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PubMed1995

PCR differential display identifies a rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine.

Neuronal plasticity associated with both short- and long-term administration of psychomotor stimulants involves alterations in specific patterns of gene expression. In order to screen for brain region specific mRNAs which are transcriptionally regulated by acute cocaine and amphetamine, PCR differential display was employed. This approach identified a previously uncharacterized mRNA whose relative levels in the striatum are induced four- to fivefold by acute psychomotor stimulant administration. Isolation and characterization of corresponding cDNA clones resulted in complete nucleotide sequence analysis, including prediction of the encoded protein product. Alternate polyA site utilization in the predicted 3' noncoding region results in the appearance of an RNA doublet, approximately 700 and 900 bases in length, following Northern analysis. A presumed alternate splicing event further generates diversity within the transcripts, and results in the presence or absence of an in-frame 39 base insert within the putative protein coding region. As a result, the predicted translation products are either 129 or 116 amino acids in length. A common hydrophobic leader sequence at the amino terminus is present within each predicted polypeptide, suggesting that the protein product is targeted for entry into the secretory pathway. Basal expression of the RNA doublet is limited to neuroendocrine tissues, further implying that the protein product plays a functional role in both neuronal and endocrine tissues.

Amino Acid Sequence↗
PubMed1979

Sequence analysis of adenovirus DNA: complete nucleotide sequence of the spliced 5' noncoding region of adenovirus 2 hexon messenger RNA.

The complete nucleotide sequence of the 5' noncoding region of the adenovirus 2 hexon messenger RNA has been established by sequence analysis of reverse transcripts. Such transcripts were generated by extension of specific single-stranded DNA primers with reverse transcriptase after hybridization to purified hexon mRNA. The total length of the 5' noncoding region was determined to be 240 nucleotides, of which the spliced tripartite leader sequence contributes 202 nucleotides including the terminal m7G. The sizes of the different segments of the tripartite leader were estimated by comparing the established mRNA sequence with the genomic sequences for the first and third leader segments, and were found to be 42 nucleotides for the first segment, 71 nucleotides for the second and 89 nucleotides for the third. The estimates are ambiguous, however, due to the presence of tandemly repeated sequences at both ends of the intervening sequence between the third leader segment and the body of the hexon mRNA. The sequence of the leader allows the formation of hydrogen-bonded interactions with the 3' end of 18S ribosomal RNA near the capped 5' end and also close to the initiator AUG.

Adenoviruses, Human↗
PubMed2005

The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation.

The untranslated leader RNA is the most conserved part of the human immunodeficiency virus type I (HIV-1) genome. It contains many regulatory motifs that mediate a variety of steps in the viral life cycle. Previous work showed that the full-length leader RNA can adopt two alternative structures: a long distance interaction (LDI) and a branched multiple-hairpin (BMH) structure. The BMH structure exposes the dimer initiation site (DIS) hairpin, whereas this motif is occluded in the LDI structure. Consequently, these structures differ in their capacity to form RNA dimers in vitro. The BMH structure is dimerization-competent, due to DIS hairpin formation, but also presents the splice donor (SD) and RNA packaging (Psi) hairpins. In the LDI structure, an extended RNA packaging (Psi(E)) hairpin is folded, which includes the splice donor site and gag coding sequences. The gag initiation codon is engaged in a long distance base pairing interaction with sequences in the upstream U5 region in the BMH structure, thus forming the evolutionarily conserved U5-AUG duplex. Therefore, the LDI-BMH equilibrium may affect not only the process of RNA dimer formation but also translation initiation. In this study, we designed mutations in the 3'-terminal region of the leader RNA that alter the equilibrium of the LDI-BMH structures. The mutant leader RNAs are affected in RNA dimer formation, but not in their translation efficiency. These results indicate that the LDI-BMH status does not regulate HIV-1 RNA translation, despite the differential presentation of the gag initiation codon in both leader RNA structures.

Base Sequence↗
PubMed1990

Diverse soybean actin transcripts contain a large intron in the 5' untranslated leader: structural similarity to vertebrate muscle actin genes.

Plant actins are encoded by complex and highly divergent multigene families. Despite the general lack of intron conservation in animal, fungal and protist actin genes, evidence is presented which indicates that higher plant actin genes have an untranslated leader exon with structural similarity to that found in vertebrate actin genes. All functional higher plant actin genes sequenced to date contain a potential intron acceptor site in the 5' untranslated region 10 to 13 nucleotides upstream of the initiator ATG. A leader specific cDNA probe hybridized to sequences over 1.0 kbp upstream from the coding region confirming the presence of an upstream exon. Primer extension of mRNA with gene-specific oligonucleotides was used to analyze the 5' untranslated exon and leader intron from four divergent soybean actin genes, SAc3, 4, 6 and 7. The 5' ends of all four mRNAs are heterogeneous. The consensus promoter elements of the SAc7 actin promoter were identified. Gene specific primer extension sequencing of actin mRNAs indicated that splicing of the 5' leader intron occurred at the predicted acceptor site in SAc6 and SAc7. The SAc6 and SAc7 5' untranslated exons are small (88-111 nt) and the leader introns are relatively large (844-1496 nt). The presence of an intron within the 5' RNA leader and an intron which splits a glycine codon at position 152 in all plant actin genes and all vertebrate muscle actin genes suggests that these structures may have been conserved due to a functional role in actin expression. The 5' regions of these two soybean actin genes contain many unusual features including (CT) repeats and long stretches of pyrimidine-rich DNA. The possible roles of the upstream exon/intron and the C + T-rich regions are discussed.

Actins↗
PubMed1983

Transcriptional organization of bovine papillomavirus type 1.

Multiple bovine papillomavirus type 1 (BPV-1)-specific polyadenylated RNA species in a BPV-1-infected bovine fibropapilloma were identified and mapped. All of the RNA species were transcribed from the same DNA strand of the BPV-1 genome. Five RNA species previously identified in BPV-1-transformed mouse cells were also present in the bovine fibropapilloma. These five species measured 1,050, 1,150, 1,700, 3,800, and 4,050 bases, mapped within the 69% transforming segment of the BPV-1 genome, and shared a 3' coterminus at 0.53 map units (m.u.). The 5' ends of the bodies of these distinct transcripts were located at ca. 0.03, 0.09, 0.34, 0.39, and 0.41 m.u. Additional polyadenylated RNA species not present in BPV-1-transformed mouse cells were specific for the BPV-1-infected bovine fibropapilloma and measured 1,700, 3,700, 3,800, 6,700, and 8,000 bases. These wart-specific species shared a 3' coterminus at 0.90 m.u. The 5' termini of the bodies of the 1,700- and 3,800-base species mapped at 0.71 and 0.42 m.u., respectively. Exonuclease VII analysis failed to reveal any internal splicing in these two species; however, the presence of small remote 5' leader sequences could not be ruled out. The 3,700-base species hybridized to DNA fragments from the 69% transforming segment as well as from the 31% nontransforming segment of the BPV-1 genome; however, this species was not precisely mapped. The 5' termini of the two largest RNA species (6,700 and 8,000 bases in size) were located at ca. 0.01 and 0.90 m.u., respectively. Since the 5' ends of these mapped adjacent to a TATAAA sequence which could possibly serve as an element of a transcriptional promoter, it is possible that one or both of these species represent nonspliced precursor RNA molecules.

Animals↗
PubMed1998

Nucleotide sequence, genome organization, and transcription map of bovine adenovirus type 3.

The complete DNA sequence of bovine adenovirus type 3 is reported here. The size of the genome is 34,446 bp in length with a G+C content of 54%. All the genes of the early and late regions are present in the expected locations of the genome. However, the late-region genes are organized into seven families, instead of five as they are in human adenovirus type 2. The deduced amino acid sequences of open reading frames (ORFs) in the late regions and early region 2 (E2) and for IVa2 show higher degrees of homology, whereas the predicted amino acid sequences of ORFs in the E1, E3, and E4 regions and the pIX, fiber, and 33,000-molecular-weight nonstructural proteins show little or no homology with the corresponding proteins of other adenoviruses. In addition, the penton base protein lacks the integrin binding motif, RGD, but has an LDV motif instead of an MDV motif. Interestingly, as in other animal adenoviruses, the virus-associated RNA genes appear to be absent from their usual location. Sequence analysis of cDNA clones representing the early- and late-region genes identified splice acceptor and splice donor sites, polyadenylation signals and polyadenylation sites, and tripartite leader sequences.

Amino Acid Sequence↗
PubMed1994

Synthesis of circular RNA in bacteria and yeast using RNA cyclase ribozymes derived from a group I intron of phage T4.

Studies on the function of circular RNA and RNA topology in vivo have been limited by the difficulty in expressing circular RNA of desired sequence. To overcome this, the group I intron from the phage T4 td gene was split in a peripheral loop (L6a) and rearranged so that the 3' half intron and 3' splice site are upstream and a 5' splice site and 5' half intron are downstream of a single exon. The group I splicing reactions excise the internal exon RNA as a circle (RNA cyclase ribozyme activity). We show that foreign sequences can be placed in the exon and made circular in vitro. Expression of such constructs (RNA cyclase ribozymes) in Escherichia coli and yeast results in the accumulation of circular RNA in these organisms. In yeast, RNA cyclase ribozymes can be expressed from a regulated promoter like an mRNA, containing 5' leader and 3' trailer regions, and a nuclear pre-mRNA intron. RNA cyclase ribozymes have broad application to questions of RNA structure and function including end requirements for RNA transport or function, RNA topology, efficacy of antisense or ribozyme gene control elements, and the biosynthesis of extremely long polypeptides.

Bacteriophage T4↗
PubMed1984

Altered mRNA splicing in monkey cells abortively infected with human adenovirus may be responsible for inefficient synthesis of the virion fiber polypeptide.

Messenger RNA encoding the fiber protein of the human adenovirus serotype 2 (Ad2) capsid is inefficiently translated in abortively infected African green monkey kidney cells. The amount of fiber mRNA present in the cytoplasm of abortively infected monkey cells is less than that in productively infected cells by a factor of 5-10 but synthesis of the fiber polypeptide is reduced by a factor of more than 100. Evidence from a variety of experiments indicates that the defect does not lie in the translational apparatus of the monkey cell but may best be explained by differences in the fiber messages made in abortively versus productively infected cells. Here we report that fiber mRNA isolated from abortively infected monkey cells is processed differently than that made in productively infected cells. Primer extension analysis of the 5' ends of fiber messages from several different productive and abortive infections shows a direct correlation between synthesis of the fiber polypeptide in vivo and the presence of the "x" and/or "y" ancillary leaders on messages encoding the fiber polypeptide. Of all the mRNAs encoded by the major late transcriptional unit of Ad2 only the fiber message can contain the x and y leaders, and the fiber protein is the only late Ad2 protein reported to be glycosylated. We speculate that these leader sequences play a role in the synthesis of this glycoprotein, as well as that of the Ad2 19-kilodalton glycoprotein encoded by early region 3, whose mRNA also contains the x and y leaders.

Adenoviruses, Human↗
PubMed1983

Structure and function of adenovirus DNA binding protein: comparison of the amino acid sequences of the Ad5 and Ad12 proteins derived from the nucleotide sequence of the corresponding genes.

The adenoviral DNA binding protein (DBP) is a multifunctional protein involved in DNA replication and gene expression. In order to investigate the relation between structure and function of DBP, the amino acid sequences of the serotypes 5 and 12 (Ad5 and Ad12) have been compared. The amino acid sequence of Ad5 DBP was previously established by nucleotide sequence analysis of the Ad5 DBP gene (W. Kruijer, F. M. A. Van Schaik, and J. S. Sussenbach, Nucl. Acids Res. 9, 4439-4457, 1981). In this study the analysis of the Ad5 DBP gene and adjacent regions by determination of the sequence of the first leader in late DBP mRNA's and the splice point between the tripartite leader and the main body of the mRNA encoding the 100-kDa protein has been extended. The nucleotide sequence of the Ad12 DBP gene is also described. From the nucleotide sequence and RNA mapping data of Ad12 DBP mRNA's (I. Saito, J. Sato H. Handa, K. Shiraki, and H. Shimojo, Virology 114, 379-398, 1981) the complete Ad12 DBP amino acid sequence could be deduced. Ad12 DBP contains 484 amino acids and has an actual Mr of 54,992. It is 45 amino acids shorter than Ad5 DBP. Comparison of the Ad12 and Ad5 DBP amino acid sequences shows that several longer deletions are present in the N-terminal 125 amino acid residues of Ad12 DBP. In contrast, only a single amino acid deletion and insertion is found in the C-terminal 359 amino acids of Ad12 DBP. The N- and C-terminal domains of Ad12 and Ad5 DBP are 45 and 80% homologous, respectively. This suggests that both domains of DBP are subjected to different evolutionary pressures. Analysis of various Ad5 mutants with an altered DBP gene, has indicated that the C-terminal domain is involved in DNA replication and early gene expression, while the N-terminal domain has a role in late gene expression in monkey cells. These results are discussed in relation to the structure and function of adenovirus DBP.

Adenoviruses, Human↗
PubMed2006

Identification of novel snRNA-specific Sm proteins that bind selectively to U2 and U4 snRNAs in Trypanosoma brucei.

In eukaryotes the seven Sm core proteins bind to U1, U2, U4, and U5 snRNAs. In Trypanosoma brucei, Sm proteins have been implicated in binding both spliced leader (SL) and U snRNAs. In this study, we examined the function of these Sm proteins using RNAi silencing and protein purification. RNAi silencing of each of the seven Sm genes resulted in accumulation of SL RNA as well as reduction of several U snRNAs. Interestingly, U2 was unaffected by the loss of SmB, and both U2 and U4 snRNAs were unaffected by the loss of SmD3, suggesting that these snRNAs are not bound by the heptameric Sm complex that binds to U1, U5, and SL RNA. RNAi silencing and protein purification showed that U2 and U4 snRNAs were bound by a unique set of Sm proteins that we termed SSm (specific spliceosomal Sm proteins). This is the first study that identifies specific core Sm proteins that bind only to a subset of spliceosomal snRNAs.

Amino Acid Sequence↗
PubMed1992

Cell-specific alternative RNA splicing of an FMRFamide gene transcript in the brain.

Individual neurons synthesize different peptide neurotransmitters and neuromodulators. In general, specificity is achieved by transcriptional regulation of neuropeptide-encoding genes. In Lymnaea, the FMRFamide and GDP/SDPFLRFamide neuropeptides are encoded by separate exons. Here we provide evidence that the two exons are part of the same gene and that in neurons expressing the gene the two exons are spliced onto a common upstream exon encoding a hydrophobic leader sequence. In addition, in situ hybridization data show that there is mutually exclusive cytoplasmic expression of each of the neuropeptide-encoding exons. Thus, differential neuropeptide synthesis is likely to be regulated by an alternative splicing mechanism. The cellular specificity of these splicing events is remarkable and suggests that cell-specific alternative splicing may be of major importance in establishing neuronal diversity in this system.

Animals↗
PubMed1985

Mitochondrial suppression of a yeast nuclear mutation which affects the translation of the mitochondrial apocytochrome b transcript.

We describe a mitochondrial suppressor mutation, which restores respiratory competence to the nuclear pet- -mutant MK2. This mutant lacks the message of the mitochondrial cob-gene and instead accumulates a partially spliced pre-mRNA which is not translated. Complete processing and translation of the cob-RNA is restored by a rearrangement of the mitochondrial DNA, leading to a fusion of the cob-coding sequences with the leader of oli1, the mitochondrial gene coding for subunit IX of the ATPase. We conclude that the nuclear gene affected in MK2 is essential to allow translation of transcripts which contain the cob-leader sequence.

Apoproteins↗
PubMed1991

Co-expression of full-length and truncated Ig mu-chains in human B lymphocytes results from alternative splicing of a single primary RNA transcript.

A survey of Ig synthesis among mu-heavy chain-producing human B cell lines indicated that roughly one-third co-express full length (mu+) and a particular type of truncated mu-chain (designated mu'). The relative molecular size of the intracellular form of this truncated mu-chain and its normal pattern of N-glycosylation suggested that mu'-chains were missing a single Ig domain at the protein level. Cell-free translation of polyA+ RNA from mu'-producing human B cell lines generated appropriate mu'-translation products, and Northern blot analysis demonstrated the presence of correspondingly truncated mu'-transcripts in these lines. These results pointed to a pretranslational basis for mu+/mu' co-expression. Sequencing of mu+- and mu'-cDNA clones from two human B cell lines showed that mu+- and mu'-transcripts derive from the same primary transcript, with mu'-mRNA formed by a direct leader-to-C mu 1 exon splice such that the heavy chain variable region exon is excluded via a cassette-type alternative splicing mechanism. Southern blot analysis of the rearranged Ig heavy chain genes in one B cell line confirmed that the co-expressed mu+- and mu'-mRNA derive from the same rearranged Ig heavy chain gene. mu'-cDNA clones were readily isolated from normal human bone marrow lymphocytes, whereas peripheral B cells do not appear to express mu'-transcripts. The frequent occurrence of mu'-mRNA in B cell lines, and its high relative expression in untransformed bone marrow lymphocytes attest to a mode of post-transcriptional control of Ig gene expression that may have implications for human B cell development.

B-Lymphocytes↗
PubMed2000

Mitochondrial aspartate aminotransferase: direction of a single protein with two distinct functions to two subcellular sites does not require alternative splicing of the mRNA.

During differentiation of mouse 3T3-L1 fibroblasts to an adipocyte phenotype, the mitochondrial isoform of aspartate aminotransferase accumulates on the plasma membrane. The determination of whether this reflects translation of an alternatively spliced message lacking the mitochondrial leader sequence required cloning of the enzyme's uncommon a allele, for which these cells are homozygous. The 1.4-kb cDNA sequence of the a allele was obtained from oligo-dT-primed reverse-transcriptase PCR products amplified from FVB mouse RNA. It differed from the b allele at only 2 bp and one amino acid. By contrast, gene-specific primers generated an additional 1.4-kb fragment that differed from the b allele by approximately 1% of nucleotides, encoding four amino acid substitutions. This sequence proved to represent a recently diverged processed pseudogene. The presence of such pseudogenes can complicate interpretation of expressed-sequence-tag data and single-nucleotide-polymorphism genotyping studies. Using probes derived from the a allele, RNase protection analyses indicated that only a single message for the enzyme was present in 3T3-L1 fibroblasts and adipocytes, despite differences in subcellular protein distribution.

3T3 Cells↗
PubMed1981

Identification of the gene and mRNA for the adenovirus terminal protein precursor.

The precursor of the 55K adenovirus terminal protein is an 87K protein that is covalently linked to viral DNA. This protein is likely to be identical to the 80,000 dalton protein described by Challberg et al. (1980). The mRNA for the 87K terminal protein precursor, like that for the E2-72K DNA binding protein, is detectable at both early and late times of infection, and its production is sensitive to protein synthesis inhibition (Lewis and Mathews, 1980). The 87K protein, together with proteins of 105,000 and 75,000 daltons, are translated from leftward transcribed (1-strand) messenger RNAs that are complementary to the viral genome between positions 11.2 and 31.5. Additional hybridization to the region between coordinates 37.3 and 41 suggests that the RNA body is spliced to sequences mapping farther right in the genome. Electron microscopic heteroduplex analysis has revealed a family of 1-strand RNAs that probably encode these proteins. The RNA bodies extend from coordinated 30, 26 and 23 to 11.1, with leaders at 39, 68.5 and 75 map units, defining a new adenovirus early region. These RNAs and region E2 RNAs share the first leader and presumably the same promoter, and may be coordinately expressed. Virions of the protease-deficient adenovirus 2 mutant ts1 grown at the restrictive temperature contain only the 87K form; when grown at the permissive temperature they contain both the 87K and 55K forms, and an additional 62K form; wild-type virions contain only the 55K form. Peptide analysis shows all these proteins to be related. The DNA-protein complex containing the 87K form is active as a template for viral DNA replication in vitro. This data supports a model of adenovirus DNA replication in which the 87K terminal protein precursor is the primary translation product and primes DNA synthesis. The 87K precursor is processed curing virus maturation to the 55K terminal protein, possibly via a 62K intermediate form, by the virus-specified Ad2ts1 protease.

Adenoviruses, Human↗
PubMed1999

Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system.

Lentiviruses infect both dividing and nondividing cells. In this study we characterized a lentiviral vector system consisting of a packaging vector (pHP) and a transducing vector (pTV) derived from a recombinant human immunodeficiency virus type 1 (HIV-1). In pHP, the long terminal repeats (LTRs), the 5' untranslated leader and portions of the env and nef genes were deleted. The leader sequence of pHP was substituted with a modified Rous sarcoma virus (RSV) 59 bp leader containing a mutated RSV gag AUG and a functional 5' splice site. The pHP construct was found to direct Gag-Pol synthesis as efficiently as wild-type HIV-1. The pTV construct contains sequences required for RNA packaging, reverse transcription and integration, but lacks viral genes. Co-transfection of pHP, pTV and a vesicular stomatitis virus G (VSV-G) envelope plasmid produced vectors at titers of 10(5)-10(6) transducing units per milliliter in 48 h. Replication-competent virus (RCV) was not detected when deletions were made in the env gene in pHP. The ability of this vector system to transduce dividing and nondividing cell in vitro and in vivo was also demonstrated. Compared with a Moloney murine leukemia virus (MLV) vector, the HP/TV vectors transduced human muscle-, kidney-, liver-derived cell lines and CD34+ primary hematopoietic progenitor cells more efficiently. Although the levels of the pTV transgene expression were high soon after transduction, the expression tended to decrease with time due either to the loss of proviral DNA or to the inactivation of promoter activity, which was found to be cell type-dependent. Analyses of extrachromosomal DNA showed that the unintegrated proviral DNA of lentiviral vectors survived much longer than that of the retroviral vectors. We demonstrate that the HP/TV vector is capable of high efficiency transduction and that long-term expression of lentiviral vectors is dependent on target cell type, the internal promoter and the transgene itself in the transducing vector.

Animals↗
PubMed2001

Expression of mouse igf2 mRNA-binding protein 3 and its implications for the developing central nervous system.

Functional analyses of neural RNA-binding proteins have focused mainly on their roles as modulators of posttranscriptional gene regulation, e.g., alternative splicing, dendritic mRNA localization, and local translation. Here we identified a mouse homologue of human IMP3, which is known to bind to and repress the translation of igf2 leader 3 mRNA. The mouse igf2 mRNA-binding protein 3 (mIMP3) is a member of the zipcode binding protein-1 (ZBP-1) family previously reported in chick fibroblast cells. mIMP3 was expressed in undifferentiated neuroepithelial cells and some postmitotic neurons at early embryonic stages (E10.5--E12.5), and its expression level decreased after the midembryonic stage (E12.5) until birth. The expression profile of mIMP3 is very similar to that of mouse igf2 leader 3 mRNA. In vitro UV cross-linking experiments showed that mIMP3 preferentially bound to igf2 leader 3 mRNA rather than igf2 leader 4 mRNA and did not bind the zipcode region of beta-actin or c-myc mRNA. Furthermore, persistent expression of mIMP3 protein in an undifferentiated P19 cell line revealed that mIMP3 inhibited neuronal differentiation morphologically and immunohistochemically. Taken together, these observations raise the possibility that mIMP3 represses neuronal differentiation through the regulation of igf2 mRNA expression.

5' Untranslated Regions↗