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PubMed1984

Characterization of leader RNA sequences on the virion and mRNAs of mouse hepatitis virus, a cytoplasmic RNA virus.

Mouse hepatitis virus, which replicates in cytoplasm, contains leader RNA sequences at the 5' end of the virus-specific mRNAs. We have sequenced this leader RNA by synthesizing cDNA from a synthetic oligodeoxyribonucleotide primer (15-mer) that is complementary to the sequences at the junction site between the leader and body sequences of the mRNAs. The leader sequences on each mRNA have exactly the same size, which span approximately equal to 70 nucleotides. Leader cDNA fragments obtained from several mRNA species were sequenced and found to be identical. Computer analysis of the leader RNA sequences shows that they share extensive sequence homology with the long-terminal-repeat region of several mammalian sarcoma viruses, suggesting possible common functions. This is a novel case of spliced leader sequences in the mRNAs of a cytoplasmic virus. An identical leader sequence is also present at the 5' end of the virion genomic RNA. The leader RNA is thus probably encoded by the virion genomic RNA template and is fused to the different body sequences of the various mRNAs. Since conventional RNA splicing is not involved, a novel mechanism for fusing two noncontiguous RNA segments in the cytoplasm must be utilized during viral transcription. Several minor cDNA bands longer than the leader were also synthesized, suggesting the possible presence of partially homologous sequences in other parts of the genome RNA.

Base Sequence↗

PubMed1996

Trypanosomatidae: a spliced-leader-derived probe specific for the genus Phytomonas.

We probed DNA from all trypanosomatid genera by slot blot hybridization with an oligonucleotide (SL3') complementary to a sequence of the Phytomonas spliced-leader or mini-exon RNA. The 19-nucleotide probe target site was previously shown to be highly conserved among a limited number of Phytomonas isolates, but diverges in other kinetoplastid genera. Our examination of 84 isolates of various genera of trypanosomatids showed hybridization of this probe exclusively with isolates from plants or insects which could, by morphological, biochemical, and molecular criteria, be considered to belong to the genus Phytomonas. In contrast, no hybridization was observed with flagellates of the genera Blastocrithidia, Crithidia, Endotrypanum, Herpetomonas, Leptomonas, Leishmania, and Trypanosoma. The method detected DNA quantities as low as 50 ng using either radioactive or nonradioactive probes, and was effective with as few as 10(4) intact flagellates. Together, these results suggest that this probe will serve as a convenient marker for taxonomic and epidemiological studies requiring reliable identification of Phytomonas spp. in plants or in putative insect vectors.

Animals↗

PubMed1996

A splice hepadnavirus RNA that is essential for virus replication.

According to the current model of hepadnavirus gene expression, the viral envelope proteins are produced from unspliced subgenomic RNAs, in contrast to the retroviral mechanism, where the subgenomic env RNA is generated by RNA splicing. We now describe and characterize a novel duck hepatitis B virus RNA species which is derived from the RNA pregenome by loss of a 1.15 kb intron. This RNA (termed spliced L RNA) codes for the large surface protein (L protein), as does the previously described unspliced mRNA (the preS RNA); however, it differs in 5' leader sequence and promoter control. Mutational analysis indicates that the spliced L RNA is functionally important for virus replication in infected hepatocytes and ducks, but not for virus formation from transfected DNA genomes. This suggests that the newly discovered second pathway for L protein synthesis plays a distinct role in an early step in the viral life cycle.

Animals↗

PubMed1980

Transcripts of the adenovirus-associated virus genome: multiple polyadenylated RNAs including a potential primary transcript.

Adenovirus-associated virus type 2 synthesizes four prominent viral transcripts, containing 4.3, 3.6, 2.6, and 2.3 kilobases (kb), in productively infected human KB cells (coinfected with adenovirus type 2). All species are polyadenylated and present in both nuclear and whole-cell RNA preparations, but only the predominant 2.3-kb (and possibly the 2.6-kb) RNA species are found on polysomes. Electrophoretic analyses under denaturing conditions of S1 nuclease-generated and exonuclease VII-generated DNA-RNA hybrids revealed, in each case, four protected DNA fragments which are equal in length (within 50 to 100 nucleotides) to the four S1 nuclease-generated hybrids resolved by electrophoresis under nondenaturing conditions. These results suggest that in the infected cell, abundant adenovirus-associated virus type 2 transcripts are present predominantly (by mass) as unspliced RNAs or, alternatively, they are spliced but contain very short (less than or equal to 50 nucleotides) leader sequences. That the 2.3-kb RNA represents such a spliced transcript is suggested by exonuclease VII mapping experiments and our more detailed RNA mapping studies (M. R. Green and R. G. Roeder, J. Virol., in press).

DNA, Viral↗

PubMed1985

Anatomy of region L1 from adenovirus type 2.

The structure of r-strand-specific RNAs encoded between coordinates 26 and 32 on the adenovirus type 2 genome was mapped by a combination of S1 endonuclease analysis, primer extension, and in vitro transcription. The region includes the third leader segment (coordinates 26.8 to 27.0), the genes for the low-molecular-weight virus-associated RNAs (VA RNAs) (coordinates 29.5 to 30.7), and the amino-terminal end of the gene for the L1 52,000-55,000 polypeptide (coordinates 30.7 to 32.1). The positions at which the tripartite leader was attached to the three longest L1 mRNAs were mapped at the nucleotide level. The leader splice junction of species L1a was located at coordinate 26.8 and coincided with the 3' splice site for the third leader segment, whereas the leader-body splice junction of species L1b and L1c were located at coordinates 29.0 and 30.7, respectively. No protein products have so far been assigned to the L1a and L1b mRNAs, although it can be predicted from the nucleotide sequence that species L1b encodes a 8,300 polypeptide. The third RNA, species L1c, encodes the well-characterized 52,000-55,000 polypeptide. It was also shown that a previously unidentified class of VA RNAs exists predominantly in the poly(A)-fraction of late RNA preparations. These RNAs are heterogeneous in length (up to 3,000 nucleotides) because of irregular transcription termination and have 5' ends which map precisely to the initiation sites for VA RNAI and VA RNAII transcription. Finally it was shown that an RNA with a 5' end coinciding with the 5' splice site for the third leader segment exists in the poly(A)-fraction of late cytoplasmic RNA. This RNA species might represent an excised intron.

Adenoviruses, Human↗

PubMed1985

Calmodulin genes in trypanosomes are tandemly repeated and produce multiple mRNAs with a common 5' leader sequence.

In Trypanosoma brucei gambiense, the Ca2+ binding protein calmodulin is encoded by three identical tandemly repeated genes. The transcripts of these genes consist of several RNA species similar in size. A 35-nucleotide spliced leader sequence is present at the 5' end of each mRNA but is not encoded by DNA contiguous to these genes. We have identified two different sites for the fusion of the leader to the mRNA. These results strongly support the idea that a novel, possibly discontinuous, transcription mechanism is used by these parasites.

Amino Acid Sequence↗

PubMed2002

Full-length-enriched cDNA libraries from Echinococcus granulosus contain separate populations of oligo-capped and trans-spliced transcripts and a high level of predicted signal peptide sequences.

The tissue-dwelling larval stages of the cestode Echinococcus granulosus are intimately associated with the host, implying that a range of molecular mediators may be secreted by the parasite into the host environment. These mediators are being sought through a transcriptome-based analysis, using recombinant cDNA libraries. Conventional cDNA libraries of E. granulosus contain high levels of mitochondrial transcripts, as well as host (bovine) genomic DNA. In particular, 60% of a conventional protoscolex stage cDNA library corresponds to the large subunit (LSU) of mitochondrial rRNA. We attribute the presence of LSU rRNA copies to its polyadenylation in E. granulosus. To circumvent this problem, we adapted the 5' Rapid Amplification of cDNA Ends (RNA-ligase mediated RACE) technique that excludes all polynucleotides missing the 7-methyl-guanosine (7MG) cap specific to the 5' end of full-length mRNA. By ligating a specific oligonucleotide (oligo-cap) to 7MG-bearing mRNA, three cDNA libraries were made by PCR from oligo-cap and oligo-dT primers. Analysis of these libraries showed that mitochondrial RNA contaminants had been excluded. Moreover, no bovine genomic sequences were detected. In parallel, we constructed three cDNA libraries using the newly described trans-spliced leader (SL) from Echinococcus. Although these represent a smaller subset of parasite genes, mitochondrial and genomic contributions were again excluded. In both cases, a majority of cDNAs (61-92%) were judged to contain the initiation ATG codon, and 11-27% of inserts included potential N-terminal signal sequences. The 5' UTR tracts of most oligo-capped cDNAs were <100 nt, although approximately 8% were longer than this. Among the trans-spliced cDNAs, 43% potentially utilise the AUG donated by the SL, and in only 6% was the SL separated from an endogenous putative start site by >60 nt. Sequence analysis of randomly selected clones shows virtually no overlap between the oligo-capped and SL libraries, indicating that trans-spliced E. granulosus mRNAs appear to be insensitive to the enzymatic treatments used to 'oligo-cap' unspliced mRNAs. The oligo-capped and SL strategies represent efficient and complementary pathways to isolate full-length cDNA clones from this cestode parasite and, possibly, from related parasitic flatworms.

5' Untranslated Regions↗

PubMed1979

Evidence for the identity of shared 5'-terminal sequences between genome RNA and subgenomic mRNA's of B77 avian sarcoma virus.

The polyribosomal fraction from chicken embryo fibroblasts infected with B77 avian sarcoma virus contained 38S, 28S, and 21S virus-specific RNAs in which sequences identical to the 5'-terminal 101 bases of the 38S genome RNA were present. The only polyadenylic acid-containing RNA species with 5' sequences which was detectable in purified virions had a sedimentation coefficient of 38S. This evidence is consistent with the hypothesis that a leader sequence derived from the 5' terminus of the RNA is spliced to the bodies of the 28S and 21S mRNA's, both of which have been shown previously to be derived from the 3' terminal half of the 38S RNA. The entire 101-base 5' terminal sequence of the genome RNA appeared to be present in the majority of the subgenomic intracellular virus-specific mRNA's, as established by several different methods. First, the extent of hybridization of DNA complementary to the 5'-terminal 101 bases of the genome to polyadenylic acid-containing subgenomic RNA was similar to the extent of its hybridization to 38S RNA from infected cells and from purified virions. Second, the fraction of the total cellular polyadenylic acid-containing RNA with 5' sequences was similar to the fraction of RNA containing sequences identical to the extreme 3' terminus of the genome RNA when calculated by the rate of hybridization of the appropriate complementary DNA probes. This suggests that most intracellular virus-specific RNA molecules contain sequences identical to those present in the 5'-terminal 101 bases of the genome. Third, the size of most of the radioactively labeled DNA complementary to the 5'-terminal 101 bases of the genome remained unchanged after the probe was annealed to either intracellular 38S RNA or to various size classes of subgenomic RNA and the hybrids were digested with S1 nuclease and denatured with alkali. However, after this procedure some DNA fragments of lower molecular weight were present. This was not the case when the DNA complementary to the 5'-terminal 101 bases of the genome was annealed to 38S genome RNA. These results suggest that, although the majority of the intracellular RNA contains the entire 101-base 5'-terminal leader sequence, a small population of virus-specific RNAs exist that contain either a shortened 5' leader sequence or additional splicing in the terminal 101 bases.

Avian Sarcoma Viruses↗

PubMed1993

Multifactorial regulation of IGF-I gene expression.

Insulin-like growth factor-I (IGF-I) is a highly conserved 70-residue circulating peptide with diverse biological effects. In mammals IGF-I is an essential mediator of normal postnatal growth and its expression is influenced by hormonal, nutritional, tissue-specific, and developmental factors. Recent studies have demonstrated that the IGF-I gene is more complicated than might have been predicted from its simple protein sequence. In rats and in humans the single-copy six-exon gene is transcribed by adjacent promoters into nascent RNAs with different 5' leader sequences that undergo both alternative RNA splicing and differential polyadenylation to yield multiple mature transcripts. These observations suggest that trophic agents may modulate expression of IGF-I at any of several nodal points. In this report we review several of the mechanisms responsible for regulating production of IGF-I in the rat. During neonatal development IGF-I gene transcription is progressively activated, leading to a rise in both hepatic IGF-I mRNA and in serum IGF-I. The induction of IGF-I expression is limited to mRNAs directed by promoter 1, the more 5' of two rat IGF-I gene promoters, and precedes the ontogenic appearance of liver growth hormone (GH) receptors, indicating that mechanisms independent of GH activate IGF-I expression during early postnatal life. By contrast, in adult GH-deficient rats, a single intraperitoneal injection of GH causes a prompt rise in IGF-I gene transcription that is mediated equivalently by promoters 1 and 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed2004

Ribozyme-mediated induction of apoptosis in human cancer cells by targeted repair of mutant p53 RNA.

A variety of mutations in the p53 tumor suppressor gene have been found in over half of human tumors. Thus, restoration of wild-type p53 activity by repair of mutant RNA has been previously suggested as an approach to cancer treatment. To explore the potential utility of RNA repair for cancer therapy, we developed a group I intron-based ribozyme that can replace mutant p53 RNA with a wild-type RNA sequence attached to the 3' end of the ribozyme by trans-splicing reaction. First, RNA mapping analysis demonstrated that the leader sequences upstream of the AUG start codon in the mutant p53 RNA appeared to be particularly accessible to the ribozymes. Then, the trans-splicing ribozyme specifically recognizing the most accessible sequence induced functional p53 activity, resulting in an 8- and a 2.6-fold induction of transactivation of p53-responsive promoters in two mutant p53-related ovarian cancer cell lines, SKOV3 cells and 2774 cells, respectively, by repairing defective p53 RNA. The repair efficiency of the mutant p53 RNA was almost 10% in 2774 cells. Moreover, the ribozyme activated the expression level of endogenous p21 and Bax genes in the cells. Furthermore, apoptosis was efficiently triggered in the human cancer cells transfected with the specific ribozyme. These results suggest that a trans-splicing ribozyme could be a potent anti-cancer agent that can revert the defective p53-related neoplastic phenotype.

Apoptosis↗

PubMed1996

Binding properties of human immunodeficiency virus type-2 (HIV-2) RNA corresponding to the packaging signal to its nucleocapsid protein.

The nucleocapsid (NC) protein of human immunodeficiency virus type-1 (HIV-1) contains two zinc finger motifs (ZFMs), and binds specifically to the packaging signal which is located in the 5' leader sequence of the viral genomic RNA between the first splice donor and the gag initiator codon (AUG). In this study, we analyzed the specificity of the binding of the corresponding region of HIV-2 (Region 3) to its NC protein (NCp8), by performing a competitive ultraviolet (UV) cross-linking assay using in vitro-synthesized 32P-labeled and unlabeled RNAs corresponding to a sequence between the primer binding site and the gag AUG (Region 1). Binding of 32P-labeled Region 1 RNA to NCp8 was inhibited specifically by adding unlabeled Region 1 and 3 RNAs and no specific binding was detected using deletion mutant peptides of NCp8. These findings suggest that the region(s) which bind(s) specifically to HIV-2 NCp8 lie(s) between the first splice donor and the gag AUG in the 5' leader sequence and that NCp8 is the minimum binding region responsible for the specific binding of the region downstream of the first splice donor site of HIV-2 RNA.

Amino Acid Sequence↗

PubMed2002

ExonHit Therapeutics.

ExonHit Therapeutics' mission is to be the leader in the application of alternative RNA splicing to the discovery and development of therapeutics and diagnostics for cancer and neurodegenerative diseases. The most advanced drugs in the Company's pipeline are treatments for amyotropic lateral sclerosis. One of these drugs entered Phase II trials in October 2002 and the other is planned for Phase I trials in 2003. Compounds for treating cancer are currently in the preclinical stage. ExonHit is also engaged in the discovery of molecular pharmacogenomic and diagnostic tests derived from RNA splicing events. ExonHit has established alliances with leading pharmaceutical and diagnostics companies, accelerating the R&D programs of its partners. The Company intends to license its pipeline of high-value drugs (after Phase IIb clinical trials) and diagnostics to these and other global partners.

Exons↗

PubMedDate not supplied

Sequences homologous to variant antigen mRNA spliced leader in Trypanosomatidae which do not undergo antigenic variation.

Trypanosomes which parasitize mammals have evolved mechanisms to evade immune attack, such as the occupation of 'safe' intracellular sites (for example, Trypanosoma cruzi), or antigenic variation, exemplified by the salivarian trypanosomes (for example, Trypanosoma brucei). Antigenic variation is mediated by sequential expression of single variant surface glycoprotein (VSG) genes, and often involves transposition of the active gene. Every VSG transcript examined shares the same 5' terminal 35-nucleotide leader sequence. In T. brucei, this leader is encoded within a 1.4-kilobase unit tandemly reiterated to form a large array. It is hypothesized that this array is distantly linked to the expressed VSG gene and functions as a multiple promoter of VSG gene transcription, restricting transcription to that gene which, through genomic rearrangement, is placed downstream from the array. Leader and structural gene sequences are presumably juxtaposed by RNA splicing. Here we show that several trypanosomatids, both those which undergo antigenic variation (Trypanosoma congolense and Trypanosoma vivax) and those which do not (T. cruzi and Leptomonas collosoma), contain reiterated sequences homologous to the T. brucei spliced leader (SL). These results suggest that the SL, although utilized in VSG gene expression, is an ancestral sequence also used in the expression of other trypanosomatid genes.

Animals↗

PubMed1982

Non-random localization of ribonucleoprotein (RNP) structures within an adenovirus mRNA precursor.

Heterogeneous nuclear protein complexes (hnRNP) containing the precursor RNA from the adenovirus early region 2 were analysed to determine the specificity of protein-RNA interaction. RNA precursor sequences were present in isolated hnRNP complexes and endogenous 30S particles. At least 20-40 bases long fragments were protected when RNase A was used to remove unprotected RNA sequences in hnRNA complexes. Similarly around 40 bases of RNA were protected in 30S particles. These sequences represent discrete regions of the adenovirus genome. Especially sequences complementary to the EcoRI-F fragment encoding the first leader and the major intron for the DNA binding protein (DBP) RNA precursor, were analysed in detail. Tentatively, sequences resistant to RNase A were located in the middle of the intron and at the splice-donor junction of the first leader of the DBP precursor RNA. The same sequences were identified irrespective whether hnRNP complexes or 30S particles were used suggesting that 30S particles originate from hnRNP complexes. A 38.000 dalton protein appears to be in direct contact with RNA sequences complementary to the EcoRI-F fragment.

Adenoviruses, Human↗

PubMedDate not supplied

Characterization of a Trypanosoma cruzi genomic fragment complementary to several species-specific mRNAs but different from the spliced leader sequence.

We have isolated a clone of Trypanosoma cruzi genomic DNA, lambda 3b2-5, which contains sequences that are reiterated in the genome. Northern blot analysis showed that clone 3b2-5 hybridizes to 1,200-5,000 bases different mRNA species. The number of mRNAs species hybridized to clone 3b2-5 exceeds its coding capacity showing that this clone carries sequences that are common to several mRNAs species and conserved in the poly A(+) RNA. These sequences are not homologous to the T. cruzi spliced leader sequence, since clone 3b2-5 does not hybridize to a synthetic 20 nucleotide complementary to the spliced leader sequence. Clone 3b2-5 does not hybridize to DNA and RNA from several genera of Trypanosomatidae and other Trypanosoma species indicating that it carries T. cruzi species-specific sequences.

Animals↗

PubMed1986

Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans splicing.

We present evidence that addition of the 35 nucleotide spliced leader (SL) to the 5' end of T. brucei mRNAs occurs via trans RNA splicing. A 100 nucleotide fragment of the 135 base SL RNA (100-mer) is revealed by S1 nuclease analysis of total and poly(A)+ RNA. This 100-mer is not detected by Northern hybridization analysis, indicating that it does not exist free in the cell. The 5' end of the 100-mer maps precisely to the conserved splice junction sequence of the SL RNA. Purified debranching enzyme releases this 100-mer RNA as a free, 100 nucleotide species. This indicates that the 100-mer is covalently linked to poly(A)+ RNA by a 2'-5' phosphodiester bond, that the branched intermediate has a discontinuous intron or Y structure (rather than a lariat), which is expected of a trans-spliced mRNA, and that the SL RNA is indeed the donor of the SL sequence to trypanosome mRNAs.

Animals↗

PubMed1995

Epstein-Barr virus latent messages with shuffled leader exons: remnants of circumgenomic transcription?

The EBNA transcription unit which is active in Epstein-Barr virus-immortalized latently infected B lymphocytes covers approximately 60% of the 172-kb genome. Since the genome exists as a circular double-stranded DNA molecule in latently infected cells, it is conceivable that complete copies are made during transcription. Rather than attempt to detect gigantic RNA molecules directly, we used RNA-PCR to detect incorporation of leader exons into mRNA in a shuffled order. The downstream U leader exon was detected spliced upstream of the internal repeat leader exons W1 and W2 in the polyadenylated RNA fraction of spontaneous lymphoblastoid cell lines, restricted phenotype BL cell lines Wanyanyi and Wewak2, and in B95-8, Raji, and Akata cells. Quantitative competitive RNA-PCR showed that the ratio of U exon-containing EBNA1 messages to U exon-shuffled leader messages was approximately 10:1, with large variation from cell line to cell line, and was not affected by induction of the lytic cycle in B95-8, Raji, or Akata cells. Messages with shuffled exons contained only the C2W1 alternative splice, which does not produce an initiator AUG methionine codon for EBNA4 gene expression. The results provide evidence for long-range exon skipping and imply that genome-length transcripts may occur and participate in viral gene expression in latency.

Adult↗

PubMed1983

Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein.

A mouse monoclonal antibody and human autoimmune sera directed against various classes of small ribonucleoprotein particles have been tested for inhibition of mRNA splicing in a soluble in vitro system. The splicing of the first and second leader exons of adenovirus late RNA was inhibited only by those sera that reacted with U1 RNP. Both U1 RNP-specific human autoimmune serum and sera directed against the Sm class of small nuclear RNPs, including a mouse monoclonal antibody, specifically inhibited splicing. Antisera specific for U2 RNP had no effect on splicing nor did antisera specific for the La or Ro class of small RNPs. These results suggest that U1 RNP is essential for the splicing of mRNA precursors.

Adenoviruses, Human↗