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At least 685 records · Page 38Linked to original sources
PubMed2006

Variants in the human insulin gene that affect pre-mRNA splicing: is -23HphI a functional single nucleotide polymorphism at IDDM2?

Predisposition to type 1 diabetes and juvenile obesity is influenced by the susceptibility locus IDDM2 that includes the insulin gene (INS). Although the risk conferred by IDDM2 has been attributed to a minisatellite upstream of INS, intragenic variants have not been ruled out. We examined whether INS polymorphisms affect pre-mRNA splicing and proinsulin secretion using minigene reporter assays. We show that IVS1-6A/T (-23HphI+/-) is a key INS variant that influences alternative splicing of intron 1 through differential recognition of its 3' splice site. The A allele resulted in an increased production of mature transcripts with a long 5' leader in several cell lines, and the extended mRNAs generated more proinsulin in culture supernatants than natural transcripts. The longer mRNAs were significantly overrepresented among beta-cell-expressed sequenced tags containing the A allele as compared with those with T alleles. In addition, we show that a rare insertion/deletion polymorphism IVS1+5insTTGC (IVS-69), which is exclusively present in Africans, activated a downstream cryptic 5' splice site, extending the 5' leader by 30 bp. These results indicate that -23HphI and IVS-69 are the most important INS variants affecting pre-mRNA splicing and suggest that -23HphI+/- is a common functional single nucleotide polymorphism at IDDM2.

Alternative Splicing↗
PubMed2005

Polymorphism of the untranslated regions of the F3/contactin mRNA in the rat nervous system.

F3/contactin is a neural adhesion molecule implicated in various physiological processes. In rat brain tissues, we cloned various mRNA with the same coding region but differing in 3' and 5'UTR. The 3'UTR presents two polyadenylation signals. At the 5' end, we identified two leader exons, multiple transcription initiation sites and splicing events, leading to at least 19 different 5'UTR. The F3/contactin rat gene differs from the mouse gene for two reasons: (1) it contains two additional untranslated exons that are alternatively spliced and (2) it lacks the homologue mouse untranslated exon 0.

Alternative Splicing↗
PubMed1994

Altered proteoglycan gene expression and the tumor stroma.

Tumor stroma is a specialized form of tissue that is associated with epithelial neoplasms. Recent evidence indicates that significant changes in proteoglycan content occur in the tumor stroma and that these alterations could support tumor progression and invasion as well as tumor growth. Our main hypothesis is that the generation of tumor stroma is under direct control of the neoplastic cells and that, via a feedback loop, altered proteoglycan gene expression would influence the behavior of tumor cells. In this review, we will focus primarily on the work from our laboratory related to the altered expression of chondroitin sulfate proteoglycan and its role in tumor development and progression. The connective tissue stroma of human colon cancer is enriched in chondroitin sulfate and the stromal cell elements, primarily colon fibroblasts and smooth muscle cells, are responsible for this biosynthetic increase. These changes can be reproduced in vitro by using either tumor metabolites or co-cultures of human colon carcinoma cells and colon mesenchymal cells. The levels of decorin, a leucine-rich proteoglycan involved in the regulation of matrix assembly and cell proliferation, are markedly elevated in the stroma of colon carcinoma. These changes correlate with a marked increase in decorin mRNA levels and a concurrent hypomethylation of decorin gene, a DNA alteration associated with enhanced gene expression. Elucidation of decorin gene structure has revealed an unexpected degree of complexity in the 5' untranslated region of the gene with two leader exons that are alternatively spliced to the second coding exon. Furthermore, a transforming growth factor beta (TGF-beta)-negative element is present in the promotor region of decorin gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Alternative Splicing↗
PubMed2002

Discontinuous and non-discontinuous subgenomic RNA transcription in a nidovirus.

Arteri-, corona-, toro- and roniviruses are evolutionarily related positive-strand RNA viruses, united in the order Nidovirales. The best studied nidoviruses, the corona- and arteriviruses, employ a unique transcription mechanism, which involves discontinuous RNA synthesis, a process resembling similarity-assisted copy-choice RNA recombination. During infection, multiple subgenomic (sg) mRNAs are transcribed from a mirror set of sg negative-strand RNA templates. The sg mRNAs all possess a short 5' common leader sequence, derived from the 5' end of the genomic RNA. The joining of the non-contiguous 'leader' and 'body' sequences presumably occurs during minus-strand synthesis. To study whether toroviruses use a similar transcription mechanism, we characterized the 5' termini of the genome and the four sg mRNAs of Berne virus (BEV). We show that BEV mRNAs 3-5 lack a leader sequence. Surprisingly, however, RNA 2 does contain a leader, identical to the 5'-terminal 18 residues of the genome. Apparently, BEV combines discontinuous and non-discontinuous RNA synthesis to produce its sg mRNAs. Our findings have important implications for the understanding of the mechanism and evolution of nidovirus transcription.

Base Sequence↗
PubMed1982

Genetic analysis of the processing of a spliced tRNA.

We analyzed the effect of 18 single nucleotide changes on the processing of the transcripts produced by cloned yeast tRNATyr genes after microinjection into the nucleus of living Xenopus oocytes. The processing step most easily blocked by mutation is the early maturation of the 5' and 3' termini of the tRNATyr primary transcript, involving removal of 5'-leader and 3'-trailer sequences and CCA addition. The enzymes seem to recognize the whole tRNA cloverleaf structure since mutations in all regions of the molecule can stop processing. Mutations that affect splicing of the 92-nucleotide precursor (which has mature ends but still contains the intervening sequence, and is the normal substrate for the splicing enzymes), are located in the vicinity of the intervening sequence. Base modification enzymes that add pseudouridine, 1-methyladenosine and 5-methylcytosine appear rather insensitive to changes in secondary and tertiary structure of early transcripts in the 16 mutants examined. These enzymes may recognize only limited regions of the precursor RNA. RNA polymerase III behaves as if able to count the number of Us added before termination; and aberrant termination products in two mutants suggest that the secondary structure of the nascent transcript can be very imortant in eukaryotic transcription termination.

Animals↗
PubMed2003

Rev binds specifically to a purine loop in the SL1 region of the HIV-1 leader RNA.

The leader RNA sequence of human immunodeficiency virus type 1 (HIV-1) consists of a complex series of stem loop structures that are critical for viral replication. Three-dimensional structural analysis by NMR of one of these structures, the SL1 stem loop of the packaging signal region, revealed a highly conserved purine rich loop with a structure nearly identical to the Rev-binding loop of the Rev response element. Using band-shift assays, surface plasmon resonance, and further NMR analysis, we demonstrate that this loop binds Rev. HIV-1 appears to have a second Rev-binding site close to the major splice donor site that may have an additional role in the viral life cycle.

5' Untranslated Regions↗
PubMed1987

A reiterated leader sequence is present in polyomavirus late transcripts produced by a transformed rat cell line.

In cells transformed by polyomavirus, the viral genome is integrated into the host DNA, and in the absence of excision, viral gene expression is limited to the early region. We report here that the ability of a unique transformed rat cell line, designated SS1A, to produce readily detectable levels of late mRNAs is due to rearrangements of the integrated viral sequences. The structure of the SS1A insertion, resulting from amplification and deletion events, allows for the formation of a primary late transcript that can subsequently be spliced to generate a reiterated leader attached to the body of the late mRNA coding sequences. The presence of transcripts containing such a leader was confirmed by sequencing the 5' end of cDNA copies of late mRNAs isolated from a library constructed with SS1A mRNA. These results suggest that a reiterated leader sequence is necessary to stabilize late mRNA.

Animals↗
PubMed1993

Occurrence of alternatively spliced leader-delta onc-poly(A) transcripts in chicken neuroretina cells infected with Rous-associated virus type 1: implication in transduction of the c-mil/c-raf and c-Rmil/B-raf oncogenes.

We previously reported that serial passaging of Rous-associated virus type 1 in nondividing chicken embryo neuroretina cells leads to reproducible generation of acutely mitogenic retroviruses that transduced the catalytic domain of c-mil/c-raf or c-Rmil/B-raf. On the basis of structural analysis of several retroviruses, we proposed that the early step of oncogene transduction is the constitution of alternatively spliced leader-delta onc-poly(A) transcripts. Here, we show that neuroretina cells do synthesize hybrid leader-delta mil and leader-delta Rmil RNAs and that these RNAs exhibit mitogenic properties and serve as templates for the generation of transducing retorviruses.

Alternative Splicing↗
PubMed2002

A maize r1 gene is regulated post-transcriptionally by differential splicing of its leader.

Anthocyanin biosynthesis in Zea mays is controlled by regulatory genes of the r1/b1 family that encode bHLH transcription factors. Analysis of the 381 nucleotide leader sequence of a member of this family, Sn, discloses the presence of five ATG triplets upstream of the coding region and three upstream open reading frames (uORFs) of 38, 15 and 13 amino acids respectively. RT-PCR studies revealed that a splicing event occurs in the leader region in the different tissues tested. Splicing deletes 146 nucleotides which include uORF2 and uORF3. By trans-activation experiments in maize protoplasts we find that the spliced leader, compared to the non-spliced one, reduces the number of pigmented protoplasts by four-fold. We suggest a multilevel regulation of the Sn transcription factor acting not only at the transcriptional but also at the post-transcriptional level.

5' Untranslated Regions↗
PubMed1996

Cloning and expression of DiT33 from Dirofilaria immitis: a specific and early marker of heartworm infection.

Dirofilaria immitis is an important filarial parasite of dogs and cats, and a useful model for human filariasis. Current diagnostic tests for heartworm infection in animals rely on the presence of fecund female worms (usually found 6.5 months post-infection or later) and therefore fail to detect pre-patent infections. Putative pepsin inhibitors from 2 filarial parasites of humans namely Onchocerca volvulus (Ov33, Oc3.6, OvD5B) and Brugia malayi (Bm33), have been shown to be useful in diagnosis of onchocerciasis and lymphatic filariasis, respectively. Previous studies have suggested that a homologue exists in D. immitis (DiT33), which may have potential in diagnosis of heartworm infection. In this study, the isolation and characterization of a cDNA clone encoding DiT33 is described. This cDNA contains 12 bases of the nematode-specific 22 nucleotide spliced leader sequence and encodes a 26.4 kDa-protein with a high level of similarity (87-89%) to other filarial members of the family. DiT33 was over-expressed in E. coli as a fusion with the maltose-binding protein and serological analysis was performed using a panel of clinically defined dog sera. The findings of this study indicate that DiT33 is a promising antigen for the early detection of D. immitis and may be a valuable tool in the control and management of heartworm infection.

Amino Acid Sequence↗
PubMedDate not supplied

Structure of three spliced mRNAs from region E3 of adenovirus type 2.

A cDNA library representing early adenovirus type 2 (Ad2) mRNA was constructed. The cDNA copies were inserted into the PstI cleavage site of the pBR322 plasmid, and clones containing sequences from region E3 of the Ad2 genome were identified by colony hybridization. Selected clones were characterized by restriction enzyme cleavage, hybridization, and partial DNA sequence analysis. The precise structure of three spliced mRNAs was established by comparing the results with the DNA sequence of region E3 from Ad2 (Herissé et al., Nucl. Acids Res. 8 (1980) 2173--2191; Herissé and Galibert, Nucl. Acids Res. 9 (1981) 1229--1249). One of the characterized mRNA species encodes the E3/19K glycoprotein, whereas the other two most likely encode the E3/14K protein. The results demonstrate, moreover, that certain splice points which are used to generate the major E3 mRNAs are also used to splice the supplementary leader segments to the fibre mRNA at late times after infection. Two separate poly(A)-addition sites were identified in region E3 by analysis of the cDNA clones; one is preceded by the hexanucleotide sequence AAUAAA, whereas the other is preceded by an altered hexanucleotide, having the sequence AUUAAA.

Adenoviruses, Human↗
PubMed1986

Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control.

Avian retroviral mRNAs contain a long 5' untranslated leader of approximately 380 nucleotides. The leader includes sequences required for viral replication and three AUG codons which precede the AUG codon used for translational initiation of the gag and env genes. We have used sensitive, quantitative assays of viral gene transcription and translation to analyze the role of this mRNA leader in viral gene expression. By substituting segments from related viruses, we had previously shown that the endogenous avian provirus ev-1 contained a defective leader segment (B. R. Cullen, A. M. Skalka, and G. Ju, Proc. Natl. Acad. Sci. USA 80:2946-2950, 1983). The sequence analysis presented here, followed by comparison with the nondefective ev-2 endogenous provirus segment, identified the critical changes at nucleotides 4 and 7 upstream of the initiator AUG. These differences do not alter the most conserved nucleotides within the consensus sequence which precedes eucaryotic initiation codons, but lie within a nine-nucleotide region that is otherwise highly conserved among avian retrovirus strains. Analysis of a series of deletion mutants indicated that other sequences within the leader are also required for efficient expression. Characterization of the altered transcripts demonstrated that the presence of the defective ev-1 segment or the deletion of a ca. 200-nucleotide leader segment did not affect the steady-state level or splicing efficiency of these mRNAs. Thus, we conclude that the reduced expression of these mRNAs is due to a translational deficiency. These results indicate that specific leader sequences, other than the previously identified consensus nucleotides which precede eucaryotic AUG initiator codons, can influence eucaryotic gene translation.

Animals↗
PubMed1995

trans-splicing: an update.

5'-end maturation of messenger RNAs via acquisition of a trans-spliced leader sequence occurs in several primitive eukaryotes, some of which are parasitic. This type of trans-splicing proceeds though a two-step reaction pathway directly analogous to that of cis-splicing and like cis-splicing it requires multiple U snRNP cofactors. This minireview attempts to provide a brief synopsis of our current understanding of the evolution and biological significance of trans-splicing. Progress in deciphering the mechanism of trans-splicing, particularly as it relates to current models of cis-splicing, is also discussed.

Animals↗
PubMed1985

Exon mutations that affect the choice of splice sites used in processing the SV40 late transcripts.

The spliced species of late SV40 RNAs present in the cytoplasm of cells infected with various wild-type and mutant strains of SV40 that differ in their leader regions were determined using a novel modification of the primer extension method and the S1 nuclease mapping technique. These data indicated that mutations within the first exon of the late RNAs can affect dramatically the utilization of downstream donor and acceptor splice sites. In one instance, a ten base pair insertion within the predominant first exon increased utilization of an infrequently utilized donor splice site such that the small alteration became part of an intervening sequence, thereby suggesting a novel mechanism for regulation of gene expression. In addition, our method enabled detection of a previously unidentified spliced species, representing less than one percent of the SV40 late 19S RNA present in cells infected with wild-type virus, that may be an intermediate in the synthesis of a known doubly spliced 16S RNA species of SV40.

Animals↗
PubMed2006

A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in Chlamydomonas reinhardtii.

In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont.

Algal Proteins↗
PubMed1993

Differential regulation of transcription initiation from insulin-like growth factor-I (IGF-I) leader exons and of tissue IGF-I expression in response to changed growth hormone and nutritional status in sheep.

In the mammalian insulin-like growth factor-I (IGF-I) gene, exons 1 and 2 are differentially spliced to exon 3 producing alternate class 1 and class 2 transcripts. The aim of this study was to investigate the tissue expression of these leader exons in lambs growing at different rates as a result of chronic manipulation of nutritional and GH status. Riboprobes were developed so that leader exon-specific and total IGF-I gene expression could be determined using a single RNase protection assay. Lambs were fed a diet containing high or low protein content either ad libitum or at a restricted intake; within these dietary groups they were treated with either saline or GH for 10 weeks. Total hepatic IGF-I messenger RNA (mRNA) transcripts were significantly increased by GH (P = 0.004), protein (P = 0.002), and energy (P < 0.001) status as were circulating IGF-I concentrations (GH, P < 0.001; protein, P = 0.026; energy, P < 0.001). Exons 1 and 2 were expressed in liver but to a variable extent. Increased dietary energy and protein induced increased expression from both class 1 and 2 transcripts, but the percent increases was at least 5-fold greater for class 2 than for class 1 mRNA. GH treatment only stimulated significant increases in expression from class 2 transcripts. In the low protein, energy-restricted, saline-treated lambs exon 1 transcripts accounted for approximately 70% of total class 1 and 2 transcripts, and this proportion declined significantly as class 1 and 2 transcripts, and this proportion declined significantly as GH and nutritional status increased to only 30% in the high protein, ad libitum-fed, GH-treated animals; class 2 transcripts therefore displayed the opposite pattern of expression. These data indicate that exon 2 may be far more sensitive than exon 1 in intact animals which have been stimulated within normal physiological limits. Muscle IGF-I gene expression was at least 20-fold less than that for the liver and consisted mainly of class 1 transcripts. Muscle total IGF-I mRNA was insensitive to changed nutritional status or to GH treatment, even though significant increases in muscle growth occurred in response to ad libitum intake and GH, indicating that hepatically derived endocrine IGF-I could have a role in the regulation of muscle growth.

Animal Nutritional Physiological Phenomena↗
PubMed1995

Male-specific lethal 2, a dosage compensation gene of Drosophila, undergoes sex-specific regulation and encodes a protein with a RING finger and a metallothionein-like cysteine cluster.

In Drosophila the equalization of X-linked gene products between males and females, i.e. dosage compensation, is the result of a 2-fold hypertranscription of most of these genes in males. At least four regulatory genes are required for this process. Three of these genes, maleless (mle), male-specific lethal 1 (msl-1) and male-specific lethal 3 (msl-3), have been cloned and their products have been shown to interact and to bind to numerous sites on the X chromosome of males, but not of females. Although binding to the X chromosome is negatively correlated with the function of the master regulatory gene Sex lethal (Sxl), the mechanisms that restrict this binding to males and to the X chromosome are not yet understood. We have cloned the last of the known autosomal genes involved in dosage compensation, male-specific lethal 2 (msl-2), and characterized its product. The encoded protein (MSL-2) consists of 769 amino acid residues and has a RING finger (C3HC4 zinc finger) and a metallothionein-like domain with eight conserved and two non-conserved cysteines. In addition, it contains a positively and a negatively charged amino acid residue cluster and a coiled coil domain that may be involved in protein-protein interactions. Males produce a msl-2 transcript that is shorter than in females, due to differential splicing of an intron of 132 bases in the untranslated leader. Using an antiserum against MSL-2 we have shown that the protein is expressed at a detectable level only in males, where it is physically associated with the X chromosome. Our observations suggest that MSL-2 may be the target of the master regulatory gene Sxl and provide the basic elements of a working hypothesis on the function of MSL-2 in mediating the 2-fold increase in transcription that is characteristic of dosage compensation.

Amino Acid Sequence↗
PubMed2002

Unique features of fowl adenovirus 9 gene transcription.

We examined the transcriptional organization of fowl adenovirus 9 (FAdV-9) and analyzed temporal transcription profiles of its early and late mRNAs. At least six early and six late transcriptional regions were identified for FAdV-9. Extensive splicing was observed in all FAdV-9 early transcripts examined. Sequence analysis of the cDNAs representing the early proteins identified untranslated leader sequences, precise locations of splice donor and acceptor sites, as well as polyadenylation signals and polyadenylation sites. A unique characteristic, compared to other adenoviruses, was the detection by RT-PCR of multiple transcripts specific for each of five late genes (protein III, pVII, pX, 100K, and fiber), suggesting that FAdV-9 late transcripts undergo more extensive splicing than reported for other adenoviruses.

Adenovirus Early Proteins↗