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Results for “RNA Splicing”

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PubMed1990

Transcription of a nematode trans-spliced leader RNA requires internal elements for both initiation and 3' end-formation.

We have used block substitution mutagenesis and in vitro transcription to define sequence elements important for efficient initiation and 3' end-formation of the trans-spliced leader RNA (SL RNA) of the parasitic nematode Ascaris lumbricoides. These experiments indicate that the SL RNA has an unusual promoter structure containing elements which include the 22 nt trans-spliced leader exon itself. Efficient transcription is correlated with the binding of a factor to the 22 nt (SL) sequence; mutations within the SL which abolish transcription lead to a loss in binding of this factor. In addition to internal sequences, synthesis of SL RNA in vitro requires an element centered 50 bases upstream of the cap site. Mutations within this element dramatically affect the level of SL RNA synthesis but do not affect accuracy of initiation. Finally, all of the information required for accurate 3' end-formation of SL RNA lies within the transcribed region. Thus, the arrangement of sequences necessary for the synthesis of SL RNAs does not resemble that of sequences important for the synthesis of vertebrate U snRNAs despite the similarities between SL RNAs and U snRNAs.

Animals↗

PubMed1999

A reverse transcriptase/maturase promotes splicing by binding at its own coding segment in a group II intron RNA.

Group II introns encode reverse transcriptases that promote RNA splicing (maturase activity) and then with the excised intron form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription (TPRT). Here, we show that the primary binding site for the maturase (LtrA) encoded by the Lactococcus lactis Ll.LtrB intron is within a region of intron domain IV that includes the start codon of the LtrA ORF. This binding is enhanced by other elements, particularly domain I and the EBS/IBS interactions, and helps position LtrA to initiate cDNA synthesis in the 3' exon as occurs during TPRT. Our results suggest how the maturase functions in RNA splicing and support the hypothesis that the reverse transcriptase coding region was derived from an independent genetic element that was inserted into a preexisting group II intron.

Bacterial Proteins↗

PubMed1995

Role of the hepatitis B virus posttranscriptional regulatory element in export of intronless transcripts.

Hepatitis B virus S transcripts contain a region, known as the posttranscriptional regulatory element (PRE), that activates their transport from the nucleus to the cytoplasm. J. Huang and T. J. Liang (Mol. Cell. Biol. 13:7476-7486, 1993) have shown that this element can partially substitute for the human immunodeficiency virus Rev-response element (RRE) in a reporter plasmid that is dependent on the RRE and Rev protein for expression and concluded that PRE exhibits Rev-RRE-like functions by inhibiting splicing. However, we have obtained additional data which indicate that the PRE functions in a novel manner that is not dependent on inhibition of splicing. Unlike Rev-RRE, the PRE functions independently of splice donor and acceptor sites and can activate cytoplasmic expression of an intronless (so-called prespliced) beta-globin transcript. Conversely, a heterologous intron can substitute for the PRE in increasing cytoplasmic expression of hepatitis B virus S transcripts. In addition, the host nuclear factor, YL2 (p32), which enhances Rev-RRE function has no effect on PRE-dependent gene expression. Since S transcripts are not normally known to be spliced and since RNA splicing and cytoplasmic transport are tightly linked processes in higher eucaryotic cells, we conclude that the PRE functions in cis to allow the export of nuclear transcripts that do not interact efficiently with the splicing pathway and hence are normally not exported well from the nucleus. Such elements are critical for the life cycle of viruses, such as hepatitis B virus, which undergo reverse transcription during replication.

Base Sequence↗

PubMed1989

Characterization and expression of a spliced leader RNA in the parasitic nematode Ascaris lumbricoides var. suum.

The parasitic nematode Ascaris spp. contains a 22-nucleotide spliced-leader (SL) sequence identical to the trans-SL previously described in Caenorhabditis elegans and other nematodes. The SL comprises the first 22 nucleotides of a approximately 110-base RNA and is transcribed by RNA polymerase II. The SL RNA contains a trimethylguanosine cap and a consensus Sm binding site. Furthermore, the Ascaris SL RNA has the potential to adopt a secondary structure which is nearly identical to potential secondary structures of similar SL RNAs in C. elegans and Brugia malayi.

Animals↗

PubMed1999

Transcription initiation at the TATA-less spliced leader RNA gene promoter requires at least two DNA-binding proteins and a tripartite architecture that includes an initiator element.

Eukaryotic transcriptional regulatory signals, defined as core and activator promoter elements, have yet to be identified in the earliest diverging group of eukaryotes, the primitive protozoans, which include the Trypanosomatidae family of parasites. The divergence within this family is highlighted by the apparent absence of the "universal" transcription factor TATA-binding protein. To understand gene expression in these protists, we have investigated spliced leader RNA gene transcription. The RNA product of this gene provides an m(7)G cap and a 39-nucleotide leader sequence to all cellular mRNAs via a trans-splicing reaction. Regulation of spliced leader RNA synthesis is controlled by a tripartite promoter located exclusively upstream from the transcription start site. Proteins PBP-1 and PBP-2 bind to two of the three promoter elements in the trypanosomatid Leptomonas seymouri. They represent the first trypanosome transcription factors with typical double-stranded DNA binding site recognition. These proteins ensure efficient transcription. However, accurate initiation is determined an initiator element with a a loose consensus of CYAC/AYR (+1), which differs from that found in metazoan initiator elements as well as from that identified in one of the earliest diverging protozoans, Trichomonas vaginalis. Trypanosomes may utilize initiator element-protein interactions, and not TATA sequence-TATA-binding protein interactions, to direct proper transcription initiation by RNA polymerase II.

Animals↗

PubMed1995

Structure and expression of novel spliced leader RNA genes in Caenorhabditis elegans.

Approximately 25% of Caenorhabditis elegans genes are organized as operons. Polycistronic transcripts are converted to monocistronic mRNAs by 3' cleavage/polyadenylation and 5' trans-splicing with untranslated, 5' termini of mRNAs encoded by downstream genes in operons are acceptors for > or = 7 recently discovered "novel" SLs and a classical SL (SL2). Diversity in SL exons is now partly explained by the discovery and characterization of five novel genes that encode C. elegans SL RNAs. These novel SL RNAs contain a 22- or 23-nucleotide SL followed by conserved splice donor and downstream sequences that are essential for catalysis of trans-splicing reactions. The SL3 alpha, SL4, and SL5 RNA genes are tightly clustered on chromosome III; their 114-nucleotide transcripts deliver three distinct SLs to mRNAs. The SL3 beta and SL3 gamma RNA genes are on chromosome I, but are not tightly linked. SL RNAs 3 alpha, 3 beta, and 3 gamma provide identical 5' leader exons, although their 3' sequences diverge. Transcription of SL 3-5 RNA genes appears to be driven by flanking DNA elements that are homologous with segments of promoters for the C. elegans SL2 RNA and small nuclear RNA genes. RNase protection assays demonstrated that novel SL RNAs are transcribed in vivo and accumulate in the poly(A-) RNA pool. SL3 exons are transferred to mRNAs as frequently as SL2 exons. In contrast, SL4 is appended to mRNAs 10% as frequently as SL3. The abundance of SL4 RNA increased 6-fold during postembryonic development, and the SL4 RNA gene promoter is active principally in hypodermal cells.

Animals↗

PubMed1979

Splicing and the formation of stable RNA.

To determine whether RNA splicing plays an obligatory role in gene expression, we have constructed a series of SV40-transducing viruses carrying various combinations of splice junctions derived from the viral genome and a mouse globin gene. All of the viruses that retain at least one functional splice junction, derived from either the viral or the mouse genome, encode stable hybrid RNAs. In contrast, a virus from which all the splice junctions have been removed fails to produce any detectable stable RNA. These results suggest that splicing is a prerequisite for stable RNA formation.

Animals↗

PubMed1997

Trypanosoma cruzi strains partition into two groups based on the structure and function of the spliced leader RNA and rRNA gene promoters.

We have previously identified a major proximal sequence element (PSE) responsible for transcription of the spliced leader (SL) gene from Trypanosoma cruzi strain CL, and showed that the sequence encompassing this PSE exhibits approximately 30% divergence between two major groups of T. cruzi isolates, but strong conservation within the groups. In this report, we show that the SL RNA gene promoter from the CL strain (group I) is efficiently expressed only in T. cruzi isolates from group I. Similarly, the sequence of the approximately 643 bp promoter region of the T. cruzi rRNA is strongly conserved within, but diverged approximately 20% between, the two groups. Reporter constructs driven by the rRNA promoter sequences from group I strains are strongly expressed after electroporation into other group I strains, but not expressed in group II strains. In contrast, constructs bearing rRNA promoter sequences from group II strains are active in strains from both groups. Phylogenetic analyses performed with both the rRNA and the SL RNA gene promoter sequences yielded similar trees, and these trees strongly reinforce the partitioning of known T. cruzi into two major groups that parallel the observed functional specificity of the promoters. Given the well-documented species specific pattern of both rRNA promoters and PSEs in higher eukaryotes, these results suggest an ancient evolutionary divergence among organisms currently classified as T. cruzi.

Animals↗

PubMed2006

An amelogenin minigene to study alternative splicing.

Diversity in gene expression is commonly observed as a result of alternative splicing of RNA transcripts. This is true in the case of amelogenin, one of the enamel matrix proteins. Our hypothesis is that additional amelogenin mRNA transcripts are generated in vivo, but these transcripts have yet to be observed because of the limitations of currently used detection methodologies. For this study our objective was to create an amelogenin minigene to study amelogenin RNA splicing events in cell lines of diverse character. Mouse genomic DNA was used as a PCR template to amplify the amelogenin DNA sequence spanning exons 2-7. The resulting PCR-generated DNA was subcloned in an expression vector. This resulting amelogenin minigene was shown to be functionally active by transfection into multiple cell lines. We have successfully cloned an amelogenin minigene, and as a result we describe and discuss novel amelogenin alternatively spliced transcripts.

Alternative Splicing↗

PubMed2003

A novel RNA splicing-mediated gene silencing mechanism potential for genome evolution.

Over 90% of the human genome consists of non-protein-coding regions. Introns constitute most of the non-coding regions located in precursor messenger RNAs (pre-mRNAs). During pre-mRNA maturation, the introns are excised out of mRNA and thought to be completely digested prior to translation. If the introns were merely metabolic "leavings," why would the genome hold such a large amount of extraneous genetic materials? Here we show a novel posttranscriptional gene silencing system identified within mammalian introns. By packaging human spliceosome-recognition sites along with an exonic insert into an artificial intron, we observed that the splicing and processing of such an exon-containing intron in either sense or antisense conformation produced equivalent gene silencing effects, while a palindromic hairpin insert containing both sense and antisense strands resulted in synergistic effects. These findings may explain how cells respond to the presence of transgenic introns that are homologous to pre-existing exons during genomic evolution.

Animals↗

PubMed1986

One binding site determines sequence specificity of Tetrahymena pre-rRNA self-splicing, trans-splicing, and RNA enzyme activity.

The specificity of reactions catalyzed by the Tetrahymena pre-rRNA intervening sequence (IVS) was studied using site-specific mutagenesis. Two sequences required for 5' splice-site selection during self-splicing were defined. Single-base changes in either a 5' exon sequence or a 5' exon-binding site within the IVS disrupt their ability to pair and result in inefficient or inaccurate splicing. Combinations that restore complementarity suppress the effect of the single-base changes. Sequence alterations in the 5' exon-binding site also change the specificity of two other reactions: intermolecular exon ligation (trans-splicing) and the enzymatic nucleotidyltransferase activity of the IVS RNA. Thus the substrate specificity of an RNA enzyme can be changed in a manner predictable by the rules of Watson-Crick base-pairing.

Base Sequence↗

PubMed1990

Processing of complementary sense RNAs of Digitaria streak virus in its host and in transgenic tobacco.

We have used a polymerase chain reaction (PCR) procedure to analyse low abundance complementary sense RNAs of Digitaria streak virus (DSV) from infected leaves of Digitaria setigera. This study has confirmed that both spliced and unspliced RNAs are synthesised by the same transcription unit. The position of the intron has been proven from sequencing cDNAs corresponding to the spliced RNA. Although the majority of cDNAs have 3' ends at coordinate 1063, downstream from a consensus polyadenylation sequence, a minor population of RNAs with heterogeneous 3' ends has also been identified. Two major RNA species with alternative splice sites or 3' ends, previously identified by nuclease S1 protection assays, could not be detected, but a cDNA species was observed with an apparent 90bp insertion at the 5' end of the intron. In transgenic tobacco containing integrated dimers of DSV DNA, the major unspliced RNA could readily be detected, but no spliced RNA was present. This may be a reason why DSV DNA did not replicate in tobacco. In addition, neither the minor population of heterogeneous RNAs nor the cDNA species with the insertion could be detected. The failure of the intron to be spliced in tobacco and its low activity in Digitaria is discussed in relation to recent studies on RNA splicing in plants and has led us to the conclusion that the geminivirus introns may be intrinsically inefficient.

Base Sequence↗

PubMed2004

Spliced leader RNA gene promoter sequence heterogeneity in CL-Brener Trypanosoma cruzi reference strain.

Trypanosoma cruzi is divided into two phylogenetic lineages, T. cruzi I and T. cruzi II, which contain different spliced leader (SL) RNA gene promoter sequences: Class I SL gene promoter sequences are found in T. cruzi II, and Class II sequences in T. cruzi I. We analysed different SL RNA promoter sequences from CL-Brener reference strain, belonging to T. cruzi II lineage, and detected sequences that differed within the -80/+1 highly conserved region. Indeed, many of these divergent SL promoters present features of T. cruzi I promoters. Some of these sequences were grouped into the T. cruzi I sequences clade by Bayesian analysis. The results presented herein show that sequence heterogeneity in SL RNA gene promoter not only exists between T. cruzi strains but also within CL-Brener strain. These CL-Brener "T. cruzi I-like" sequences could be considered a molecular trace of a hybrid origin of the SL RNA gene and a new evidence for the presence of sequences of T. cruzi I origin into a T. cruzi II strain. The possible origins of these sequences are discussed.

Animals↗

PubMed1994

trans-spliceosomal U6 RNAs of Crithidia fasciculata and Leptomonas seymouri: deviation from the conserved ACAGAG sequence and potential base pairing with spliced leader RNA.

U6 RNA genes from the trypanosomatids Crithidia fasciculata and Leptomonas seymouri have been isolated and sequenced. As in Trypanosoma brucei, the U6 RNA genes in both C. fasciculata and L. seymouri are arranged in close linkage with upstream tRNA genes. The U6 RNA sequences from C. fasciculata and L. seymouri deviate in five and three positions, respectively, from the published T. brucei sequence. Interestingly, both C. fasciculata U6 RNA genes carry a C-->T change at the second position of the ACAGAG hexanucleotide sequence, which is important for splicing function and has been considered phylogenetically invariable. A compensatory base change of the C. fasciculata spliced leader RNA at the highly conserved 5' splice site position +5, G-->A, suggests that an interaction between the 5' splice site region and U6 RNA recently proposed for the yeast cis-splicing system may also occur in trans splicing.

Animals↗

PubMed1986

cDNA clones of the neural cell adhesion molecule (N-CAM) lacking a membrane-spanning region consistent with evidence for membrane attachment via a phosphatidylinositol intermediate.

In embryonic chicken brains, the neural cell adhesion molecule N-CAM is expressed mainly as two polypeptides, the large intracellular-domain polypeptide (ld) (Mr = 160,000) and the small intracellular-domain polypeptide (sd) (Mr = 130,000) chains, that differ in their cytoplasmic domains and that arise by alternative splicing of RNA transcribed from a single gene. There is evidence for a minor N-CAM polypeptide of Mr = 120,000 that is similar to the ld and sd chains for most of its amino-terminal sequence, but which lacks a cytoplasmic domain. We report here the isolation and characterization of a cDNA clone, lambda N151, that appears to encode this third N-CAM polypeptide, which we designate the ssd (small surface-domain) polypeptide chain. The cDNA insert of lambda N151 consists of 2437 base pairs (bp). DNA hybridization and sequencing indicate that the first 1721 bp are nearly identical to the corresponding sequences of clone lambda N208, which encodes the ld chain. Following in the same reading frame, lambda N151 encodes 25 amino acids not present in lambda N208. The rest of lambda N151 consists of a 637-bp noncoding region containing an AATACA polyadenylylation sequence and a 55-bp poly(A) tract. Messenger RNAs complementary to lambda N151 appear later in development than those complementary to the ld and sd chains, and their appearance is correlated with the appearance of the ssd polypeptide. Although the polypeptide encoded by lambda N151 lacks a membrane region that would define a cytoplasmic domain, it does contain at its carboxyl end a relatively hydrophobic stretch of amino acids similar to those seen in precursors of membrane proteins that are attached to membranes via the lipid phosphatidylinositol. We show here that the ssd chain of chicken N-CAM can be released from brain vesicles by treatment with phospholipase C, suggesting that it too may have a phosphatidylinositol anchor. These results define two additional modes by which N-CAM expression can be modulated: by RNA splicing at a new site and by differential membrane attachment of the resulting polypeptide through a lipid intermediate.

Amino Acid Sequence↗

PubMed1989

Functional domains of the Drosophila melanogaster muscle myosin heavy-chain gene are encoded by alternatively spliced exons.

The single-copy Drosophila muscle myosin heavy-chain (MHC) gene, located at 36B(2L), has a complex exon structure that produces a diversity of larval and adult muscle MHC isoforms through regulated alternative RNA splicing. Genomic and cDNA sequence analyses revealed that this 21-kilobase MHC gene encodes these MHC isoforms in 19 exons. However, five sets of these exons, encoding portions of the S1 head and the hinge domains of the MHC protein, are tandemly repeated as two, three, four, or five divergent copies, which are individually spliced into RNA transcripts. RNA hybridization studies with exon-specific probes showed that at least 10 of the 480 possible MHC isoforms that could arise by alternative RNA splicing of these exons are expressed as MHC transcripts and that the expression of specific members of alternative exon sets is regulated, both in stage and in muscle-type specificity. This regulated expression of specific exons is of particular interest because the alternatively spliced exon sets encode discrete domains of the MHC protein that likely contribute to the specialized contractile activities of different Drosophila muscle types. The alternative exon structure of the Drosophila MHC gene and the single-copy nature of this gene in the Drosophila genome make possible transgenic experiments to test the physiological functions of specific MHC protein domains and genetic and molecular experiments to investigate the mechanisms that regulate alternative exon splicing of MHC and other muscle gene transcripts.

Amino Acid Sequence↗

PubMed1991

The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA.

Vascular endothelial growth factor (VEGF) was recently identified as a secreted, direct-acting mitogen specific for vascular endothelial cells and capable of stimulating angiogenesis in vivo. Molecular cloning revealed multiple forms of VEGF, apparently arising from alternative splicing of its RNA transcript. We have examined various human cDNA libraries by the polymerase chain reaction technique and discovered a fourth molecular form, VEGF206. This form contains a 41-amino acid insertion relative to the most abundant form, VEGF165, and includes the highly basic 24-amino acid insertion found in VEGF189. Southern blot analysis revealed that a single gene encoded these various forms, and nucleic acid sequence analysis of a portion of the VEGF gene revealed an intron/exon structure compatible with alternative splicing of RNA as a mechanism for their generation. Transient transfection of human embryonic kidney 293 cells showed that, like VEGF189, VEGF206 was predominately cell-associated and only very poorly secreted despite the presence of the signal peptide identical to that found in VEGF121 and VEGF165, both of which are efficiently exported from the cell. Vascular permeability activity was detected in the medium of 293 cells transfected with all four forms of VEGF; however, endothelial cell mitogenic activity was apparent only with VEGF121 and VEGF165. Thus, alternative splicing of VEGF RNA can produce four polypeptides with strikingly different secretion patterns, which suggests multiple physiological roles for this family of proteins.

Amino Acid Sequence↗

PubMed2000

Identification of alternative splicing and negative splicing activity of a nonsegmented negative-strand RNA virus, Borna disease virus.

Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus that belongs to the Mononegavirales. Unlike other animal viruses of this order, BDV replicates and transcribes in the nucleus of infected cells. Previous studies have shown that BDV uses RNA splicing machinery for its mRNA expression. In the present study, we identified spliced RNAs that use an alternative 3' splice site, SA3, in BDV-infected cell lines as well as infected animal brain cells. Transient transfection analysis of cDNA clones of BDV RNA revealed that although SA3 is a favorable splice site in mammalian cells, utilization of SA3 is negatively regulated in infected cells. This negative splicing activity of the SA3 site is regulated by a putative cis-acting region, the exon splicing suppressor (ESS), within the polymerase exon of BDV. The BDV ESS contains similar motifs to other known ESSs present in viral and cellular genes. Furthermore, our results indicated that a functional polyadenylation signal just upstream of the BDV ESS is also involved in the regulation of alternative splicing of BDV. These observations represent the first documentation of complex RNA splicing in animal RNA viruses and also provide new insight into the mechanism of regulation of alternative splicing in animal viruses.

Alternative Splicing↗