About 30% of minute virus of mice RNA is spliced out following polyadenylation.
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Cytoplasm↗
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Results for “RNA Splicing”
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Sites of recombination between the transforming gene of avian myeloblastosis virus and its helper virus.
The sites of recombination between the transforming gene of avian myeloblastosis virus (AMV) and its natural helper myeloblastosis-associated virus (MAV) have been determined. In AMV, the cellular sequence substituting for the viral envelope (env) gene gives rise to a different carboxyl terminus of the DNA polymerase. The 5'-recombination site coincides with the RNA splice acceptor site for the production of env mRNA in MAV-infected cells. The 3'-recombination site reveals that the last 11 amino acids including the termination codon are shared by the env protein and AMV transforming protein. The RNA splice acceptor site for the generation of subgenomic v-myb mRNA is located 84 nucleotides downstream from the 5'-recombination site. The AMV transforming protein consists of helper virus-related sequences at both of its amino and carboxyl termini, and all but 84 nucleotides of the cell-derived v-myb sequence. The comparison of MAV gp85 amino acid sequence with those of subgroups B, C, and E indicates that the MAV present in clone lambda 10A2-1 belongs to subgroup B. The high degree of homology among different avian retroviruses of the same subgroup indicates that the amino acid sequence of gp85 is important in determining the conformation of the envelope glycoprotein.
Amino Acid Sequence↗
Reported in vivo splice-site mutations in the factor IX gene: severity of splicing defects and a hypothesis for predicting deleterious splice donor mutations.
Small consensus sequences have been defined for RNA splicing, but questions about splicing in humans remain unanswered. Analysis of germline mutations in the factor IX gene offers a highly advantageous system for studying the mutational process in humans. In a sample of 860 families with hemophilia B, 9% of independent mutations are likely to disrupt splicing as their primary mode of action. This includes 26 splicing mutations reported herein. When combined with the factor IX splice mutations reported by others, at least 104 independent mutations have been observed, 80 of which are single base substitutions within the splice donor and splice acceptor consensus sequences. After analysis of these mutations, the following inferences emerge: (1) the susceptibility of a splice donor sequence to deleterious mutation depends on the degree of similarity with the donor consensus sequence, suggesting a simple "5-6 hypothesis" for predicting deleterious vs. neutral mutations; (2) the great majority of mutations that disrupt the splice donor or splice acceptor sequences result in at least a 100-fold decrement in factor IX coagulant activity, indicating that the mutations at these sites generally function as an on/off switch; (3) mutations that create cryptic splice junctions or that shorten but do not interrupt the polypyrimidine tract in the splice acceptor sequence can reduce splicing by a variable amount; and (4) there are thousands of potential donor-acceptor consensus sequence combinations in the 38-kb factor IX gene region apparently not reduced by evolutionary selective pressure, presenting an apparent paradox; i.e., mutations in the donor and acceptor consensus sequences at intron/exon splice junctions can dramatically alter normal splicing, yet, appropriately spaced, good matches to the consensus sequences do not predispose to significant amounts of alternative splicing.
Computer Simulation↗
Reverse transcriptase activity associated with maturase-encoding group II introns in yeast mitochondria.
Group II introns al1 and al2 of the yeast mtDNA cox1 gene encode reverse transcriptase-like proteins that function in RNA splicing and may play a role in intron mobility and excision. We find that ribonucleoprotein particles from yeast mitochondria contain a reverse transcriptase activity that is likely encoded by al1 and al2 and is highly specific for the introns and their flanking exons. Using a mutant strain with elevated activity, we show that the reverse transcriptase uses either excised intron RNA or cox1 pre-mRNA as template and initiates cDNA synthesis near the 3' end of al2 and immediately downstream in E3. Our results suggest that introns al1 and al2 are retroelements, which encode reverse transcriptases that have adapted to function in RNA splicing.
Base Sequence↗
Alternatively spliced exons encode the tissue-specific 5' termini of leukocyte pp52 and stromal cell S37 mRNA isoforms.
The pp52 gene encodes an intracellular, F-actin-binding phosphoprotein (also designated LSP1 and WP34) postulated to function in cytoskeleton dynamics and cell motility. We previously reported that different mRNA isoforms are expressed from this gene in cells of the leukocyte lineage versus mesodermally derived cells. These tissue-specific mRNA isoforms are identical except for 5'-untranslated regions and sequences coding for unique N-termini of 23 and 21 amino acids, respectively. As this is a single-copy gene, we predicted that these tissue-specific mRNA isoforms would be generated by alternative RNA splicing. We report that the unique 5' sequences in these mRNA isoforms are encoded in two separate exons containing ATG initiation codons. These features confirm that the pp52 and S37 mRNA isoforms are generated by alternative RNA splicing and establish that they are independently translated. Other results presented here indicate that the differential expression of these exons in leukocytes versus mesodermally derived cells is regulated at the level of transcription by tissue-specific promoters.
Alternative Splicing↗
Molecular basis for two forms of the G protein that stimulates adenylate cyclase.
Most cells contain two forms of the alpha subunit of the G protein (Gs) that stimulates adenylate cyclase; their apparent molecular weights are 45,000 and 52,000. Two cDNAs that correspond to distinct mRNAs for the alpha subunit of Gs have been cloned from a bovine adrenal library and sequenced. The sequences of the two cDNAs, designated pGs-l and pGs-S, are identical except for a single stretch of 46 nucleotides in the coding region, where four are altered and 42 are deleted in pGs-S. Expression of pGs-S and pGs-l in COS-m6 cells yields protein products with apparent molecular weights of 45,000 and 52,000, respectively, based on their mobility in sodium dodecyl sulfate-polyacrylamide gels. We conclude that pGs-S and pGs-l encode the 45- and 52-kDa forms of Gs alpha, respectively, and propose that the mRNAs encoding these proteins arise from a single gene by internal alternative RNA splicing.
Adenylyl Cyclases↗
The genetics of nuclear pre-mRNA splicing: a complex story.
The occurrence of introns in nuclear precursor RNAs (pre-mRNAs) is widespread in eukaryotes, and the splicing process that removes them is basically the same in yeasts as it is in higher eukaryotes. Splicing takes place in a very large, multi-component complex, the splicesome, and biochemical studies have been complicated by the large number of splicing factors involved. This review describes how genetic approaches used to study RNA splicing in Saccharomyces cerevisiae have complemented the biochemical studies and led to rapid advances in the field.
RNA Precursors↗
Splicing of adenovirus RNA in a cell-free transcription system.
A soluble whole-cell extract prepared accurately from HeLa cells splices 2-3% of the RNA transcribed from a DNA template containing the first and second leader exons of late adenovirus RNA. The spliced RNA was detected by a sensitive technique using hybridization to a single-stranded phage M13 cDNA clone, followed by binding to nitrocellulose filters. The identity of the spliced RNA was established by RNase T1 and pancreatic RNase two-dimensional peptide mapping. The bond formed during the in vitro splicing reaction appears to be a typical 3',5'-phosphodiester bond as judged by its sensitivity to RNase T1. The splicing reaction is specifically inhibited by KCl at concentrations greater than 50 mM and by the addition of cellular RNA. Three features of this system may account for the detection of splicing in a soluble extract: (i) the sensitive and unambiguous hybridization assay, (ii) the high transcriptional activity of the major late promoter of adenovirus, and (iii) the use of the first and second leader exon splice of adenovirus, which may be unusually rapid.
Adenoviruses, Human↗
Trans-splicing of pre-mRNA in plants, animals, and protists.
Messenger RNA maturation in eukaryotes typically involves the removal of introns from long precursor molecules. An unusual form of RNA splicing in which separate precursor transcripts contribute sequences to the mature mRNA through intermolecular reactions has now been documented in a number of diverse organisms. In this review, the phenomenon of pre-mRNA trans-splicing has been divided into two categories. The "spliced leader" type, found in protozoans such as trypanosomes and lower invertebrates such as nematodes, results in the addition of a short, capped 5' noncoding sequence to the mRNA. The "discontinuous group II intron" form of trans-splicing, found in plant/algal chloroplasts and plant mitochondria, involves the joining of independently transcribed coding sequences, presumably through interactions between "intronic" RNA pieces. Both categories of trans-splicing are mechanistically similar to conventional nuclear pre-mRNA cis-splicing; potential evolutionary relationships are discussed.
Animal Population Groups↗
The polyprotein nature of substance P precursors.
Substance P and related tachykinin peptides probably act as neurotransmitters or modulators of neurotransmission, and regulate biological processes as diverse as salivary secretion and transmission of pain signals. Substance P peptide sequences are expressed in three distinct mRNAs that are generated from one gene by differential RNA splicing. In addition to substance P, as many as three other tachykinin peptides can be generated from the polyprotein precursors by differential posttranslational processing. Three tachykinin receptor subtypes have been extensively characterized which differentially interact with the naturally occurring tachykinin peptides. Therefore, the generation of diversity of tachykinin peptides results from differential precursor RNA splicing and differential posttranslational processing. The specificity of peptide responses is the result of selective receptor subtype expression.
Amino Acid Sequence↗
Molecular structure of the human desmoplakin I and II amino terminus.
Desmoplakins (DPs) I and II are closely related proteins found in the innermost region of the desmosomal plaque, which serves as a cell surface attachment site for cytoplasmic intermediate filaments. Overlapping cDNA clones comprising 9.2 kilobases of DP-I, predicted to encode a full-length 310-kDa polypeptide (2677 amino acid residues), have now been identified. Here we report the predicted protein sequence and structural analysis of the N terminus of DP, extending our previous study of the rod and carboxyl domains. The N terminus contains groups of heptad repeats that are predicted to form at least two major alpha-helical-rich bundles. Unlike the rod and carboxyl domains, the N terminus did not display a periodic distribution of charged residues. Northern blot mapping and genomic sequence analysis were also undertaken to examine the organization of the DP mRNAs. A 1-kilobase intron was located at the 3' boundary of a DP-I-specific region; however, instead of an intron at the 5' junction, a possible splice donor site was observed within a potential coding sequence, suggesting alternative RNA splicing from an internal donor site.
Amino Acid Sequence↗
Expression of the mutagenic peptide of herpes simplex virus type 1 in virus-infected cells.
A fragment of DNA from within the minimum transforming region (mtr-1) of herpes simplex virus type 1 (HSV-1) is known to raise the mutation frequency of cells. This activity has been attributed to a viral protein whose properties are largely unknown. Antiserum was raised to a synthetic peptide of a predicted amino acid sequence from the protein, and was found to react with cells that were infected by HSV-1 in an ELISA and by immunocytochemical staining. A combination of immunoprecipitation and immunoblotting techniques confirmed that the epitope is located at the carboxy terminus of the UL26 gene product and is downstream of epitopes that are recognized by two monoclonal antibodies. The mutagenic peptide was different from the conventional gene product of UL26 in that: (a) It was expressed from a different reading frame, (b) It was expressed earlier in infection, and (c) It bound DNA, and thus could be separated by DNA-cellulose chromatography. An RT-PCR experiment revealed two deletions in the cDNA, suggesting that RNA splicing could account for the frameshift. Examination of the DNA sequence of the region also revealed a potential ribosomal frame-shift site. The mutagenic peptide of HSV-1 is therefore a product of the UL26 gene which is expressed with a different carboxy terminus early in infection, and this could be due either to RNA splicing or to ribosomal frame-shifting.
Amino Acid Sequence↗
Role of the branch site/3'-splice site region in adenovirus-2 E1A pre-mRNA alternative splicing: evidence for 5'- and 3'-splice site co-operation.
The adenovirus E1A gene encodes five overlapping mRNAs which are processed by alternative RNA splicing from a common pre-mRNA. To characterize cis-acting sequence elements which are of importance for the alternative 5'-splice site selection deletion and substitution mutants within the intron that is common to all E1A mRNAs were constructed. Deletion of the wild-type E1A branch site/polypyrimidine tract resulted in activation of a functionally redundant sequence located within an A/T rich sequence just upstream of the normal E1A lariat branch site. Removal of both regulatory sequences abolished in vivo splicing completely and did not lead to activation of cryptic 3'-splice sites at other locations in the E1A pre-mRNA. Furthermore we show that the sequence around the E1A branch site/3'-splice site region may have a more direct effect on the efficiency by which the alternative E1A 5'-splice sites are selected. Replacing the E1A branch site/3'-splice site region with the corresponding sequence from the second intron of the rabbit beta-globin gene or the first intron of the major late transcription unit resulted in drastic changes in E1A 5'-splice site selection. For example, with the E1A/beta-globin hybrid gene the 9S mRNA became the most abundant E1A mRNA to accumulate. This contrasts with the wild-type E1A gene in which almost undetectable levels of 9S mRNA were produced in transient expression assays. Our results strongly suggest that a cooperative interaction between 5'- and 3'-splice sites on a pre-mRNA determines the outcome of alternative splicing.
Adenoviridae↗
Tissue-specific alternative splicing of ascidian troponin I isoforms. Redesign of a protein isoform-generating mechanism during chordate evolution.
In vertebrates, troponin I (TnI) exists as shorter and longer isoforms encoded by distinct genes expressed in skeletal and cardiac muscle, respectively. We report that the protochordate ascidian Ciona intestinalis expresses a homologous set of shorter and longer TnI isoforms in body wall muscle and heart, respectively. The heart-specific segment of the ascidian longer TnI isoform shares several sequence features with vertebrate cardiac TnI but lacks the protein kinase A phosphorylation sites implicated in sympatho-adrenal control of cardiac function. In contrast with vertebrates, the ascidian longer and shorter TnI isoforms are produced from a single gene by tissue-specific alternative RNA splicing; remarkably, the molecular mechanism of TnI isoform generation has been entirely reworked during ascidian/vertebrate evolution. Because alternative splicing is the more probable chordate ancestral condition, the long/cardiac versus short/somatic muscle pattern of TnI isoforms likely existed before the occurrence of the gene duplication events that created the vertebrate TnI gene family. Thus, gene duplication was apparently not the primary engine of isoform diversity in this aspect of TnI gene family evolution; rather, it simply provided an alternative (transcriptional) means of maintaining a previously established system of isoform diversity and tissue specificity based on alternative RNA splicing.
Alternative Splicing↗
The optional E. coli prr locus encodes a latent form of phage T4-induced anticodon nuclease.
The optional Escherichia coli prr locus restricts phage T4 mutants lacking polynucleotide kinase or RNA ligase. Underlying this restriction is the specific manifestation of the T4-induced anticodon nuclease, an enzyme which triggers the cleavage-ligation of the host tRNALys. We report here the molecular cloning, nucleotide sequence and mutational analysis of prr-associated DNA. The results indicate that prr encodes a latent form of anticodon nuclease consisting of a core enzyme and cognate masking agents. They suggest that the T4-encoded factors of anticodon nuclease counteract the prr-encoded masking agents, thus activating the latent enzyme. The encoding of a tRNA cleavage-ligation pathway by two separate genetic systems which cohabitate E. coli may provide a clue to the evolution of RNA splicing mechanisms mediated by proteins.
Amino Acid Sequence↗
Alternative splicing of a novel glycophorin allele GPHe(GL) generates two protein isoforms in the human erythrocyte membrane.
The Henshaw antigen (synonym: He or MNS6) is carried by an altered form of glycophorin B (GPB), but the molecular basis for its variable expression or quantitative polymorphism remains largely undefined. We report here the identification and analysis of a novel glycophorin He allele, GPHe(GL), which gives rise to the expression of two protein isoforms in the erythrocyte membrane. In addition to the nucleotide changes defining the epitopic sequence of He, a single C-to-G nucleotide transversion in exon V coding for the membrane domain was found to cause aberrant RNA splicings by creating a new acceptor splice site. In addition, a T-to-G transversion at -6 position of the acceptor splice site for exon IV was identified. Both full-length and truncated transcripts of GPHe(GL) were detected as the result of partial activation of the new acceptor splice site and partial inactivation of the normal splice sites. The full-length cDNA encoded He, S, and U antigens, whereas the three truncated ones lacked either the sequence for S and U antigens or a large portion of the membrane domain or both. The GPB gene on the other chromosome was apparently normal and its transcript encoded N, s, and U antigens. These results correlate alternative RNA splicing with the expression of two GPHe isoforms and thus delineate a new mechanism for the phenotypic diversity of membrane glycophorins.
Alleles↗
RNA editing: trypanosomes rewrite the genetic code.
The understanding of how genetic information is stored and expressed has advanced considerably since the "central dogma" asserted that genetic information flows from the nucleotide sequence of DNA to that of messenger RNA (mRNA) which in turn specifies the amino acid sequence of a protein. It was found that genetic information can be stored as RNA (e.g. in RNA viruses) and can flow from RNA to DNA by reverse transcriptase enzyme activity. In addition, some genes contain introns, nucleotide sequences that are removed from their RNA (by RNA splicing) and thus are not represented in the resultant protein. Furthermore, alternative splicing was found to produce variant proteins from a single gene. More recently, the study of trypanosome parasites revealed an unexpected and indeed counter-intuitive genetic complexity. Genetic information for a single protein can be dispersed among several (DNA) genes in these organisms. One of these genes specifies an encrypted precursor mRNA that is converted to a functional mRNA by a process called RNA editing that inserts and deletes uridylate nucleotides. The sequence of the edited mRNA is specified by multiple small RNAs, named guide RNAs, (gRNAs) each of which is encoded in a separate gene. Thus, edited mRNA sequences are assembled from multiple genes by the transfer of information from one type of RNA to another. The existence of editing was surprising but has stimulated the discovery of other types of RNA editing. The Stuart laboratory has been exploring RNA editing in trypanosomes from the time of its discovery. They found dramatic differences between the mitochondrial gene sequences and those of the corresponding mRNAs, which indicated editing by the insertion and deletion of uridylates. Some editing was modest; simply eliminating shifts in sequence register of minimally extending the protein coding sequence. However, editing of many mRNAs was startingly extensive. The RNA sequence was essentially entirely remodeled with its sequence more the result of editing than the gene sequence. The identities of genes for such extensively edited RNA were not recognizable from the DNA sequence but they were readily identifiable from the edited mRNA sequence. Thus, despite the complex and extensive editing the resultant mRNA sequence is precise. Characterization of partially edited RNAs indicated that editing proceeds in the direction opposite to that used to specify the protein which reflects the use of the gRNAs. The numerous gRNAs that are used for editing are encoded in the DNA molecules whose role was previously a mystery. Using information gained in our earlier studies, the Stuart group developed an in vitro system that reproduces the fundamental process of editing in order to resolve the mechanism by which it occurs. They determined that editing entails a series of enzymatic steps rather than the mechanism used in RNA splicing. They also showed that chimeric gRNA-mRNA molecules are aberrant by-products of editing rather than intermediates in the process as had been proposed. Additional studies are exploring precisely how the number of added and deleted uridylates is specified by the gRNA. The Stuart laboratory showed that editing is performed by an aggregation of enzymes that catalyze the separate steps of editing. It also developed a method to purify this multimolecule complex that contains several, perhaps tens of, proteins. This will allow the study of its composition and the functions of its component parts. Indeed, the gene for one component has been identified and its detailed characterization begun. These studies are developing tools to explore related processes. An early finding in the lab was that the various mRNAs are differentially edited during the life cycle of the parasite. The pattern of this editing indicates that editing serves to regulate the alternation between two modes of energy generation. This regulation is coordinated with other events that are occurring during the life c
Animals↗
Plant intron sequences: evidence for distinct groups of introns.
In vivo and in vitro RNA splicing experiments have demonstrated that the intron splicing machineries are not interchangeable in all organisms. These differences have prevented the efficient in vivo expression of monocot genes containing introns in dicot plants and the in vitro excision of some plant introns in HeLa cell in vitro splicing extracts. We have analyzed plant introns for sequence differences which potentially account for the functional splicing differences. Three classes of plant introns can be differentiated by the purine or pyrimidine-richness of sequences upstream from the 3' splice site. The frequency of these three types of introns in monocots and dicots varies significantly. The degree of variability in the 5' and 3' intron boundaries is evaluated for each of these classes in monocots and dicots. The 5' splice site consensus sequences developed for the monocot and dicot introns differ in their ability to base pair with conserved nucleotides present at the 5' end of many U1 snRNAs.
Base Sequence↗