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At least 1,423 records · Page 79Linked to original sources
PubMed1989

U2 snRNA sequences that bind U2-specific proteins are dispensable for the function of U2 snRNP in splicing.

Previously we showed that microinjection of purified U2 snRNA from HeLa cells into Xenopus laevis oocytes, depleted of their endogenous U2 snRNPs by oligonucleotide-targeted degradation, led to assembly of hybrid snRNPs that were fully functional for splicing of SV40 late pre-mRNA. We have extended these results by examining features of U2 RNA that are required for its role in splicing. Injection of Xenopus U2 snRNA transcribed in vitro by T7 RNA polymerase, differing in sequence from authentic U2 by only one nucleotide, although capable of efficient assembly into snRNP-like particles, did not complement U2-predepleted oocytes for splicing. However, when injected into pretargeted oocytes, a plasmid containing Xenopus U2 snRNA sequences resulted in synthesis of U2 snRNA that was assembled into snRNPs capable of mediating splicing of SV40 late pre-mRNA. This allowed us to test several U2 RNA mutants for their function in splicing. Mutants with sequences deleted within U2 stem-loops I and II, although efficiently assembled into snRNP-like particles upon injection, failed to restore splicing. Interestingly, however, injection of a mutant that lacks the binding site for the U2-specific proteins A' and B", restored pre-mRNA splicing. These data suggest that the direct binding of U2-specific proteins with snRNA is not essential for the function of U2 snRNPs in splicing of pre-mRNA.

Animals↗
PubMed1991

Autoregulation of the splicing of transcripts from the transformer-2 gene of Drosophila.

The Drosophila transformer-2 gene uses alternative promoters and splicing patterns to generate four different mRNAs that together encode three putative RNA-binding polypeptides. The transformer-2 products expressed in somatic tissues function to regulate the RNA splicing of the sex determination gene doublesex, whereas products expressed in the male germ line play an unknown, but essential, role in spermatogenesis. Two alternatively spliced transformer-2 transcripts, each encoding a different putative RNA-binding protein, are found only in the male germ line. These male germ line-specific mRNAs differ from each other by the presence or absence of a single intron called M1. We show that M1-containing transcripts make up a majority of transformer-2 germ-line transcripts in wild-type males but fail to accumulate in males homozygous for transformer-2 null mutations. Germ-line transformation experiments using a variety of reporter gene constructs demonstrate that specific polypeptide products of the transformer-2 gene itself normally repress M1 splicing in the male germ line. Thus, in addition to its role in the sex-specific control of doublesex RNA splicing in somatic tissues, the transformer-2 gene also regulates the splicing of its own transcripts in the male germ line. We propose that this autoregulatory function may serve in negative feedback control of transformer-2 activity during spermatogenesis. The finding that transformer-2 controls multiple splicing decisions suggests that a variety of different alternative splicing choices could be regulated by a relatively limited number of trans-acting factors.

Animals↗
PubMed1994

A tertiary interaction in the Tetrahymena intron contributes to selection of the 5' splice site.

The utilization of cryptic splice sites has been observed in a number of RNA splicing reactions. In the self-splicing group I intron of Tetrahymena thermophila, point mutations of either A57 or A95 promote cleavage at two sites other than the normal 5' splice site, suggesting that these nucleotides are involved in a common tertiary interaction. These results are unusual since A57 and A95 are neither at nor near the 5' splice site in the sequence or secondary structure. Cleavage at the alternative sites appears to occur by intron cyclization, a reaction with well-established structural and mechanistic similarities to the first step of RNA self-splicing. Alternative docking of P1 (the helix containing the 5' splice site paired to the internal guide sequence of the intron) into the catalytic core accounts for cleavage at the cryptic reaction sites. We propose that the A57/A95 interaction, along with an element implicated previously (J1/2), provide structural connectivity from the reaction site in P1 to the catalytic core of the Tetrahymena intron. It seems likely that RNA splicing in general will require such tertiary interactions to position RNA helices.

Adenine↗
PubMed2002

Alu-containing exons are alternatively spliced.

Alu repetitive elements are found in approximately 1.4 million copies in the human genome, comprising more than one-tenth of it. Numerous studies describe exonizations of Alu elements, that is, splicing-mediated insertions of parts of Alu sequences into mature mRNAs. To study the connection between the exonization of Alu elements and alternative splicing, we used a database of ESTs and cDNAs aligned to the human genome. We compiled two exon sets, one of 1176 alternatively spliced internal exons, and another of 4151 constitutively spliced internal exons. Sixty one alternatively spliced internal exons (5.2%) had a significant BLAST hit to an Alu sequence, but none of the constitutively spliced internal exons had such a hit. The vast majority (84%) of the Alu-containing exons that appeared within the coding region of mRNAs caused a frame-shift or a premature termination codon. Alu-containing exons were included in transcripts at lower frequencies than alternatively spliced exons that do not contain an Alu sequence. These results indicate that internal exons that contain an Alu sequence are predominantly, if not exclusively, alternatively spliced. Presumably, evolutionary events that cause a constitutive insertion of an Alu sequence into an mRNA are deleterious and selected against.

Alternative Splicing↗
PubMed2002

Selecting for functional alternative splices in ESTs.

The expressed sequence tag (EST) collection in dbEST provides an extensive resource for detecting alternative splicing on a genomic scale. Using genomically aligned ESTs, a computational tool (TAP) was used to identify alternative splice patterns for 6400 known human genes from the RefSeq database. With sufficient EST coverage, one or more alternatively spliced forms could be detected for nearly all genes examined. To identify high (>95%) confidence observations of alternative splicing, splice variants were clustered on the basis of having mutually exclusive structures, and sample statistics were then applied. Through this selection, alternative splices expected at a frequency of >5% within their respective clusters were seen for only 17%-28% of genes. Although intron retention events (potentially unspliced messages) had been seen for 36% of the genes overall, the same statistical selection yielded reliable cases of intron retention for <5% of genes. For high-confidence alternative splices in the human ESTs, we also noted significantly higher rates both of cross-species conservation in mouse ESTs and of validation in the GenBank mRNA collection. We suggest quantitative analytical approaches such as these can aid in selecting useful targets for further experimental characterization and in so doing may help elucidate the mechanisms and biological implications of alternative splicing.

Alternative Splicing↗
PubMed2003

Structure and expression of phosphoenolpyruvate carboxylase kinase genes in solanaceae. A novel gene exhibits alternative splicing.

Phosphorylation of phosphoenolpyruvate carboxylase (PEPc; EC 4.1.1.31) plays an important role in the control of central metabolism in higher plants. Two PPCK (PEPc kinase) genes have been identified in tomato (Lycopersicon esculentum cv Alicante), hereafter termed LePPCK1 and LePPCK2. The function of the gene products has been confirmed by transcription of full-length cDNAs, translation, and in vitro assay of kinase activity. Previously studied PPCK genes contain a single intron. LePPCK2 also contains a novel second intron that exhibits alternative splicing. The correctly spliced transcript encodes a functional PEPc kinase, whereas unspliced or incorrectly spliced transcripts encode a truncated, inactive protein. The relative abundance of the transcripts depends on tissue and conditions. Expression of LePPCK2 was markedly increased during fruit ripening. In ripe Alicante fruit, the locule and seeds contained only the correctly spliced LePPCK2 transcripts, whereas in ripe fruit of the tomato greenflesh mutant, they contained correctly and incorrectly spliced transcripts. Potato (Solanum tuberosum) contains genes that are very similar to LePPCK1, and LePPCK2; StPPCK2 exhibits alternative splicing. Aubergine (Solanum melongena) and tobacco (Nicotiana tabacum) also contain a PPCK2 gene; the sequence of the alternatively spliced intron is highly conserved between all four species. The data suggest that the two PPCK genes have different roles in tissue-specific regulation of PEPc and that the alternative splicing of PPCK2 transcripts is functionally significant.

Alternative Splicing↗
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↗
PubMed1991

Control of alternative splicing by the differential binding of U1 small nuclear ribonucleoprotein particle.

Cellular factors controlling alternative splicing of precursor messenger RNA are largely unknown, even though this process plays a central role in specifying the diversity of proteins in the eukaryotic cell. For the identification of such factors, a segment of the rat preprotachykinin gene was used in which differential expression of neuropeptides gamma and K is dependent on alternative splicing of the fourth exon (E4). Sequence variants of the three-exon segment, (E3-E4-E5) were created, resulting in a sensitive assay for factors mediating the splicing switch between E4-skipping and E4-inclusion. A dinucleotide mutation in the 5' splice site of E4 that increase base-pairing of this site to U1 small nuclear RNA resulted in uniform selection of E4, whereas a control mutation that destroyed base-pairing resulted in uniform E4-skipping. Affinity selection of spliceosomes formed on these functionally distinct substrates revealed that the extreme difference in splicing was mediated by differential binding of the U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site of E4. These data show that, apart from its established role in selecting 5' splice sites, U1 snRNP plays a fundamental role in 3' exon selection and provides insight into possible mechanisms of alternative splicing.

Animals↗
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↗
PubMed2005

Quantitative analysis of group II intron expression and splicing in Lactococcus lactis.

The group II intron Ll.ltrB is found within the ltrB relaxase gene of the conjugative element pRS01 in Lactococcus lactis. Precise splicing of the intron is essential for pRS01 transfer. The transcription regulation and in vivo splicing activity of Ll.ltrB have not been investigated thoroughly in L. lactis in the natural pRS01 context. We developed absolute quantitative real-time reverse transcription-PCR assays to quantify RNA levels of the 5' exon (ltrBE1) and the spliced relaxase (ltrB) and intron-encoded protein (ltrA) genes, as well as Ll.ltrB splicing activity under different physiological conditions. The mRNA levels for the ATP-binding protein OppD were assayed for comparison to the ltrB transcripts. The oppD mRNA ranged from 10- to 10,000-fold higher than ltrB region genes. ltrBE1 expression was growth-phase dependent. The mRNA level of ltrA was almost constant during all growth phases and in all media tested. Ll.ltrB in vivo splicing activity ranged from (6.5 +/- 2.1)% to (22.1 +/- 8.0)%. Acid challenge significantly decreased both ltrB region mRNA levels and intron splicing activity. The presence of recipient cells, different mating environments, and temperature stress had no significant effects on expression and splicing. Western blotting showed that the level of LtrB protein expressed from an intronless ltrB gene was much higher (about 20-fold) than the level of protein expressed from an intron-containing construct. Interestingly, LtrB protein showed a tendency to function in cis on its oriT target. The low level of ltrB transcript and relatively inefficient splicing of the intron may limit Ll.ltrB mobility and dissemination in nature.

Bacterial Proteins↗
PubMed1986

Three splicing patterns are used to excise the small intron common to all minute virus of mice RNAs.

We identified three splicing patterns used to excise the small intron common to all three transcripts encoded by minute virus of mice. Sequence analysis of minute virus of mice-specific cDNAs indicated that two donor and two acceptor splice sites were used: in pattern 1, the most frequent, nucleotide 2280 was spliced to nucleotide 2377; in pattern 2, nucleotides 2317 and 2399 were joined. Oligonucleotide probes, each specific for one of the four possible splice junction sequences, were synthesized and hybridized to viral mRNAs immobilized on nitrocellulose filters. The probes specific for splice patterns 1 and 2 hybridized to all three viral mRNAs, as did a third oligomer specific for a splicing pattern in which nucleotides 2280 and 2399 were joined. The fourth potential splicing pattern, linking nucleotides 2317 and 2377, was not detected. The presence of three splicing patterns in the transcripts designated R2 and R3 would allow the translation of five distinct polypeptides from these two mRNAs.

Base Sequence↗
PubMed1987

A small deletion distant from a splice or polyadenylation site dramatically alters pre-mRNA processing in region E3 of adenovirus.

The E3 complex transcription unit of adenovirus encodes overlapping mRNAs (a to i) with different exon structures. The major mRNAs are a (approximately 40% of the total) and c (approximately 15%), which are spliced once, and f (approximately 15%) and h (approximately 25%), which are spliced twice. mRNA a uses the upstream E3A polyadenylation site, and the other mRNAs use the downstream E3B polyadenylation site. We analyzed virus deletion mutants to identify sequences important in alternative pre-mRNA processing in region E3. Our main finding is that a 64-base-pair deletion in dl742 causes mainly mRNAs f and h to be formed. mRNAs a and c are barely made. dl742 does not delete either a splice site or a polyadenylation site. Thus, the sequences deleted must function in alternative pre-mRNA processing independently of the signals at the actual splice and polyadenylation sites. The lack of synthesis of mRNA a by dl742 does not appear to result from a defect in the E3A polyadenylation signal but rather from an increase in splicing activity which results in the synthesis of doubly spliced mRNAs f and h at the expense of singly spliced mRNAs a and c. This suggests, in the wild-type situation, that the frequency of use of the E3A versus the E3B polyadenylation site may be determined by the rate of splicing, as well as, presumably, the rate of cleavage-polyadenylation at the E3A site.

Adenoviridae↗
PubMed1991

Analysis of splicing patterns of human spumaretrovirus by polymerase chain reaction reveals complex RNA structures.

Mapping of transcripts of the human foamy virus genome was carried out in permissive human embryonic fibroblast cells by Northern blot hybridization and S1 nuclease analysis. Since several splice sites that are localized within a relatively narrow genomic region were detected, the polymerase chain reaction (PCR) was employed, and cloning and sequencing of the splice site junctions of the corresponding viral cDNAs were subsequently performed. All spumavirus transcripts have a common but relatively short leader RNA. Genomic, singly spliced env mRNAs and several singly and multiply spliced subgenomic transcripts were identified. The multiply spliced viral mRNAs consist of various exons located in the central or 3' part of the viral genome. At least four novel gene products, termed Bet, Bes, Beo, and Bel3, are predicted to exist. The poly(A) addition site that defines the boundary of the R and U5 region in the 3' long terminal repeat was determined. The pattern of spumavirus splicing is more complex than that of oncoviruses and more similar to that of lentiviruses. One of the characteristic features of spumavirus transcription is the existence of singly spliced bel1 and bel2 mRNAs that alternatively are multiply spliced, thereby generating a complexity comparable to, but different from, that of lentiviruses and from that of other known retroviruses. The complex spumavirus transcriptional pattern of human spumavirus and the coding potential of the 10 exons identified are discussed.

Amino Acid Sequence↗
PubMed1991

Characterization and genetic analysis of alternatively spliced transcripts of hepatitis B virus in infected human liver tissues and transfected HepG2 cells.

The transcriptional map of hepatitis B virus (HBV) has been expanded recently by the discovery of a singly spliced transcript in hepatoma cell lines transfected with cloned viral DNA and a doubly spliced one in naturally infected human liver tissues. By the use of reverse transcription and a subsequent polymerase chain reaction, the two spliced HBV RNAs were shown to be present in both types of cells. As further evidence, an HBV mutant was constructed and found to exclusively express the singly spliced RNA. This mutant was also used to quantitate the two spliced species in transfected HepG2 cells; they were found to be equally abundant, and each represented about 30% of the pregenomic RNA. The HBV mutant could still produce replication-competent HBV virions when transfected into HepG2 cells, indicating that the doubly spliced transcript, just like the singly spliced one, was not essential for HBV replication. However, the two abundant spliced HBV transcripts were detected in most naturally infected human liver tissues, suggesting that they may have biologic functions in vivo.

Base Sequence↗
PubMed1992

Nucleotide 880 splice donor site required for efficient transformation and RNA accumulation by human papillomavirus type 16 E7 gene.

Mutations within coding sequences of the various human papillomavirus type 16 (HPV-16) genes have been used to demonstrate that the HPV-16 E7 gene is necessary and sufficient for transformation of rodent cells. We now provide evidence that, in addition to E7 coding sequences, a small cis-acting region immediately flanking the 3' end of E7 coding sequences is also required for transformation. This was shown by translation termination linker insertion, progressive deletion analysis, and site-directed mutagenesis. Disruption of the nucleotide (nt) 880 splice donor site within the 3'-flanking region by deletion of as few as 4 nt or substitution of 3 nt totally abolished transformation. Regeneration of the wild-type sequence in a previously transformation-incompetent splice site mutant restored transformation. Mutating the wild-type splice donor site to the consensus splice site resulted in a stronger transformation phenotype, while mutating the +2 position of the consensus sequence significantly reduced the frequency of transformation. It was shown with RNase protection assays that the amount of E7 mRNA in transformation-deficient splice site mutants was much lower. Nuclear runoff experiments revealed that there was no change in the rate of synthesis of E7 message in the nt 880 splice site mutant. Furthermore, mutations of HPV-16 sequences indicated that the two other early region splice donor sites have no more than minor roles in transformation and efficient RNA accumulation. These results indicate that the specific integrity of the nt 880 splice donor site is essential for both accumulation of E7 RNA and efficient E7-mediated transformation.

Animals↗
PubMed1993

Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus.

The polymerase chain reaction was used to detect and characterize low-abundance bovine leukemia virus (BLV) mRNAs. In infected cattle we could detect spliced mRNA with a splice pattern consistent with a Tax/Rex mRNA, as well as at least four alternatively spliced RNAs. Two of the alternatively spliced mRNAs encoded hitherto unrecognized BLV proteins, designated RIII and GIV. The Tax/Rex and alternatively spliced mRNAs could be detected at their highest levels in BLV-infected cell cultures; the next highest levels were found in samples from calves experimentally infected at 6 weeks postinoculation. Alternatively spliced mRNAs were also expressed, albeit at lower levels, in naturally infected animals; they were detected by a nested polymerase chain reaction. Interestingly, the GIV mRNA was specifically detected in naturally infected cows with persistent lymphocytosis and in two of five calves at 6 months after experimental infection with BLV. Furthermore, the calf with the strongest signal for GIV had the highest lymphocyte counts. These data may suggest a correlation between expression of the GIV product and development of persistent lymphocytosis. Some of the donor and acceptor sites in the alternatively spliced mRNAs were highly unusual. The biological mechanisms and significance of such a choice of unexpected splice sites are currently unknown.

Alternative Splicing↗
PubMed2005

Kaposi's sarcoma-associated herpesvirus K8beta is derived from a spliced intermediate of K8 pre-mRNA and antagonizes K8alpha (K-bZIP) to induce p21 and p53 and blocks K8alpha-CDK2 interaction.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a lymphotropic DNA tumor virus that induces Kaposi's sarcoma and AIDS-related primary effusion lymphoma. KSHV open reading frame 50 and K8 genes in early viral lytic infection express, respectively, a tricistronic and a bicistronic pre-mRNA, which undergo alternative splicing to create two major spliced mRNA isoforms, alpha and beta, by inclusion (beta) or exclusion (alpha) of an intron at nucleotides 75563 to 75645. This intron contains some suboptimal features, which cause the intron 5' splice site (ss) to interact weakly with U1 snRNA and the 3' ss to bind a U2 auxiliary factor, U2AF, with low affinity. Optimization of this intron in K8 (K8 intron 2) promoted the interaction of the 5' ss with U1 and the 3' ss with U2AF, resulting in a substantial increase in intron splicing. Splicing of K8 intron 2 has also been shown to be stimulated by the splicing of a downstream intron. This was confirmed by the insertion of a human beta-globin intron into the K8beta exon 3-exon 4 splice junction, which promoted splicing of K8beta intron 2 and conversion of the K8beta mRNA to the K8alpha mRNA that encodes a K-bZIP protein. Intron 2 contains a premature termination codon, yet the K8beta mRNA is insensitive to nonsense-mediated mRNA decay, suggesting that the truncated K8beta protein may have a biological function. Indeed, although the truncated K8beta protein is missing only a C-terminal leucine zipper domain from the K-bZIP, its expression antagonizes the ability of the K-bZIP to induce p53 and p21 and blocks K-bZIP-CDK2 interaction through interfering K8alpha mRNA production.

Alternative Splicing↗
PubMed1990

A highly specific phosphatase from Saccharomyces cerevisiae implicated in tRNA splicing.

We identified and partially purified a phosphatase from crude extracts of Saccharomyces cerevisiae cells that can catalyze the last step of tRNA splicing in vitro. This phosphatase can remove the 2'-phosphate left over at the splice junction after endonuclease has removed the intron and ligase has joined together the two half-molecules. We suggest that this phosphatase is responsible for the completion of tRNA splicing in vivo, based primarily on its specificity for the 2'-phosphate of spliced tRNA and on the resistance of the splice junction 2'-phosphate to a nonspecific phosphatase. Removal of the splice junction 2'-phosphate from the residue adjacent to the anticodon is likely necessary for efficient expression of spliced tRNA. The phosphatase appears to be composed of at least two components which, together with endonuclease and ligase, can be used to reconstitute the entire tRNA-splicing reaction.

Phosphoric Monoester Hydrolases↗