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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↗
PubMed1985

Alternative splicing caused by RNA secondary structure.

mRNA precursors with stable hairpins were constructed by inserting inverted repeats into an adenovirus transcriptional template that encoded the three late leader exons. When the loop of the hairpin contained the second exon and the flanking splice sites, most of the RNA spliced in vitro had the first exon joined directly to the third exon. The remainder was spliced normally. The same types of alternatively spliced RNAs were formed when a similar template was introduced into HeLa cells by transfection. Thus both in extracts and in cells, an exon became optional when sequestered in a hairpin loop. Perhaps a related mechanism creates the alternative splicing patterns of complex transcription units.

Adenoviruses, Human↗
PubMed1994

A common pyrimidine-rich motif governs trans-splicing and polyadenylation of tubulin polycistronic pre-mRNA in trypanosomes.

In trypanosomes, the generation of monocistronic mRNAs from polycistronic precursors is achieved via RNA processing, namely trans-splicing of the spliced leader sequence at the 5' end and cleavage/polyadenylation at the 3' end of the mRNA coding region. Recent evidence raised the intriguing possibility that these two reactions are coupled. To begin a dissection of the signals required for mRNA 5'-end and 3'-end formation and to uncover potential interactions between trans-splicing and polyadenylation, we mutagenized the intergenic region between the beta- and alpha-tubulin genes of Trypanosoma brucei. Block substitutions identified the pyrimidine-rich sequences at the alpha-tubulin 3'-splice-acceptor site as a major determinant for accurate trans-splicing downstream and 3'-end formation upstream. In addition to the utilization of cryptic 3'-splice sites, obliteration of the polypyrimidine tracts led to aberrant poly(A)+ site choice, even in the presence of the wild-type poly(A)+ site and neighboring sequences. Taken together, these results indicate that the polypyrimidine-rich sequences act as a bifunctional element that affects RNA processing both upstream and downstream from itself. This is consistent with the possibility that the polypyrimidine tract is recognized by both the trans-splicing and polyadenylation machineries, either sequentially or simultaneously.

Animals↗
PubMed1987

Transcription map of the African green monkey lymphotropic papovavirus.

The S1 endonuclease mapping technique was employed to characterize polyadenylated RNA molecules encoded by the African green monkey lymphotropic papovavirus (LPV) in B-lymphoblastoid cells of human origin infected with the virus. Two groups of stable transcripts of opposite polarities were identified and mapped. By analogy with other papovaviruses, these molecules were designated as "early" and "late" RNAs. The early transcripts include two RNA molecules consisting of a colinear 3' "main body" of about 1.9 kb spliced to 5' "leaders" of 0.26 and 0.60 kb, respectively. These molecules are presumably mRNAs encoding the LPV large T and small t antigens. Shorter, less abundant early RNA molecules have also been detected but have not been mapped. The late RNAs include three molecules, of which two consist of a common leader of 0.22 kb spliced to main bodies of 1.2 and 1.85 kb, respectively. The third late transcript of 2.3 kb is colinear. These late transcripts are presumably mRNAs encoding the LPV capsid proteins. A survey of the LPV DNA sequence published by M. Pawlita, A. Clad, and H. zur Hausen (1985, Virology 143, 196-211) allowed tentative assignment of the termini and splice sites of the various RNAs to nucleotides in the DNA sequence. The LPV transcription map is similar but not entirely analogous to those of polyoma virus and simian virus 40. These distinctions support a previous classification of LPV as a member of a new subgroup of the polyomaviruses.

Animals↗
PubMed1998

Human immunodeficiency virus type 2 lentivirus vectors for gene transfer: expression and potential for helper virus-free packaging.

In addition to the long-term expression of the transgene provided by all retroviral vectors, lentiviruses present the opportunity to transduce nondividing cells and potentially achieve regulated expression. The development of lentiviral vectors requires the design of transfer vectors to ferry the transgene with efficient encapsidation of the transgene RNA and with full expression capability, and of a packaging vector to provide packaging machinery in trans but without helper virus production. For both vectors, a knowledge of packaging signal is required-the signal to be included in the transfer vector but excluded from the packaging vector. Among the human lentiviruses, human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2), we think HIV-2 is better suited for gene transfer than HIV-1. It is less pathogenic and thus safer during design and production; its desirable nuclear import and undesirable cell-cycle arrest functions are segregated on two separate genes. In HIV-1 infection, it is less likely to recombine with the resident HIV-1, and it may itself downregulate HIV-1 expression. Evidently, elements located both upstream and downstream of the splice donor site in the leader sequence participated in RNA encapsidation and these sequences appeared necessary and sufficient. Deletion of both sequence elements resulted in a dramatic curtailment of RNA encapsidation and helper virus production. This was accompanied by some but acceptable loss of gene expression capability. The helper virus-free phenotype and expression capability of the double mutant was maintained upon replacement of its 3' long terminal repeat with a minigene cassette containing a transcriptional termination signal and a drug resistance marker gene. Deletion of the splice donor site itself had a dramatic negative effect on gene expression, supporting the important role of this element in the life of RNA.

Cell Line↗
PubMed1995

Characterization of the manganese superoxide dismutase cDNA and gene from the human parasite Onchocerca volvulus.

The manganese-containing superoxide dismutase (MnSOD) is a major component of the cellular defence mechanisms against the toxic effects of the superoxide radical. Within the framework of studies on anti-oxidant enzymes and their protective role in the human parasitic nematode Onchocerca volvulus, sequences encoding the MnSOD were isolated and examined in this study. Degenerate primers were designed based upon conserved regions of MnSOD sequences from other organisms, and were used in PCR on reverse-transcribed O. volvulus total RNA and genomic DNA to identify partial cDNA and genomic DNA fragments encoding the O. volvulus MnSOD (OvMnSOD). The genomic DNA PCR product was used to screen an O. volvulus adult worm lambda unizap II cDNA library and the nucleotide sequence of the longest clone determined. The complete 5'-end of the OvMnSOD cDNA was obtained using the rapid amplification of cDNA ends (RACE) procedure with O. volvulus total RNA and was found to possess a spliced leader sequence at the 5'-terminus. The deduced primary sequence encodes a 25 kDa protein, which has the conserved residues required for enzyme activity and metal binding. The 24 N-terminal amino acids encoded by the OvMnSOD cDNA comprise a putative mitochondrial transit peptide. The OvMnSOD gene was also isolated from an O. volvulus adult worm lambda fix II genomic library, a restriction map was constructed and the nucleotide sequence determined. The OvMnSOD gene was found to possess five exons and four introns with consensus splice-site junctions. Potential regulatory elements were identified in the 5' genomic flanking sequence. Southern-blot analysis with total worm genomic DNA indicates a single-copy gene, with a restriction pattern consistent with that of the isolated gene.

Amino Acid Sequence↗
PubMed1997

A dynamin GTPase mutation causes a rapid and reversible temperature-inducible locomotion defect in C. elegans.

Drosophila shibire and its mammalian homologue dynamin regulate an early step in endocytosis. We identified a Caenorhabditis elegans dynamin gene, dyn-1, based upon hybridization to the Drosophila gene. The dyn-1 RNA transcripts are trans-spliced to the spliced leader 1 and undergo alternative splicing to code for either an 830- or 838-amino acid protein. These dyn-1 proteins are highly similar in amino acid sequence, structure, and size to the Drosophila and mammalian dynamins: they contain an N-terminal GTPase, a pleckstrin homology domain, and a C-terminal proline-rich domain. We isolated a recessive temperature-sensitive dyn-1 mutant containing an alteration within the GTPase domain that becomes uncoordinated when shifted to high temperature and that recovers when returned to lower temperatures, similar to D. shibire mutants. When maintained at higher temperatures, dyn-1 mutants become constipated, egg-laying defective, and produce progeny that die during embryogenesis. Using a dyn-1::lacZ gene fusion, a high level of dynamin expression was observed in motor neurons, intestine, and pharyngeal muscle. Our results suggest that dyn-1 function is required during development and for normal locomotion.

Amino Acid Sequence↗
PubMed1989

Isolation and sequence of four small nuclear U RNA genes of Trypanosoma brucei subsp. brucei: identification of the U2, U4, and U6 RNA analogs.

Trypanosomes use trans splicing to place a common 39-nucleotide spliced-leader sequence on the 5' ends of all of their mRNAs. To identify likely participants in this reaction, we used antiserum directed against the characteristic U RNA 2,2,7-trimethylguanosine (TMG) cap to immunoprecipitate six candidate U RNAs from total trypanosome RNA. Genomic Southern analysis using oligonucleotide probes constructed from partial RNA sequence indicated that the four largest RNAs (A through D) are encoded by single-copy genes that are not closely linked to one another. We have cloned and sequenced these genes, mapped the 5' ends of the encoded RNAs, and identified three of the RNAs as the trypanosome U2, U4, and U6 analogs by virtue of their sequences and structural homologies with the corresponding metazoan U RNAs. The fourth RNA, RNA B (144 nucleotides), was not sufficiently similar to known U RNAs to allow us to propose an identify. Surprisingly, none of these U RNAs contained the consensus Sm antigen-binding site, a feature totally conserved among several classes of U RNAs, including U2 and U4. Similarly, the sequence of the U2 RNA region shown to be involved in pre-mRNA branchpoint recognition in yeast, and exactly conserved in metazoan U2 RNAs, was totally divergent in trypanosomes. Like all other U6 RNAs, trypanosome U6 did not contain a TMG cap and was immunoprecipitated from deproteinized RNA by anti-TMG antibody because of its association with the TMG-capped U4 RNA. These two RNAs contained extensive regions of sequence complementarity which phylogenetically support the secondary-structure model proposed by D. A. Brow and C. Guthrie (Nature [London] 334:213-218, 1988) for the organization of the analogous yeast U4-U6 complex.

Animals↗
PubMed1992

Molecular organization of Leishmania RNA virus 1.

The complete 5284-nucleotide sequence of the double-stranded RNA genome of Leishmania RNA virus 1 (LRV1) was determined and contains three open reading frames (ORFs) on the plus (+) (mRNA) strand. The predicted amino acid sequence of ORF3 has motifs characteristic of viral RNA-dependent RNA polymerases. ORF2, which may encode the major viral coat protein, overlaps ORF3 by 71 nucleotides, suggesting a +1 translational frameshift to produce a gag-pol type of fusion protein. Two alternative models for the frameshift are presented. The 5' splice leader sequence of kinetoplastid mRNAs is not in LRV1 RNA. This suggests that the 450-base region at the 5' end of the LRV1 (+)-strand, which contains ORF1 and is highly conserved among viral strains, does not encode protein but has a role in initiation of translation and/or RNA stability. The similarity of LRV1 genomic organization, replication cycle, and RNA-dependent RNA polymerase sequence to those of the yeast virus ScV L-A suggests a common ancestral origin. The possibility that LRV1 affects pathogenesis in leishmaniasis is intriguing.

Amino Acid Sequence↗
PubMed1998

Oligonucleotide-mediated inhibition of genomic RNA dimerization of HIV-1 strains MAL and LAI: a comparative analysis.

An essential step in the replication cycle of retroviruses is the dimerization of two copies of the genomic RNA. In vitro and in vivo studies have demonstrated that dimerization is mediated at least partially by RNA-RNA interactions. In HIV-1, the cis-element most important for dimerization is the dimerization initiation site (DIS), a stem-loop structure with an autocomplementary loop located between the primer binding site and the splice donor site in the 5' leader region of genomic RNA. We have studied the inhibition of dimerization of RNA corresponding to the first 615 nt of HIV-1 strains MAL and LAI in vitro using RNA and DNA oligonucleotides. The oligonucleotides were identical to or complementary to the DIS of the MAL and LAI strains, which are representative of the two most common DIS motifs found in natural isolates. The loop sequence of the DIS of the MAL isolate is AGGUGCACA, and that of the LAI sequence is AAGCGCGCA (the autocomplementary sequences are GUGCAC and GCGCGC, respectively). Several of the oligonucleotides were very efficient inhibitors of dimerization. However, homologous oligonucleotides displayed vastly different inhibition efficiencies between the two strains despite relatively modest sequence differences. Some of the oligonucleotides bound the viral RNA via a loop-loop interaction alone, whereas others recruited stem nucleotides to form an extended duplex even in the absence of loop complementarity. Furthermore, oligonucleotide inhibition was ineffective at low temperature, suggesting that a conformational change in the DIS is necessary for disruption of the dimeric structure of the DIS or binding of oligonucleotide or both.

Base Sequence↗
PubMed1992

Genomic organisation and expression of a differentially-regulated gene family from Leishmania major.

We have isolated and characterised a differentially-regulated gene family in the protozoan parasite Leishmania major. The family contains 5 genes linked within a 10Kb region of the genome: three of the genes are closely related in DNA sequence, the other two have only limited homology. Post-transcriptional control of the differential expression pattern is suggested by detection of precursor RNA molecules containing intergenic sequences and evidence that mature mRNA molecules contain a 35nt spliced leader sequence at their 5' ends. These features support a model of polycistronic transcription in which the stability and differential processing of precursor RNA molecules determine the steady state levels of mature mRNA. We have identified several DNA sequence motifs within the gene family that have potential roles in differential processing and/or RNA stability: an alternative 5' splice acceptor site for trans-splicing; a putative polyadenylation site; and a region of potential secondary structure within 3' flanking sequences. The 3' sequence elements are conserved in those genes that share the same pattern of differential regulation. To our knowledge, this is the first example of coordinated differential-regulation of a non-identical gene cluster in Leishmania.

Animals↗
PubMed2002

The genes pme-1 and pme-2 encode two poly(ADP-ribose) polymerases in Caenorhabditis elegans.

Poly(ADP-ribose) polymerases (PARPs) are an expanding, well-conserved family of enzymes found in many metazoan species, including plants. The enzyme catalyses poly(ADP-ribosyl)ation, a post-translational modification that is important in DNA repair and programmed cell death. In the present study, we report the finding of an endogenous source of poly(ADP-ribosyl)ation in total extracts of the nematode Caenorhabditis elegans. Two cDNAs encoding highly similar proteins to human PARP-1 (huPARP-1) and huPARP-2 are described, and we propose to name the corresponding enzymes poly(ADP-ribose) metabolism enzyme 1 (PME-1) and PME-2 respectively. PME-1 (108 kDa) shares 31% identity with huPARP-1 and has an overall structure similar to other PARP-1 subfamily members. It contains sequences having considerable similarity to zinc-finger motifs I and II, as well as with the catalytic domain of huPARP-1. PME-2 (61 kDa) has structural similarities with the catalytic domain of PARPs in general and shares 24% identity with huPARP-2. Recombinant PME-1 and PME-2 display PARP activity, which may partially account for the similar activity found in the worm. A partial duplication of the pme-1 gene with pseudogene-like features was found in the nematode genome. Messenger RNA for pme-1 are 5'-tagged with splice leader 1, whereas those for pme - 2 are tagged with splice leader 2, suggesting an operon-like expression for pme - 2. The expression pattern of pme-1 and pme-2 is also developmentally regulated. Together, these results show that PARP-1 and -2 are conserved in evolution and must have important functions in multicellular organisms. We propose using C. elegans as a model to understand better the functions of these enzymes.

Animals↗
PubMed1984

Splice junctions in adenovirus 2 early region 4 mRNAs: multiple splice sites produce 18 to 24 RNAs.

We localized the splice junctions in adenovirus 2 early region 4 (E4) mRNAs. Processing of the E4 precursor RNA positioned the donor splice site of the 5' leader sequence adjacent to acceptor sites near the 5' ends of five of the six open reading regions in the E4 transcription unit. Of particular interest among the E4 mRNAs is an extensively spliced class which includes multiple species with sizes ranging from 1.1 to 0.75 kilobases (kb). Purified 1.1- to 0.75-kb mRNAs specified at least 10 polypeptides in vitro. We detected eight acceptor and two donor splice sites utilized in the deletion of the intron from the 3' portion of these mRNAs. E4 RNAs were isolated from the cytoplasm of infected cells at 5, 9, 12, and 18 h after infection. The E4 mRNAs were present throughout infection, but different members of the 1.1- to 0.7-kb class were predominant at each time assayed. Alternate splicing of the 3.0-kb E4 precursor RNA can generate as many as 25 mRNAs that encode at least 16 polypeptides.

Adenoviruses, Human↗
PubMed1988

Maturation of the major ubiquitin gene transcript in Caenorhabditis elegans involves the acquisition of a trans-spliced leader.

Recently, studies on the 5'-ends of actin mRNAs in the nematode, Caenorhabditis elegans, have demonstrated that three of the four mature actin transcripts contain a 22-nucleotide leader sequence which is acquired by trans-splicing from a novel 100-nucleotide RNA. In the course of our studies of the ubiquitin genes in C. elegans (R. W. Graham and E. P. M. Candido, manuscript in preparation) we were led to the possibility that the major ubiquitin transcript in this organism might also contain a trans-spliced leader sequence. The 5'-noncoding region of the major ubiquitin gene (UbiA) contains a 3'-splice consensus sequence six nucleotides upstream of the initiation codon; however, sequencing of a further 1.6 kilobases upstream failed to reveal the existence of any potential 5'-splice sites in the correct relationship to known promoter elements. Furthermore, S1 and Northern blot analyses using probes complementary to upstream regions failed to detect the ubiquitin transcript. Primer extension experiments on total RNA using an oligonucleotide which hybridized to the ubiquitin transcript downstream of the 3'-splice site demonstrated the existence of a 22-nucleotide leader sequence not present in the ubiquitin gene 5'-region. Thus we show here that the 2500-nucleotide mRNA encoding a polyubiquitin precursor protein contains a 22-nucleotide leader sequence identical to that found in several C. elegans actin mRNAs and which is most likely acquired by a trans-splicing mechanism. In addition we have localized the site of transcript initiation for UbiA by S1 nuclease mapping.

Amino Acid Sequence↗
PubMed1980

Transcripts of the adeno-associated virus genome: mapping of the major RNAs.

The four major adeno-associated virus type 2 (AAV2)-specific RNAs were mapped on the linear viral genome by a variety of biochemical techniques, including S1 nuclease and exonuclease VII mapping, RNA gel-transfer hybridization, and analysis of reverse transcriptase extension products. All the major AAV2 RNAs were derived from the minus DNA strand and had 3' termini at position 96. The nucleus-specific 4.3- and 3.6-kilobase (kb) RNAs had 5' termini at positions 6 and 19, respectively. The 5' terminus of the 2.6-kb RNA mapped to position 38.5. The predominant 2.3-kb AAV2 mRNA was spliced and contained a short leader sequence (approximately 50 nucleotides) which mapped to position 38.5, coincident with the 5' terminus of the 2.6-kb RNA. The 5' end of the body of the 2.3-kb RNA mapped to position 46.5. These results are discussed in terms of the involvement of single versus multiple promoters (for transcription) and RNA splicing mechanisms in the generation of the AAV2 RNAs.

Chromosome Mapping↗