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Results for “RNA, Spliced Leader”

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PubMed1991

RNA-protein complexes mediate in vitro capping of the spliced-leader primary transcript and U-RNAs in Trypanosoma cruzi.

A 39-nucleotide spliced leader (SL) is joined to the 5' ends of trypanosome mRNAs in a bimolecular or trans-splicing process. The SL in Trypanosoma cruzi is transcribed as an approximately 110-nucleotide RNA (SL-RNA or SL primary transcript) bearing the 39-nucleotide SL at the 5' end. The SL-RNA is 5' capped by a guanylyltransferase activity prior to trans-splicing and trypanosome mRNAs thus obtain their mature caps from the SL by trans-splicing. We have previously characterized a guanylyltransferase activity from T. cruzi nuclear extracts and shown that this capping activity has an unusual ATP dependence and an apparent specificity for the SL-RNA and U-RNAs. Herein, we show that the capping activity sediments as a 12-15S particle during velocity sedimentation in glycerol gradients and fractionates as a greater than 150-kDa particle during large-pore gel filtration chromatography. Moreover, the endogenous substrate RNAs--the SL-RNA and U-RNAs--consistently copurify with the capping activity, suggesting that the activity and the substrates form a ribonucleoprotein particle. The capping activity and substrate RNAs are not dissociated in isopycnic Cs2SO4 gradients and band at a density expected for an RNA-protein complex, confirming the existence of ribonucleoprotein particles bearing both the activity and its substrate RNAs. Finally, we partially purified these ribonucleoprotein particles and showed that the capping activity remains ATP dependent and highly specific for the SL-RNA and the U-RNAs. These observations are consistent with the hypothesis that one of the functions of trans-splicing is for mRNA capping.

Animals↗

PubMed1987

Trypanosome mRNAs have unusual "cap 4" structures acquired by addition of a spliced leader.

A single capped oligonucleotide is released from Trypanosoma brucei poly(A)+ RNA upon digestion with RNase T2. This observation supports the hypothesis that all T. brucei mRNAs share a common leader sequence. Digestion of the T2-resistant species with nucleotide pyrophosphatase shows that the capping nucleotide is 7-methylguanosine 5'-monophosphate (pm7G). Additional characterization of the T2-resistant fragment indicates that modifications are present on the first four transcribed nucleotides; the 5' termini of T. brucei mRNAs can, therefore, be described as "cap 4" structures. Identical 5'-cap structures are found on the T. brucei spliced leader (SL) RNA; an observation compatible with the hypothesis that the small SL RNA acts as a donor of the SL for the mRNA. However, we find that within a population of purified SL RNAs are species that are capped but incompletely modified. The presence of these unmodified and partially modified species allowed us to analyze the 5' sequence of the SL RNA transcript. The results indicate that transcription begins four nucleotides upstream of the reported 5' end. Therefore, the T. brucei SL transcript is actually 39 rather than 35 nucleotides long. We have also analyzed the capped oligonucleotide of a distantly related Trypanosomatid, Leptomonas collosoma and find it to be identical to that of T. brucei. The potential significance of these results is discussed in light of observations of trypanosome gene expression.

Animals↗

PubMed1983

Splicing of adenovirus RNA in a cell-free transcription system.

A soluble whole-cell extract prepared accurately from HeLa cells splices 2-3% of the RNA transcribed from a DNA template containing the first and second leader exons of late adenovirus RNA. The spliced RNA was detected by a sensitive technique using hybridization to a single-stranded phage M13 cDNA clone, followed by binding to nitrocellulose filters. The identity of the spliced RNA was established by RNase T1 and pancreatic RNase two-dimensional peptide mapping. The bond formed during the in vitro splicing reaction appears to be a typical 3',5'-phosphodiester bond as judged by its sensitivity to RNase T1. The splicing reaction is specifically inhibited by KCl at concentrations greater than 50 mM and by the addition of cellular RNA. Three features of this system may account for the detection of splicing in a soluble extract: (i) the sensitive and unambiguous hybridization assay, (ii) the high transcriptional activity of the major late promoter of adenovirus, and (iii) the use of the first and second leader exon splice of adenovirus, which may be unusually rapid.

Adenoviruses, Human↗

PubMed1996

5' sequences essential for trans-splicing of msp (gp63) RNAs in Leishmania chagasi.

The 5' sequences essential for spliced leader (SL) addition to RNA encoding the abundant Leishmania surface protease gp63 were determined. A DNA segment found upstream of all Leishmania chagasi gp63 genes was cloned in front of a luciferase gene, and luciferase activity was measured in transiently transfected L. chagasi promastigotes. Two hundred and twenty bp of the upstream region was needed for optimal luciferase activity. Deletions and point mutations were placed in this segment to determine which nucleotides participate in the splicing events. A region containing 87% pyrimidines located between 31 and 69 bp upstream of the splice acceptor dinucleotide and containing three potential branch point A residues was highly beneficial for reported gene activity. In contrast, a 30-nt pyrimidine-rich sequence immediately upstream of the splice acceptor site did not by itself enhance luciferase activity. Luciferase activity was associated with the presence of trans-spliced luciferase mRNA. The results suggest that sequences 31-69 bp upstream of gp63 genes enhance gene expression through provision of signals for efficient trans-splicing.

Animals↗

PubMed1987

Tandem linkage and unusual RNA splicing of the T-cell receptor beta-chain variable-region genes.

The variable-region (V) genes of the murine T-cell receptor beta chain exist largely as single-element subfamilies. The V beta 5 and V beta 8 genes belong to the only two known three-member V beta subfamilies. We present studies on the linkage of these six genes and show that the genomic organization is that of alternating V beta 5 and V beta 8 genes. Our analysis suggests that these genes were tandemly duplicated, the unit of duplication being a pair of V beta 5 and V beta 8 genes. This tandem organization permits transcripts to initiate from the promoter of an unrearranged V beta located upstream of the rearranged V beta gene. These transcripts can generate functional beta-chain gene messages by novel RNA splicing of the upstream leader exon to the V beta coding exon of the downstream rearranged gene. We extend the analysis of the T-cell receptor genomic organization to include 12 V beta genes and suggest that all V beta genes are closely linked on chromosome 6. In addition, we discuss the possible implications of the close linkage of the V beta genes on the development of the T-cell receptor beta-chain gene repertoire.

Amino Acid Sequence↗

PubMed1986

Leader sequences of murine coronavirus mRNAs can be freely reassorted: evidence for the role of free leader RNA in transcription.

Mouse hepatitis virus (MHV), which replicates in cytoplasm of infected cells, contains an identical leader RNA sequence at the 5' end of each of the virus-specific mRNAs. Previous studies suggested that the synthesis of these mRNAs does not involve conventional RNA splicing and may instead require priming by a free leader RNA. In this communication, we demonstrate that, during a mixed infection with two different MHVs, the leader RNA sequences from one virus could be detected on the mRNAs of the coinfecting virus at a high frequency, as if the leader sequence and mRNAs were joined together from two randomly segregating RNA segments. This finding demonstrates that MHV mRNA transcription utilizes independently transcribed leader RNA species that possess the trans-acting property. This study thus provides further evidence in support of the unique model of "leader-primed transcription" for coronavirus mRNA synthesis.

Murine hepatitis virus↗

PubMed1992

Role of TAR RNA splicing in translational regulation of simian immunodeficiency virus from rhesus macaques.

The untranslated leader sequences of rhesus macaque simian immunodeficiency virus mRNAs form a stable secondary structure, TAR. This structure can be modified by RNA splicing. In this study, the role of TAR splicing in virus replication was investigated. The proportion of viral RNAs containing a spliced TAR structure is high early after infection and decreases at later times. Moreover, proviruses containing mutations which prevent TAR splicing are significantly delayed in replication. These mutant viruses require approximately 20 days to achieve half-maximal virus production, in contrast to wild-type viruses, which require approximately 8 days. We attribute this delay to the inefficient translation of unspliced-TAR-containing mRNAs. The molecular basis for this translational effect was examined in in vitro assays. We found that spliced-TAR-containing mRNAs were translated up to 8.5 times more efficiently than were similar mRNAs containing an unspliced TAR leader. Furthermore, these spliced-TAR-containing mRNAs were more efficiently associated with ribosomes. We postulate that the level of TAR splicing provides a balance for the optimal expression of both viral proteins and genomic RNA and therefore ultimately controls the production of infectious virions.

Animals↗

PubMed1995

Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity.

A splicing event essential for the infectivity of a plant pararetrovirus has been characterized. Transient expression experiments using reporter constructs revealed a splice donor site in the leader sequence of the cauliflower mosaic virus (CaMV) 35S RNA and three additional splice donor sites within open reading frame (ORF) I. All four donors use the same splice acceptor within ORF II. Splicing between the leader and ORF II produces an mRNA from which ORF III and, in the presence of the CaMV translational transactivator, ORF IV can be translated efficiently. The other three splicing events produce RNAs encoding ORF I-II in-frame fusions. All four spliced CaMV RNAs were detected in CaMV-infected plants. Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays an essential role in the CaMV life cycle. The results presented here suggest a model for viral gene expression in which RNA splicing is required to provide appropriate substrate mRNAs for the specialized translation mechanisms of CaMV.

Base Sequence↗

PubMed1987

Trypanosoma cruzi exhibits inter- and intra-strain heterogeneity in molecular karyotype and chromosomal gene location.

Molecular karyotypes of 6 strains and 6 clones of Trypanosoma cruzi were determined using orthogonal-field-alternation gel electrophoresis. At least 15 different chromosome-sized DNA molecules, ranging in size from less than 200 kilobase pairs to greater than 2000 kilobase pairs, were resolved for each of the isolates examined. Many of the bands were present in different relative intensities suggesting that the number of individual chromosomes per organism may be considerably higher. Significant inter- and intra-strain differences in molecular karyotype and in the chromosomal locations of the genes for the spliced leader, tubulins, 5S ribosomal RNA and a heat shock protein were found. These marked chromosomal differences among T. cruzi strains and clones may be related to the high degree of phenotypic heterogeneity previously found in this parasite.

Animals↗

PubMed1993

Trans-splicing of nematode premessenger RNA.

In nematodes, many mRNAs contain a common 5' terminal 22-nt sequence. This sequence, the spliced leader (SL), is acquired from a small (approximately 100 nt) SL RNA via trans-splicing. Parallel in vitro and in vivo experiments have begun to clarify both the mechanism and biological role of trans-splicing. In vitro analysis (in cell free extracts) has shown that trans-splicing is remarkably similar to the snRNP mediated removal of intervening sequences from pre-mRNAs (cis-splicing). Additionally, this analysis has suggested a mechanism that may explain how the two substrates of trans-splicing (the SL RNA and pre-mRNA) efficiently associate with one another in the absence of sequence complementarity. In vivo experiments suggest that a major biological function of trans-splicing in nematodes may be to process polycistronic transcription units. Results obtained from the study of both parasitic and free-living species are discussed, and trans-splicing in nematodes is compared and contrasted to the analogous process in trypanosomatid protozoans.

Animals↗

PubMed2004

[Screening of cDNA clone for putative RNA polymerase subunit of Cysticercus cellulosae].

OBJECTIVE: To obtain related genes of Cysticercus cellulosae from spliced leader (SL) cDNA library. METHODS: Spliced leader library of Cysticercus cellulosae was constructed using SL specific primer and oligo (dT) 15 with M13M4 primer, and positive clones were then screened randomly, identified with enzyme restriction, followed by sequencing and homologous analysis. RESULTS: The amino acid sequence, encoded by the positive clone with a poly (A)22 tail and a complete open reading frame (ORF), was with homology of RNA polymerase subunit genes of human, B. napus, fission yeast, A. thaliana, C. elegans and fruit fly up to 71.6%. CONCLUSION: The protein, RNA polymerase subunit encoded putatively by the clone, is high conservative in different species.

Amino Acid Sequence↗

PubMed1984

Expression of dihydrofolate reductase, and of the adjacent EIb region, in an Ad5-dihydrofolate reductase recombinant virus.

A gene with the Ad2 MLP and first leader, and appropriate RNA processing signals (splicing, polyadenylation) positioned around a mouse DHFR cDNA clone was substituted for the EIa region of Ad5, and virus stocks of Ad5 (DHFR-I) were prepared on 293 cells. A DHFR RNA of the expected size and structure was expressed late after infection of 293 cells by Ad5 (DHFR-I), at levels comparable to that of other Ad5 late messages. Although this DHFR mRNA was translated as efficiently as other Ad late mRNAs in vitro, it was only poorly translated in vivo. The substitution of the DHFR gene for the Ad5 EIa region results in aberrant transcriptional activity in the adjacent EIb sequences. The transcriptional levels of the EIb 1kb message were down approximately 10-fold. In addition, a novel pIX-encoding mRNA was produced, generated by the splicing of the Ad first late leader onto sequences 14 bp upstream from the pIX initiation codon. This new mRNA was found to be potently efficient for translation both in vivo and in vitro.

Adenoviruses, Human↗

PubMed1990

A spliced leader is present on a subset of mRNAs from the human parasite Schistosoma mansoni.

We present evidence that a subset of mRNAs in the human parasitic trematode Schistosoma mansoni contain an identical 36-nucleotide spliced leader (SL) sequence at their 5' termini. The SL is derived from a 90-nucleotide nonpolyadenylylated RNA (SL RNA), presumably by trans-splicing. Neither the SL nor the SL RNA share significant sequence identity with previously described trans-spliced leaders and SL RNAs in trypanosomatid protozoans or nematodes. However, several features, such as predicted secondary structure, trimethylguanosine cap, and potential Sm binding site, suggest similarities among SL RNAs in widely divergent organisms. Our evidence also indicates that the exon 3 acceptor site of the 3-hydroxy-3-methylglutaryl-CoA reductase gene can be spliced either to the SL by trans-splicing or to an upstream exon, 2, by cis-splicing. The presence of a SL sequence in S. mansoni, a member of the phylum Platyhelminthes, suggests that transplicing may be a common feature of other lower invertebrates.

Animals↗

PubMed1989

Leader-to-leader splicing is required for efficient production and accumulation of polyomavirus late mRNAs.

Polyomavirus late mRNA molecules contain multiple, tandem copies of a noncoding 57-base "late leader" exon at their 5' ends. This exon is encoded only once in the genome. Leader multiplicity arises from leader-leader splicing in giant primary transcripts, which are the result of multiple circuits of the viral genome by RNA polymerase II. We have been interested in learning more about the role of the leader exon in late viral gene expression. We recently showed that an abbreviated-leader mutant virus (ALM) with a 9-base leader exon is nonviable (G. R. Adami and G. G. Carmichael, Nucleic Acids Res. 15:2593-2610, 1987) and has a severe defect in both late pre-mRNA splicing and stability. However, a mutant virus with a different, substituted leader sequence of 51 nucleotides (SLM/MP8) is viable and has no apparent defects. Here we examined further the role of the late leader exon in late pre-mRNA processing. When the leader exon length was gradually reduced from 51 nucleotides to 9 nucleotides in a series of mutants, RNA splicing and stability defects were coupled. In this system there was a minimum exon size of between 33 and 27 nucleotides. Next, a number of mutations were introduced into the 3' splice site which precedes the late leader. Such mutations blocked leader-leader splicing. Surprisingly, they also interfered with leader-mVP1 body splicing and resulted in unstable primary transcripts. Thus, polyomavirus leader-leader splicing appears to be important for the efficient accumulation of late viral mRNA molecules.

Animals↗

PubMed2005

Dcp2 Decaps m2,2,7GpppN-capped RNAs, and its activity is sequence and context dependent.

Hydrolysis of the mRNA cap plays a pivotal role in initiating and completing mRNA turnover. In nematodes, mRNA metabolism and cap-interacting proteins must deal with two populations of mRNAs, spliced leader trans-spliced mRNAs with a trimethylguanosine cap and non-trans-spliced mRNAs with a monomethylguanosine cap. We describe here the characterization of nematode Dcp1 and Dcp2 proteins. Dcp1 was inactive in vitro on both free cap and capped RNA and did not significantly enhance Dcp2 activity. Nematode Dcp2 is an RNA-decapping protein that does not bind cap and is not inhibited by cap analogs but is effectively inhibited by competing RNA irrespective of RNA sequence and cap. Nematode Dcp2 activity is influenced by both 5' end sequence and its context. The trans-spliced leader sequence on mRNAs reduces Dcp2 activity approximately 10-fold, suggesting that 5'-to-3' turnover of trans-spliced RNAs may be regulated. Nematode Dcp2 decaps both m(7)GpppG- and m(2,2,7)GpppG-capped RNAs. Surprisingly, both budding yeast and human Dcp2 are also active on m(2,2,7)GpppG-capped RNAs. Overall, the data suggest that Dcp2 activity can be influenced by both sequence and context and that Dcp2 may contribute to gene regulation in multiple RNA pathways, including monomethyl- and trimethylguanosine-capped RNAs.

Animals↗

PubMed2006

Trypanosoma brucei: composition, organisation, plasticity, and differential transcription of NlaIII repeat elements in drug-resistant and sensitive isolates.

A Trypanosoma brucei brucei DNA repeat sequence termed NlaIII repeat (NR) was originally isolated from a multidrug-resistant field isolate CP547 [Jamnadass, R., 1995. Identification and characterisation of an extrachromosomal element from a multidrug-resistant isolate of T. brucei brucei, Ph.D. thesis, Brunel University, UK]. Subsequently studied in a laboratory strain (Tb427) [Alsford, N.S., Navarro, M., Jamnadass, H.R., Dunbar, H., Ackroyd, M., Murphy, N.B., Gull, K., Ersfeld,K., 2003. The identification of circular extrachromosomal DNA in the nuclear genome of T. brucei. Molecular Microbiology 47, 277-288], NRs were exclusively episomal. Here we show that NR sequences in CP547 are present on linear chromosomes as well as on episomal circular elements. Sequence analysis shows that NRs are composed of three classes of sub-repeat arranged in a specific order. Heterogeneity in size and sequence of an episomal 6.6kbp element was shown in successive passages of the original CP547 isolate and derived clones in mice. Its copy number was unstable and was affected by selective pressure with the trypanocide diminazene aceturate. Some of the extrachromosomal elements appear to be composed of RNA-DNA hybrids. NR sequences were transcribed in a developmentally regulated manner but transcripts did not contain the spliced-leader sequence found on all trypanosome mRNAs.

Animals↗

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↗