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Results for “RNA Splicing”

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PubMed1993

In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit.

Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

3T3 Cells↗

PubMed2004

Protein displacement by DExH/D "RNA helicases" without duplex unwinding.

Members of the DExH/D superfamily of nucleic acid-activated nucleotide triphosphatases are essential for virtually all aspects of RNA metabolism, including pre-messenger RNA splicing, RNA interference, translation, and nucleocytoplasmic trafficking. Physiological substrates for these enzymes are thought to be regions of double-stranded RNA, because several DExH/D proteins catalyze strand separation in vitro. These "RNA helicases" can also disrupt RNA-protein interactions, but it is unclear whether this activity is coupled to duplex unwinding. Here we demonstrate that two unrelated DExH/D proteins catalyze protein displacement independently of duplex unwinding. Therefore, the essential functions of DExH/D proteins are not confined to RNA duplexes but can be exerted on a wide range of ribonucleoprotein substrates.

Adenosine Triphosphate↗

PubMed1989

Rat pyruvate kinase M gene. Its complete structure and characterization of the 5'-flanking region.

Genomic clones containing the rat pyruvate kinase M gene, which encodes the M1- and M2-type isozymes, were isolated and their exon sequences were determined. This gene contains 12 exons and 11 introns and is 20 kilobases (kb) long. The sequences specific to the M1- and M2-types exist in exons 9 and 10, respectively (Noguchi, T., Inoue, H., and Tanaka, T. (1986) J. Biol. Chem. 261, 13807-13812). The seventh intron begins with the GC dinucleotide instead of the consensus GT dinucleotide, but other exon-intron boundaries are consistent with the "GT-AG" rule. S1 mapping analysis showed that M1- and M2-type mRNAs had multiple, but the same transcription initiation sites. Thus, the M1- and M2-type isozyme mRNAs are concluded to be produced from the same M gene transcript by alternative RNA splicing. RNA blot hybridization analysis indicated that developmental changes of the isozymes in brain and skeletal muscle were regulated at the level of RNA splicing. The 5'-flanking region of the gene has no "TATA box" or "CAAT box," but contains potential Sp1 binding sites. Bacterial chloramphenicol acetyltransferase assay revealed that a fragment of about 0.5 kb of the 5'-flanking region of the gene was sufficient for promotor activity in the rat hepatoma cell line, dRLh-84. This activity was not present in adult rat hepatocytes, indicating that the 0.5-kb fragment has tissue-specific promoter activity. A processed-type pseudogene that resembles the M2-type pyruvate kinase cDNA was also characterized.

Animals↗

PubMed2005

Exposure of RNA templates and encapsidation of spliced viral RNA are influenced by the arginine-rich domain of human hepatitis B virus core antigen (HBcAg 165-173).

Previously, human hepatitis B virus (HBV) mutant 164, which has a truncation at the C terminus of the HBV core antigen (HBcAg), was speculated to secrete immature genomes. For this study, we further characterized mutant 164 by different approaches. In addition to the 3.5-kb pregenomic RNA (pgRNA), the mutant preferentially encapsidated the 2.2-kb or shorter species of spliced RNA, which can be reverse transcribed into double-stranded DNA before virion secretion. We observed that mutant 164 produced less 2.2-kb spliced RNA than the wild type. Furthermore, it appeared to produce at least two different populations of capsids: one encapsidated a nuclease-sensitive 3.5-kb pgRNA while the other encapsidated a nuclease-resistant 2.2-kb spliced RNA. In contrast, the wild-type core-associated RNA appeared to be resistant to nuclease. When arginines and serines were systematically restored at the truncated C terminus, the core-associated DNA and nuclease-resistant RNA gradually increased in both size and signal intensity. Full protection of encapsidated pgRNA from nuclease was observed for HBcAg 1-171. A full-length positive-strand DNA phenotype requires positive charges at amino acids 172 and 173. Phosphorylation at serine 170 is required for optimal RNA encapsidation and a full-length positive-strand DNA phenotype. RNAs encapsidated in Escherichia coli by capsids of HBcAg 154, 164, and 167, but not HBcAg 183, exhibited nuclease sensitivity; however, capsid instability after nuclease treatment was observed only for HBcAg 164 and 167. A new hypothesis is proposed here to highlight the importance of a balanced charge density for capsid stability and intracapsid anchoring of RNA templates.

Amino Acid Sequence↗

PubMed2002

Naturally occurring heterologous trans-splicing of adenovirus RNA with host cellular transcripts during infection.

The impact of viral infection on normal host RNA processing remains largely unexplored. We postulated that the high abundance of virally derived nuclear RNA in infected cells could impact host cell RNA splicing and viability. To test for aberrant RNA splicing we examined trans-splicing following infection with the replication-competent adenovirus mutant d11520 that lacks E1B 55 kDa protein. Trans-splicing was observed between viral RNA and several cellular precursor mRNAs, including beta-actin and glyceraldehyde-3-phosphate dehydrogenase. Using a tetracycline-inducible model system simulating viral trans-splicing activity we observed that overexpression of a trans-splicing RNA specifically inhibited cell proliferation. These results demonstrate that heterologous trans-splicing occurs naturally during adenovirus infection and suggest that trans-splicing may contribute to disruption of cell function.

3T3 Cells↗

PubMed1991

Mutations in the regions of the Rous sarcoma virus 3' splice sites: implications for regulation of alternative splicing.

Retrovirus RNA is synthesized as a single primary transcript that is differentially processed by RNA splicing. Three species of viral RNA (spliced env, spliced src, and unspliced full-length RNA) are produced in chicken embryo fibroblasts infected with Rous sarcoma virus, an avian retrovirus. The env and src mRNAs are synthesized by the alternative use of two 3' splice sites. The mechanism by which balanced splicing at the two sites is maintained was investigated in this report. Mutants that increase or decrease splicing at one of the two 3' splice sites were analyzed for the effect on splicing at the other site. The two splice sites differed in their response. Mutations that caused a specific increase in the level of spliced env mRNA were associated with reciprocal changes in the levels of src mRNA but with no change in overall splicing. In contrast, mutants in which the src 3' splice site was inactivated demonstrated a decreased overall level of spliced RNA but had little or no effect on the level of spliced env mRNA. Mutations that caused specific increases in the level of spliced src mRNA had variable effects on env mRNA levels. Deletion of regions in gag, which was previously shown to contain a cis-acting negative regulator of splicing, resulted in a corresponding increase of both spliced viral mRNAs and a decrease in unspliced RNA, suggesting that this element suppressed both env and src splicing. Several models are discussed which are possible mechanisms for regulation of alternative splicing of Rous sarcoma virus RNA, but none of these models appear to be consistent with all of the data.

Animals↗

PubMed1996

Spliced leader RNA trans-splicing in metazoa.

Spliced leader trans-splicing is a form of RNA processing originally described and studied in parasitic kinetoplastida. This mechanism of gene expression also occurs in parasitic and free-living metazoa. In this review, Dick Davis describes current knowledge of the distribution, substrates, specificity and functional significance of trans-splicing in metazoa.

Journal Article↗

PubMed1997

Implication of a cis-acting element in the cytoplasmic accumulation of unspliced Borna disease virus RNAs.

Borna disease virus (BDV), the prototype of a new family within the order Mononegavirales, is unusual in its nuclear localization for replication and transcription and use of RNA splicing for gene expression. The BDV antigenome contains three transcription units and six major open reading frames. Multicistronic RNAs containing two introns are elaborated from the third transcription unit. Differential splicing of the two introns and cytoplasmic accumulation of the unspliced and partially spliced RNA are critical for the balanced expression of the putative matrix protein, glycoprotein, and polymerase. To investigate the mechanisms for cytoplasmic expression of unspliced and partially spliced BDV transcripts, the levels of these transcripts were measured in the cytoplasm of infected COS-7 cells and noninfected COS-7 cells transfected with plasmids containing 2.8-kb cDNA inserts representing either wild-type or mutant BDV RNA from the third transcription unit. Analysis of truncation mutations allowed the identification of a cis-acting element present within the 3' end of the BDV 2.8-kb transcript that facilitated the cytoplasmic accumulation of unspliced BDV transcripts through nucleocytoplasmic transport. The nucleocytoplasmic transport activity was not dependent on the presence of BDV proteins. Gel-shift assays revealed that the cis-acting element binds specifically to host cytoplasmic and nuclear proteins.

Animals↗

PubMed1995

Cloning, sequencing and expression of two isoforms of the murine oct-1 transcription factor.

Oct-1 is a ubiquitously expressed regulatory gene of the POU domain family. The Oct-1 protein binds to the octamer motif present in the control regions of a variety of genes such as the immunoglobulins, histone H2B and snRNAs. To learn about Oct-1 and its possible role in B-cell maturation, we have used oct-2 cDNA to screen a murine pre-B cell, cDNA library. Two cDNA clones were identical in their POU-homeo box DNA binding domain, but differed in their 3'-region. Whereas one clone (oct-1a) was very similar to its human oct-1 homologue, the other (oct-1b), contained an additional 72 bp sequence (designated E1) at the serine threonine rich coding region (position 1485 of the human oct-1), and a deletion of another 72 bp sequence (designated E2) downstream (position 1920). These changes preserve the protein reading frame. DNA blot analysis indicates that murine oct-1 is a single copy gene and that the two oct-1 isoforms oct-1 is expressed as a large approximately 10 kb transcript in all the cell are generated by alternative RNA splicing. RNA blots showed that oct-1 is expressed as a large approximately 10 kb transcript in all the cell lines tested. PCR analysis of the E1 and E2 72 bp regions, indicated the presence of a third isoform containing both E1 and E2 (Oct-1c). Oct-1a and Oct-1b were present in all cell types examined, but the level of expression was lower in liver and spleen as compared to testis, thymus and kidney. The ratio of Oct-1b to Oct-1a ranged between 0.2 to 0.5, for all tissues examined except for testis which expressed higher amounts of oct-1b and/or oct-1c. Our findings thus show that the pattern of expression of the oct-1 gene is more complex than hitherto thought.

Amino Acid Sequence↗

PubMed1991

Identification of spliced baculovirus RNAs expressed late in infection.

Previous to this study, the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) was known to express only one spliced RNA (spliced IE1 or IE0). We have conducted an analysis of RNA expressed during infection of Spodoptera frugiperda cells with AcMNPV and have identified a set of five additional spliced RNAs expressed late in infection. A reverse transcription-polymerase chain reaction analysis was used to confirm the identification of the LS (late, spliced) RNAs. S1 nuclease and primer extension analyses were used to map the transcription initiation sites of LS RNAs. LS1 and LS2 initiated at positions -138 and -117, respectively (relative to the IE0 +1 transcription start site). Both LS1 and LS2 contain an additional cistron potentially encoding a small, highly basic polypeptide. LS3 (-79), LS4 (-22), and LS5 (+51/52) RNAs encode only the predicted downstream IE0 ORF. Although several baculovirus late gene consensus transcription initiation sites (ATAAG) were identified within this region, only LS5 initiated at one of these conserved motifs. An S1 nuclease analysis was done to determine whether unspliced precursors of LS RNAs could be detected. Early in infection, a greater proportion of IE0 RNA was detected in the spliced form; however, during the late phase of infection a significantly greater amount of unspliced precursor forms of LS RNAs was observed.

Amino Acid Sequence↗

PubMed1992

Trypanosoma brucei spliced-leader RNA methylations are required for trans splicing in vivo.

The Trypanosoma brucei spliced leader (SL) RNA donates its 5' leader sequence to all nuclear pre-mRNAs via trans RNA splicing. The SL RNA is a small-nuclear U RNA-like molecule which is present in the cell as part of a small ribonucleoprotein particle. However, unlike the trimethylguanosine-capped small nuclear U RNAs, the SL RNA has a highly modified 5' terminus containing an m7G cap and methylations on the first four transcribed nucleotides. Here, we show that incubation of procyclic-form T. brucei in the presence of the S-adenosylmethionine analog, sinefungin, leads to a rapid inhibition of SL RNA methylation. A concomitant inhibition of trans splicing and an accumulation of high-molecular-weight tubulin transcripts were also observed. The effects of sinefungin on SL RNA methylation and on trans splicing were correlated by labeling of cells incubated in the presence of the antibiotic. The results indicate that 5' modifications of the SL RNA are necessary for it to participate in trans splicing. SL RNA modification is not required for assembly of the core SL ribonucleoprotein, as these Cs2SO4-resistant particles can be formed with either methylated or undermethylated SL RNA.

Adenosine↗

PubMed2004

Differential effects of the SR proteins 9G8, SC35, ASF/SF2, and SRp40 on the utilization of the A1 to A5 splicing sites of HIV-1 RNA.

Splicing is a crucial step for human immunodeficiency virus, type 1 (HIV-1) multiplication; eight acceptor sites are used in competition to produce the vif, vpu, vpr, nef, env, tat, and rev mRNAs. The effects of SR proteins have only been investigated on a limited number of HIV-1 splicing sites by using small HIV-1 RNA pieces. To understand how SR proteins influence the use of HIV-1 splicing sites, we tested the effects of overproduction of individual SR proteins in HeLa cells on the splicing pattern of an HIV-1 RNA that contained all the splicing sites. The steady state levels of the HIV-1 mRNAs produced were quantified by reverse transcriptase-PCR. For interpretation of the data, transcripts containing one or several of the HIV-1 acceptor sites were spliced in vitro in the presence or the absence of one of the tested SR proteins. Both in vivo and in vitro, acceptor sites A2 and A3 were found to be strongly and specifically regulated by SR proteins. ASF/SF2 strongly activates site A2 and to a lesser extent site A1. As a result, upon ASF/SF2 overexpression, the vpr mRNA steady state level is specifically increased. SC35 and SRp40, but not 9G8, strongly activate site A3, and their overexpression ex vivo induces a dramatic accumulation of the tat mRNA, to the detriment of most of the other viral mRNAs. Here we showed by Western blot analysis that the Nef protein synthesis is strongly decreased by overexpression of SC35, SRp40, and ASF/SF2. Finally, activation by ASF/SF2 and 9G8 was found to be independent of the RS domain. This is the first investigation of the effects of variations of individual SR protein concentrations that is performed ex vivo on an RNA containing a complex set of splicing sites.

Alternative Splicing↗

PubMed2001

Two sequence classes of kinetoplastid 5S ribosomal RNA gene revealed among bodonid spliced leader RNA gene arrays.

The spliced leader RNA genes of Bodo saltans, Cryptobia helicis and Dimastigella trypaniformis were analyzed as molecular markers for additional taxa within the suborder Bodonina. The non-transcribed spacer regions were distinctive for each organism, and 5S rRNA genes were present in Bodo and Dimastigella but not in C. helicis. Two sequence classes of 5S rRNA were evident from analysis of the bodonid genes. The two classes of 5S rRNA genes were found in other Kinetoplastids independent of co-localization with the spliced leader RNA gene.

Animals↗

PubMed2007

Nuclear HBcAg and histology activity index as independent predictors of the expression of singly spliced HBV-RNA.

Although hepatitis B virus (HBV) RNA splicing has been reported by many researchers, the clinical significance of this event remains illusive. The present study was designed to investigate the clinical roles of singly spliced HBV-RNA. Liver biopsy tissues obtained from 32 consecutive patients were subjected to reverse transcriptase-polymerase chain reaction for the detection of singly spliced and unspliced HBV-RNA. Stepwise linear regression model was used to estimate the ratio of singly spliced to unspliced (S/US) HBV-RNA in the presence of the following variables: age, gender, aspartate aminotransferase, alanine aminotransferase, total bilirubin, alpha-foetoprotein, status of HBV e antigen (HBeAg), status of antibody to HBeAg, HBV-DNA, histology activity index (HAI), fibrotic score, grade of cytoplasmic HBV core antigen (HBcAg), grade of nuclear HBcAg, genotype, status of precore-stop-mutation, basal core promoter mutation, previous lamivudine therapy and superinfection by other hepatitis viruses. The results showed that HAI (beta = -0.2616; P = 0.011) and grade of nuclear HBcAg expression (beta = 0.5599; P =0.0067) were two independent predictors for the expression of singly spliced HBV-RNA. Further categorical analysis showed that patients with HAI score or=2 have significantly higher S/US ratios. In conclusion, nuclear HBcAg and HAI are two independent predictors for the expression of singly spliced HBV-RNA.

Adult↗

PubMed1995

A cap-binding protein complex mediating U snRNA export.

Cap structures are added cotranscriptionally to all RNA polymerase II transcripts. They affect several processes including RNA stability, pre-messenger RNA splicing, RNA export from the nucleus and translation initiation. The effect of the cap on translation is mediated by the initiation factor eIF-4F, whereas the effect on pre-mRNA splicing involves a nuclear complex (CBC) composed of two cap binding proteins, CBP80 and CBP20. A role for CBC in the nuclear export of capped RNAs has also been proposed. We report here the characterization of human and Xenopus CBP20s. Antibodies against recombinant CBP20 prevent interaction of CBC with capped RNAs in vitro. Following microinjection into Xenopus oocytes, the antibodies inhibit both pre-mRNA splicing and export of U small nuclear RNAs to the cytoplasm. These results demonstrate that CBC mediates the effect of the cap structure in U snRNA export, and provide direct evidence for the involvement of a cellular RNA-binding factor in the transport of RNA to the cytoplasm.

Amino Acid Sequence↗

PubMed1991

Trans splicing in trypanosomes requires methylation of the 5' end of the spliced leader RNA.

Trypanosoma brucei spliced leader (SL) RNA contains an unusual cap 4 structure consisting of 7-methylguanosine linked to four modified nucleosides. During RNA maturation, trans splicing transfers the first 39 nucleotides of the SL RNA including the cap structure to the 5' end of all mRNAs. Here we show that exposure of permeable trypanosome cells to S-adenosyl-L-homocysteine inhibits methylation of the nucleosides adjacent to 7-methylguanosine of newly synthesized SL RNA and prevents utilization of the SL RNA in trans splicing. However, trans splicing of the SL RNA preexisting in the cells is not inhibited by S-adenosyl-L-homocysteine as shown by the observation that newly synthesized alpha-tubulin RNA is trans spliced at the same level as in control cells. Therefore, it appears that the newly synthesized SL RNA is the only known component of the trans-splicing machinery that is impaired in its function by inhibition of methylation. Undermethylation does not alter either the stability of the SL RNA or the electrophoretic mobility and chromatographic behavior of the core SL ribonucleoprotein particle. Taken together, our data suggest that the cap 4 structure of the SL RNA plays an essential role in the trans-splicing process.

Animals↗

PubMedDate not supplied

Participation of nuclear genes in chloroplast gene expression.

The expression of the plastid genome is dependent on a large number of nucleus-encoded factors. Some of these factors have been identified through biochemical assays, and many others by genetic screens in Arabidopsis, Chlamydomonas and maize. Nucleus-encoded factors function in each step in plastid gene expression, including transcription, RNA editing, RNA splicing, RNA processing, RNA degradation, and translation. Many of the factors discovered via biochemical approaches play general roles as components of the basic gene expression machinery, whereas the majority of those identified by genetic approaches are specifically required for the expression of small subsets of chloroplast genes and are involved in post-transcriptional steps. Some of the nucleus-encoded factors may play regulatory roles and modulate chloroplast gene expression in response to developmental or environmental cues. They may also serve to couple chloroplast gene expression with the assembly of the protein products into the large complexes of the photosynthetic apparatus. The convergence of biochemical approaches with those of classical and reverse genetics, and the contributions from large scale genomic sequencing should result in rapid advances in our understanding of the regulatory interactions that govern plastid gene expression.

Cell Nucleus↗

PubMed1999

Aminoacyl-tRNA synthetases: a new image for a classical family.

The aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes well known for their role in protein synthesis. More recent investigations have discovered that this classic family of enzymes is actually capable of a broad repertoire of functions which not only impact protein synthesis, but extend to a number of other critical cellular activities. Specific aaRSs play roles in cellular fidelity, tRNA processing, RNA splicing, RNA trafficking, apoptosis, transcriptional and translational regulation. A recent EMBO workshop entitled 'Structure and Function of Aminoacyl-tRNA Synthetases' (Mittelwihr, France, October 10-15, 1998), highlighted the diversity of the aaRSs' role within the cell. These novel activities as well as significant advances in delineating mechanisms of substrate specificity and the aminoacylation reaction affirm the family of aaRSs as pharmaceutical targets.

Amino Acyl-tRNA Synthetases↗