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PubMed1978

The spliced structures of adenovirus 2 fiber message and the other late mRNAs.

All late adenovirus 2 messenger RNAs, except perhaps that for peptide IX, are composite molecules with sequences derived from at least two to five or more separate portions of the genome, as determined by electron microscopic analyses of polysomal RNA hybridized to single- or double-stranded DNA. We previously reported that eight different rightward-transcribed mRNAs contain, at their 5' ends, a common three-part leader sequence derived from coordinates 16.6, 19.6 and 26.6, and that a ninth mRNA consists of sequences from 4.6--6.1 linked directly to those from 9.7--11.0 (Chow et al., 1977a; revised coordinates). We now demonstrate that four additional RNA species also have the same tripartite leader joined to messages with 5' ends located at coordinates at 29.1, 30.5, 33.9 and 42.8. The RNA transcript extending from 29.1--39.0 covers most or all of the viral associated (VA) RNA genes (29.0--30.1). The late RNAs can be grouped into several families of transcripts in which two or three mRNAs have the same 3' end and 3' proximal sequences, but differ substantially in length at the 5' end and, therefore, the coordinate at which the leader sequences are attached to the main body of the message. The number of RNA species transcribed from the Ad2 chromosome between coordinates 29 and 50 exceeds the number of identified Ad2 proteins. Several pairs of messages differing in length by a few percent may thus encode the same protein or have a precursor-product relationship. We have also found that a subpopulation of the polysomal mRNAs for the fiber protein, the most distal gene (86.3--91.2) in the rightward transcription unit, has a fourth leader component "y" (78.6--79.1) in addition to the common tripartite leader sequences. Less frequent fiber RNA species have fifth "x" (76.9--77.3) and/or sixth "z" (84.7--85.1) leader segments. Transcripts with x, y or z segments might be processing intermediates, and it is possible to arrange these species into an array suggestive of multiple pathways for the maturation of fiber mRNA. mRNA transcribed at late times from the L strand between coordinates 15.0 and 11.0 has a single component leader located 200 bases from the main body of the gene from coordinate 16.1 to 15.7. This leader originates from a site very close to the first leader component (16.5--16.6) of the R strand messages and suggests the presence of adjacent L and R strand promoters from which transcription diverges. Taken together, these results emphasize the diversity of splicing patterns and the variety of recombined sequences generated during the synthesis of late Ad2 mRNAs.

Adenoviruses, Human↗
PubMed1993

Complete cDNA sequence of a rainbow trout IgM gene and evolution of vertebrate IgM constant domains.

A complete cDNA clone encoding secreted IgM molecules was isolated from a spleen cDNA library of rainbow trout (Oncorhynchus mykiss). It encodes the leader peptide, VH, DH, JH, and the whole constant region of the secreted IgM molecule. We also studied the copy number of the IgM gene and an unusual RNA splicing mechanism which generates membrane IgM lacking the CH4 domain in rainbow trout. Amino acid sequence comparison of IgM protein sequences from this fish and other vertebrates indicates that some domains of IgM have evolved at a relatively constant rate. The evolution of salmonid fish including divergence time is discussed.

Amino Acid Sequence↗
PubMed1990

Characterization of a 5'-flanking region of the human liver/bone/kidney alkaline phosphatase gene: two kinds of mRNA from a single gene.

We have cloned the human liver/bone/kidney alkaline phosphatase (ALPL) gene using a liver-type ALPL cDNA as a probe. The gene is divided into 12 exons, and is likely to exist as a single copy in haploid genome. As compared with the gene isolated using a bone-type ALPL cDNA (Weiss et al., J. Biol. Chem. 263, 12002-12010, 1988), another leader exon specific for the liver-type ALPL mRNA was assigned about 3.4 kb upstream from exon 2 and the alternative splicing in the first exon was indicated. RNA blot analysis showed that three species of mRNA of 2.5, 4.1 and 4.7 kilobases were detected in liver and developmentally regulated.

Alkaline Phosphatase↗
PubMed1988

The tropomyosin mRNAs of mouse striated muscles: molecular cloning of beta-tropomyosin.

The nucleotide sequence corresponding to the complete coding region and much of the 5' and 3' untranslated regions of the skeletal muscle-specific mouse beta-tropomyosin mRNA was determined from overlapping cDNA clones isolated from a library of recombinants in pBR322. When one of these was used as a probe to detect tropomyosin mRNAs expressed after fusion of mouse myoblasts in culture, species were detected in addition to those corresponding to known tropomyosins of striated muscle. One of these was also detected in the leg muscle of 12 day-old mice. The identities of these species are uncertain, but they may correspond to alternatively spliced products of the same RNA transcripts that give rise to the predominant tropomyosin isoforms of striated muscle.

Amino Acid Sequence↗
PubMed1988

Immunoglobulin VH genes are transcribed by T cells in association with a new 5' exon.

We previously detected mRNAs in a number of human T cell lines with a probe from within the Ig VH gene locus. We now show these mRNAs consist of Ig VH genes expressed in T cells. In one human T cell line, two RNA species have been studied and found to come from transcripts of unrearranged VH segments in which the leader exon, normally associated with VH transcripts in B cells, is replaced by a novel 5' exon (ET) not encoding a hydrophobic leader peptide. In genomic DNA, this new ET exon is adjacent to a pseudo-VH gene that has not been observed in mature mRNA. This implies that RNA splicing controls association of the new exon with the expressed VH segments. Hence, VH transcription does indeed occur in T cells, but is qualitatively different from that in B cells.

Amino Acid Sequence↗
PubMed1982

Assembly of the mitochondrial membrane system. Processing of the apocytochrome b precursor RNAs in Saccharomyces cerevisiae D273-10B.

The DNA sequence of the apocytochrome b gene in Saccharomyces cerevisiae D273-10B contains two intervening sequences (Nobrega, F. G., and Tzagoloff, A. (1980) J. Biol. Chem. 255, 9828-9837). The exon-intron boundaries of the gene have been determined in this study from the sequence of the DNA which was copied from the mRNA. A protein of 385 amino acid residues is predicted from the 1155-nucleotide long coding regions. Northern blot analysis of total mitochondrial RNA, probed with restriction fragments from both exon and intron regions of the gene, reveals a 4.3-kilobase (kb) transcript containing both introns and two partially spliced intermediates, one (2.9 kb) lacking the first intron and the other (3.6 kb) lacking the second intron. The most abundant transcript (2.1 kb) hybridizes only to exon probes and is presumed to the fully spliced mRNA. S1 nuclease mapping of the purified mRNA indicates existence of two separate RNAs with identical 3' termini but differing by approximately 217 nucleotides at their 5' ends. The larger transcript has a 950-nucleotide nontranslated leader. Analyses of the RNA species present in various rho- and mit- mutants indicate that: 1) exon mutants process both introns, albeit not as efficiently as wild type, 2) intron mutants blocked in the excision of the first or second intron are capable of processing the alternate intron, suggesting a non-obligatory order of excision of the two intervening sequences, and 3) excision of the second intron occurs in rho- mutants and therefore does not require a mitochondrial translation product.

Base Sequence↗
PubMed1978

Nucleotide sequence at the junction between the coding region of the adenovirus 2 hexon messenger RNA and its leader sequence.

We have determined a 139-base-pair sequence of adenovirus 2 DNA that is located immediately leftwards of the cleavage site for endonuclease Sma I at position 51.1. The established sequence includes the hexon AUG initiator codon, located 75--77 nucleotides leftwards of this cleavage site, and codons for the first 26 amino acids of the hexon polypeptide. By the use of purified hexon mRNA as a template and separated strands of small restriction enzyme fragments as specific primers, the complete 5' noncoding region of the hexon mRNA was synthesized and part of its sequence was determined. The tripartite leader sequence of the hexon mRNA starts 39 nucleotides upstream from the initiator AUG triplet and the total length of the 5' noncoding part of the hexon mRNA was estimated to be 235 nucleotides. The sequence at the junction of the leader sequence permits the formation of secondary structures that may be of importance for the splicing reaction.

Adenoviruses, Human↗
PubMed1995

The length of 5'-untranslated leader sequences influences distribution of 3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA in polysomes: effects of lovastatin, oxysterols, and mevalonate.

Transcripts for hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase are heterogeneous in length. This heterogeneity is due to variations in the length of 5'-untranslated leader (UTL) sequences, which are generated by both alternate splicing within the first exon as well as alternate transcription start sites. Because mRNA 5'-UTL sequences have a role in regulating translational efficiency, the level and distribution of HMG-CoA reductase transcripts were measured in both total cellular RNA and polysomes from the Syrian hamster cell line C100. Cells were treated with either lovastatin alone, lovastatin and 25-hydroxycholesterol (25-OH C), or lovastatin, 25-OH C, and mevalonate, three treatment regimens used in an earlier study to demonstrate nonsterol-mediated translational control of HMG-CoA reductase synthesis [D. M. Peffley (1992) Somat. Cell Mol. Genet. 18, 19-32]. When reductase mRNA was measured by 5'-extension analysis under the same conditions, levels of transcripts with 5'-UTL regions ranging from 41 to 81 bases were reduced approximately four- to eightfold. In contrast, transcripts with 5'-UTL regions 93 to 100 bases in length were not reduced, and transcripts with 5'-UTL regions approximately 300-400 bases in length increased twofold. The addition of 25-OH C alone or both 25-OH C and mevalonate to lovastatin-treated cells lowered HMG-CoA reductase mRNA levels fivefold in total cellular RNA as determined by RNase protection assay. No comparable change was observed with control ribosomal protein S17 mRNA. Postmitochondrial supernatants representing both translationally inactive monosomes and translationally active polysomes were prepared by sucrose gradient fractionation from cells incubated with the standard three treatments. Because 5'-UTL sequences of many mRNAs have a role in regulating translational efficiency we isolated RNA from each fraction and measured levels of reductase transcripts by 5'-extension analysis. Under all three conditions, transcripts with 5'-UTL sequences 41-103 bases in length were primarily associated with dense sucrose fractions that contain polysomes. In contrast, reductase transcripts with leader sequences 300 to 400 bases were almost exclusively associated with the less dense sucrose fractions containing monosomes. These results indicate that both the level and polysome distribution of individual reductase transcripts are influenced by the length of 5'-UTL sequences.

Alternative Splicing↗
PubMed1992

Identification of a novel Brugia pahangi beta-tubulin gene (beta 2) and a 22-nucleotide spliced leader sequence on beta 1-tubulin mRNA.

We have examined the expression of beta-tubulin genes in the parasitic nematode, Brugia pahangi. A genomic library was constructed and screened by hybridization with a Haemonchus contortus beta-tubulin cDNA fragment which recognizes several B. pahangi beta-tubulin sequences, including sequences which correspond to the previously characterized beta 1-tubulin gene. The B. pahangi beta 2-tubulin gene was isolated by selecting clones which hybridize to the H. contortus beta-tubulin gene but which do not hybridize to the beta 1-tubulin gene. A partial sequence of the beta 2-tubulin gene confirms that it codes for a distinct beta-tubulin. Southern hybridization analyses show that the beta 2-tubulin sequence exists as a single copy gene within the B. pahangi genome. Expression of the beta 2-tubulin gene is developmentally regulated and the message is found predominantly in adult male worms, whereas the beta 1-tubulin gene is expressed in microfilariae and approximately equal levels of the transcript are found in male and female adult worms. During mRNA maturation the beta 1-tubulin mRNA of microfilariae and adult worms acquires a trans-spliced leader identical to the SL1 of Caenorhabditis elegans.

Amino Acid Sequence↗
PubMed2001

In vitro evidence for a long range pseudoknot in the 5'-untranslated and matrix coding regions of HIV-1 genomic RNA.

The 5'-untranslated leader region of human immunodeficiency virus type 1 (HIV-1) RNA contains multiple signals that control distinct steps of the viral replication cycle such as transcription, reverse transcription, genomic RNA dimerization, splicing, and packaging. It is likely that fine tuned coordinated regulation of these functions is achieved through specific RNA-protein and RNA-RNA interactions. In a search for cis-acting elements important for the tertiary structure of the 5'-untranslated region of HIV-1 genomic RNA, we identified, by ladder selection experiments, a short stretch of nucleotides directly downstream of the poly(A) signal that interacts with a nucleotide sequence located in the matrix region. Confirmation of the sequence of the interacting sites was obtained by partial or complete inhibition of this interaction by antisense oligonucleotides and by nucleotide substitutions. In the wild type RNA, this long range interaction was intramolecular, since no intermolecular RNA association was detected by gel electrophoresis with an RNA mutated in the dimerization initiation site and containing both sequences involved in the tertiary interaction. Moreover, the functional importance of this interaction is supported by its conservation in all HIV-1 isolates as well as in HIV-2 and simian immunodeficiency virus. Our results raise the possibility that this long range RNA-RNA interaction might be involved in the full-length genomic RNA selection during packaging, repression of the 5' polyadenylation signal, and/or splicing regulation.

5' Untranslated Regions↗
PubMed1989

Post-transcriptional allelic exclusion of two functionally rearranged T cell receptor alpha genes.

We cloned and sequenced T cell receptor (TCR) alpha and beta chain cDNA from a lambda gt10 library obtained from a murine I-Ak autoreactive helper T cell clone MS202. Two types of cDNA clones for the alpha chain and one for the beta chain were obtained. The two alpha chain transcripts used two different V alpha genes: V alpha 4, joined to J alpha 11.2; and V alpha 5, J alpha TA13. The four V alpha 4 cDNA clones obtained did not have a complete sequences, lacking the leader portion. The V alpha 4 genomic gene segment of MS202 was revealed to contain two exons corresponding to the V alpha 4.MD13 cDNA sequence, and the potential RNA splicing signals between the two exons were intact. Both of the alpha chain cDNA clones showed in-frame rearrangements. Immunoprecipitation of 125I-surface-labeled lysate of MS202 with anti-TCR antiserum and subsequent electrophonetic analyses indicated that only one of the alpha chain polypeptides was expressed on the cell surface. Thus, allelic exclusion of the alpha chain in MS202 is achieved by post-transcriptional regulation rather than rearrangements.

Alleles↗
PubMed2005

Alternative initiation and splicing in dicer gene expression in human breast cells.

INTRODUCTION: Dicer is a ribonuclease that mediates RNA interference both at the transcriptional and the post-transcriptional levels. Human dicer gene expression is regulated in different tissues. Dicer is responsible for the synthesis of microRNAs and short temporal (st)RNAs that regulate the expression of many genes. Thus, understanding the control of the expression of the dicer gene is essential for the appreciation of double-stranded (ds)RNA-mediated pathways of gene expression. Human dicer mRNA has many upstream open reading frames (uORFs) at the 5'-leader sequences (the nucleotide sequence between the 5'-end and the start codon of the major ORF), and we studied whether these elements at the 5'-leader sequences regulate the expression of the dicer gene. METHOD: We determined the 5'-leader sequences of the dicer mRNAs in human breast cells by 5'-RACE and S1-nuclease protection analysis. We have analyzed the functions of the 5'-leader variants by reporter gene expression in vitro and in vivo. RESULTS: We found that the dicer transcripts in human breast cells vary in the sequence of their 5'-leader sequences, and that alternative promoter selection along with alternative splicing of the 5'-terminal exons apparently generate these variations. The breast cell has at least two predominant forms of dicer mRNAs, one of which has an additional 110 nucleotides at the 5'-end. Sequence comparison revealed that the first 80 nucleotides of these mRNA isoforms are encoded by a new exon located approximately 16 kb upstream of the reported start site. There are 30 extra nucleotides added to the previously reported exon 1. The human breast cells studied predominantly express two 5'-leader variants of dicer mRNAs, one with the exons 2 and 3 (long form) and the other without them (short form). By reporter gene expression analysis we found that the exon 2 and 3 sequences at the 5'-leader sequences are greatly inhibitory for the translation of the mRNA into protein. CONCLUSION: Dicer gene expression in human breast cells is regulated by alternative promoter selection to alter the length and composition of the 5'-leader sequence of its mRNA. Furthermore, alternative splicing of its exon 2 and 3 sequences of their pre-mRNA creates a more translationally competent mRNA in these cells.

Alternative Splicing↗
PubMed1985

A 5' spliced leader is added in trans to both alpha- and beta-tubulin transcripts in Trypanosoma brucei.

The approximately 15 alpha- and 15 beta-tubulin genes of Trypanosoma brucei are arranged in a tandem array of alternating alpha- and beta-tubulin genes. We have examined the structure of mRNA transcripts from the tubulin gene family and have found at the 5' ends of both alpha- and beta-tubulin messages a 35-nucleotide spliced leader (SL) identical to that identified previously on the 5' ends of variant surface glycoprotein (VSG) mRNAs. No 35-nucleotide SL sequences were encoded in the tubulin intergenic regions; instead, the SL sequence apparently originates from small RNA transcripts encoded at sites that are unlinked to the tubulin genes. The tandem arrangement of the tubulin genes and the presence of the SL at the 5' end of both alpha- and beta-tubulin transcripts establishes that RNA joining between the SL and tubulin mRNA occurs in trans.

Animals↗
PubMed1986

The Drosophila melanogaster actin 5C gene uses two transcription initiation sites and three polyadenylation sites to express multiple mRNA species.

At least six mRNAs are made from the Drosophila melanogaster act5C gene. We investigated the structures of these RNAs in detail and determined that they are heterogeneous at both their 5' and 3' ends. At the 5' end there were two nonhomologous leader exons which were alternately spliced to the remainder of the gene. These leader exons mapped to 1.7 and 0.7 kilobases, respectively, upstream of a common splice acceptor site which was eight base pairs 5' to the translation initiator AUG. Exon 1 is 147 bases in length, while exon 2 is 111 bases. A consensus TATA sequence was found roughly 30 base pairs upstream from exon 1, but none was found in the analogous position upstream of exon 2. The transcript length diversity arose principally from the use of three polyadenylation sites. This gave rise to RNA molecules with 3'-untranslated regions of roughly 375, 655, and 945 base pairs. With two start sites and three termination sites, this gene has the potential to produce six different transcripts. All six possible transcripts were present in whole fly mRNA. Transcripts containing the two different leader exons were found in roughly the same relative quantities through development. In contrast, the various 3' ends were differentially represented through development.

Actins↗
PubMed1992

Nucleotide sequence of prothrombin gene in abnormal prothrombin-producing hepatocellular carcinoma cell lines.

A protein induced by vitamin K absence or antagonist II, PIVKA-II is synthesized in the liver and possesses a structure similar to prothrombin except that ten glutamic acid residues in amino-terminal Gla domain are not completely gamma-carboxylated and are functionally inactive. This protein can be detected in the plasma of patients with hepatocellular carcinoma (HCC) and used as a new tumor marker. To analyze the mechanism of PIVKA-II production in HCC tissue, the prothrombin gene of PIVKA-II-secreting HCC cell lines was sequenced to detect the mutation in the Gla domain and carboxylase recognition site of leader sequence located on exons I and II that may cause the inhibition of carboxylation. Exons I and II and donor and acceptor site of intron I of the prothrombin gene in two HCC cell lines, PLC/PRF/5 and huH-2, were analyzed by polymerase chain reaction (PCR), and the product was sequenced directly. In addition, RNA samples of these cell lines were used for complementary DNA synthesis, followed by PCR and sequencing. The nucleotide sequences of the Gla domain in both HCC cell lines were conserved. One nucleotide change was detected at nt.554 (adenine to guanine), but this did not influence the amino acid sequence. Splicing sites between exons I and II, the leader sequence of the precursor prothrombin, and protease target sites also were conserved as the reported prothrombin gene, and mutations reported for other des-gamma-carboxy coagulation factors were not detected. These results also were confirmed by DNA analysis of seven human fresh-frozen samples (three PIVKA-II-positive HCC samples and four control specimens). The mechanism of PIVKA-II production in HCC is still unclear, but it is not caused by mutation in the prothrombin gene.

Amino Acid Sequence↗
PubMed1982

Leader arrangement in the adenovirus fiber mRNA.

Four oligonucleotides, complementary to the adenovirus 2 (ad2) fiber mRNA were chemically synthesized and used as primers to study the 5' end of that messenger. The oligonucleotides which were 8, 10, 12, and 14 nucleotides long had a common 3'-terminal sequence, TAC, complementary to the initiating codon of the fiber mRNA. The 5'-32P-labeled oligonucleotides were hybridized to polyadenylated mRNA from ad2-infected cells and the primer was extended, using reverse transcriptase. When the resulting products were analyzed by polyacrylamide gel electrophoresis multiple distinct bands could be identified by autoradiography. The extension products were characterized by hybridization and sequence analysis. The most prominent band contained the well-known tripartite lead which is attached to many different late adenovirus mRNAs. In addition, four differently spliced leaders were characterized which, besides the three segments of the tripartite leader, contain one or two auxilliary segments. A kinetic analysis of the different leaders during lytic adenovirus infection shows no temporal variation.

Adenoviridae↗
PubMed1981

Structure and organization of the gene coding for the DNA binding protein of adenovirus type 5.

We have determined the nucleotide sequence of a region of adenovirus type 5 (Ad5) DNA located between map positions 61.7 and 71.4, which covers the gene form the 72 kD DNA binding protein (DBP) and the sequence encoding the amino-terminal part of the 100 kD protein. Sequence analysis of cDNA copies of DBP mRNA revealed the existence of two abundant species of spliced mRNA molecules. One species consists of two short leader sequences from positions 75.2 (67 and 68 nucleotides long) and 68.8 (77 nucleotides long), respectively, and the main body of the RNA molecules. The other species contains only the leader sequence from position 75.2 and the main body. The amino acid sequence of DBP is encoded entirely by a long open reading frame of 1587 nucleotides in the main body of DBP mRNA. From the nucleotide sequence of the DBP gene it can be derived that DBP contains 529 amino acid residues and has an actual molecular weight of 59,049 daltons. The sites of mutation in the mutants H5hr404 and H5ts125 were determined at the nucleotide level. Single nucleotide alterations were detected in H5hr404 and H5ts125 in the sequences corresponding to the amino-terminal part and the carboxy-terminal part of DBP, respectively. The implications of these mutations are discussed.

Adenoviruses, Human↗