%) of proper splicing is detectable. Branch point mapping indicates that the mutant branch is formed at the normal location--i.e., to cytidine rather than adenosine. The mutant branch is also a very poor substrate for the HeLa cell debranching enzyme. Although splicing of the mutant transcripts is very poor, the cells contain a high level of mutant intron because these excised introns are remarkably stable. The results imply that the normal branch point is important not only for branch formation and splicing but also for intron turnover.

Base Sequence↗
PubMed1990

RNA splicing in Chlamydomonas chloroplasts. Self-splicing of 23 S preRNA.

The 23 S rRNA gene of the Chlamydomonas reinhardtii chloroplast contains an 888-base pair intron with structural features characteristic of Group I introns. The nuclear, chloroplast ribosome-deficient mutant of C. reinhardtii, ac20, overaccumulates a approximately 3.6-kilobase unspliced 23 S preRNA compared to wild-type cells. We have used [alpha-32P]GTP labeling of total RNA preparations from ac20 to rapidly determine that 23 S preRNA is capable of self-splicing. The ability of the 23 S intron (with flanking exon sequences) to correctly catalyze its own splicing was confirmed using RNA produced by in vitro transcription of cloned DNA. These results identify the first example of a self-splicing RNA of chloroplast origin.

Base Sequence↗
PubMed1992

Equine infectious anemia virus gene expression: characterization of the RNA splicing pattern and the protein products encoded by open reading frames S1 and S2.

The utilization of predicted splice donor and acceptor sites in generating equine infectious anemia virus (EIAV) transcripts in fetal donkey dermal cells (FDD) was examined. A single splice donor site identified immediately upstream of the gag coding region joins the viral leader sequence to all downstream exons of spliced EIAV transcripts. The predominant 3.5-kb transcript synthesized in EIAV-infected FDD cells appears to be generated by a single splicing event which links the leader sequence to the first of two functional splice acceptor sites near the 5' end of the S1 open reading frame (ORF). The translation products encoded by the 3.5-kb transcript were examined by producing in vitro transcripts from a cDNA corresponding to this RNA followed by in vitro translation in wheat germ extracts. These transcripts directed the synthesis of three proteins: the virus trans-activator protein (EIAV Tat) encoded by ORF S1, a protein of unknown function encoded by ORF S2, and the virus envelope glycoprotein. When transfected into FDD cells, this cDNA also directed expression of EIAV Tat. Amino-terminal sequence analysis of the in vitro-synthesized S1 protein supports the suggestion that translation of EIAV Tat is initiated at a CUG codon within the virus leader region. Both in vitro-synthesized S2 protein and synthetic peptides corresponding to S2 are shown to react positively with sera obtained from EIAV-infected horses, providing the first direct evidence of expression of this protein in infected animals.

Amino Acid Sequence↗
PubMed1997

Involvement of the MRE2 gene of yeast in formation of meiosis-specific double-strand breaks and crossover recombination through RNA splicing.

BACKGROUND: The mre2 mutant of Saccharomyces cerevisiae is defective in meiotic recombination and produces inviable spores, but the sensitivities to DNA damaging agents, methyl methanesulphonate and ultraviolet light are not altered by the mutation. Mre2 has two copies of RNA recognition motif (RRM), suggesting its participation in RNA metabolism in meiosis. RESULTS: An amino acid substitution in the N-terminal RRM of Mre2 confers a meiotic recombination defect. Using this mre2N strain, the MER2 gene was isolated as a multi-copy suppressor of the recombination defect. Meiosis-specific splicing of MER2 pre-mRNA was impaired in the mre2 deletion (mre2delta) mutant. The mre2delta mutant was defective in the formation of meiosis-specific double-strand breaks (DSBs) and crossover and noncrossover recombinants. When the chromosomal MER2 gene was replaced with the intronless derivative of MER2 gene, cMER2, the formation of DSBs and of noncrossover recombinants were restored in the mre2delta mutant. However, the amount of crossover recombinants produced in the mre2delta cMER2 strain was approximately 30% that in the wild-type. In addition, the mre2delta cMER2 mutant was defective in chromosome segregation and in viable spore formation. CONCLUSIONS: Mre2 participates in the formation of DSBs through meiosis-specific splicing of MER2 pre-mRNA. Besides, Mre2 is also involved in crossover recombination, possibly through splicing of RNA from another gene(s).

Binding Sites↗
PubMed1990

Characterization and expression of novel singly spliced RNA species of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) expresses the Vif, Vpr, Vpu, and Env proteins through complex differential splicing of a single full-length RNA precursor. We used HIV-1-specific oligonucleotide primer pairs in a quantitative polymerase chain reaction procedure on RNA from fresh peripheral blood lymphocytes infected with HIV-1JR-CSF to detect and characterize the singly spliced RNA species which might encode these proteins. The nucleotide sequences at the junctions of splice donor and acceptor sites of these RNAs were determined. One of these RNAs, which has not been previously described, appears to be a novel HIV-1 RNA encoding Env and/or Vpu proteins.

Base Sequence↗