RESUMO
A genomic library of Brugia malayi was constructed and screened by hybridization with a cDNA clone corresponding to a potentially protective antigen of 63 kDa. The antigen is encoded by a multicopy gene family. Five distinct gene copies were isolated and one was characterized in detail by nucleotide sequence analysis. An apparent pseudogene was also characterized. The organization of genes encoding the antigen is typical of higher eukaryotes in exon/intron organization although the introns have an unusually high A+T content (75%). Organization of the genomic sequence along with S1 nuclease and primer extension analyses indicate that a short untranslated exon is spliced to the 5' end of the mRNAs encoding the antigen.
Assuntos
Antígenos de Helmintos/genética , Brugia/genética , Família Multigênica , Animais , Bacteriófago lambda , Sequência de Bases , Mapeamento Cromossômico , DNA , Enzimas de Restrição do DNA , Éxons , Genes Reguladores , Íntrons , Microfilárias/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , PseudogenesAssuntos
Ascaris lumbricoides/genética , Proteínas de Ligação a DNA/metabolismo , Sinais Direcionadores de Proteínas/metabolismo , Splicing de RNA , Animais , Sequência de Bases , Cromatografia de Afinidade , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Transcrição GênicaRESUMO
The trans-spliced leader RNA (SL RNA) of nematodes resembles U snRNAs both in cap structure and in the presence of a consensus Sm binding site. We show here that synthetic SL RNA, synthesized by in vitro transcription, is efficiently used as a spliced leader donor in trans-splicing reactions catalyzed by a cell free extract prepared from developing embryos of the parasitic nematode, Ascaris lumbricoides. Efficient utilization of synthetic SL RNA requires a functional Sm binding site. Mutations within the Sm binding sequence that prevent immunoprecipitation by Sm antisera and prevent cap trimethylation abolish trans-splicing. The effect on trans-splicing is not due to undermethylation of the cap structure.
Assuntos
Ascaris/genética , Splicing de RNA , Ribonucleoproteínas/fisiologia , Animais , Autoantígenos/fisiologia , Sequência de Bases , Sítios de Ligação , Análise Mutacional de DNA , Técnicas In Vitro , Dados de Sequência Molecular , Precursores de Ácido Nucleico/metabolismo , Oligonucleotídeos , Capuzes de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas Nucleares Pequenas , Proteínas Centrais de snRNPRESUMO
In nematodes, a fraction of mRNAs contains a common 22 nucleotide 5' terminal spliced leader (SL) sequence derived by trans splicing. Here, we show that a cell-free extract prepared from developing embryos of the parasitic nematode Ascaris lumbricoides catalyzes accurate and efficient SL addition to a synthetic pre-mRNA at an authentic trans splice acceptor site. SL addition occurs via a trans splicing reaction that proceeds through Y-branched intermediates. The branchpoint is located at either of two adenosine residues located 18 and 19 nucleotides upstream of the splice acceptor site.
Assuntos
Ascaris/genética , Precursores de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , Animais , Sequência de Bases , Endorribonucleases , Dados de Sequência Molecular , Mapeamento de Nucleotídeos , Sondas de Oligonucleotídeos , Ribonuclease H , Ribonuclease T1RESUMO
Some pre-mRNAs in nematodes are processed by trans-splicing. In this reaction, a 22-nt 5' terminal exon (the spliced leader, SL) and its associated 2,2,7-trimethylguanosine cap are acquired from a specialized Sm snRNP, the SL RNP. Although it has been evident for many years that not all nematode mRNAs contain the SL sequence, the prevalence of trans-spliced mRNAs has, with the exception of Caenorhabditis elegans, not been determined. To address this question in an organism amenable to biochemical analysis, we have prepared a message-dependent protein synthesis system from developing embryos of the parasitic nematode, Ascaris lumbricoides. Using this system, we have used both hybrid-arrest and hybrid-selection approaches to show that the vast majority (80-90%) of A. lumbricoides mRNAs contain the SL sequence and therefore are processed by trans-splicing. Furthermore, to examine the effect of SL addition on translation, we have measured levels of protein synthesis in extracts programmed with a variety of synthetic mRNAs. We find that the SL sequence itself and its associated hypermethylated cap functionally collaborate to enhance translational efficiency, presumably at the level of initiation of protein synthesis. These results indicate that trans-splicing plays a larger role in nematode gene expression than previously suspected.
Assuntos
Ascaris lumbricoides/genética , Biossíntese de Proteínas , Splicing de RNA , RNA Mensageiro/genética , Animais , Ascaris lumbricoides/embriologia , Sistema Livre de Células , Genes de Helmintos , Hibridização de Ácido Nucleico , Capuzes de RNA , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA Mensageiro/metabolismoRESUMO
The mRNAs encoding a 63-kDa antigen in the human parasitic nematode Brugia Malayi contain a spliced leader sequence of 22 nucleotides (nt) that is identical to the trans-spliced leader found on certain actin mRNAs in the distantly related nematode Caenorhabditis elegans. The 22-nt sequence does not appear to be encoded near the 63-kDa genes but is present in multiple copies in several locations within the parasite genome, including the 5S rRNA gene repeat. The 5S-linked copies of the 22-nt sequence are transcribed to yield a 109-nt nonpolyadenylated RNA with the 22-nt leader sequence at its 5' end. We suggest that the 22-nt leader is acquired by 63-kDa antigen mRNAs through trans-splicing. These results indicate that trans-splicing is widespread in nematodes and argue for the functional significance of the 22-nt spliced leader exon in nematode mRNA metabolism.
Assuntos
Brugia/genética , Caenorhabditis/genética , Splicing de RNA , Animais , Sequência de Bases , Humanos , Dados de Sequência Molecular , Peso Molecular , RNA Ribossômico 5S/análise , Sequências Repetitivas de Ácido Nucleico , Transcrição GênicaRESUMO
DNA sequence analysis of genes encoding 5S rRNA in the human parasitic nematode Brugia malayi (B. malayi) indicates a surprising degree of heterogeneity. This variation in coding sequence is not accompanied by corresponding heterogeneity in flanking regions which are highly conserved. Six out of eight potential 5S coding regions differed; of these sequence variants, two were abundant in the B. malayi genome. Direct RNA sequence analysis indicated that one of these abundant variants accounts for most if not all of expressed 5S RNA at two stages of development.
Assuntos
Brugia/genética , Genes , RNA Ribossômico 5S/genética , RNA Ribossômico/genética , Animais , Sequência de Bases , Southern Blotting , Deleção Cromossômica , Clonagem Molecular , DNA/genética , Variação Genética , Dados de Sequência MolecularRESUMO
The 5' exon donor in nematode trans-splicing, the SL RNA, is a small (approximately 100 nt) RNA that resembles cis-spliceosomal U snRNAs. Extensive analyses of the RNA sequence requirements for SL RNA function have revealed four essential elements, the core Sm binding site, three nucleotides immediately downstream of this site, a region of Stem-loop II, and a 5' splice site. Although these elements are necessary and sufficient for SL RNA function in vitro, their respective roles in promoting SL RNA activity have not been elucidated. Furthermore, although it has been shown that assembly of the SL RNA into an Sm RNP is a prerequisite for function, the protein composition of the SL RNP has not been determined. Here, we have used oligoribonucleotide affinity to purify the SL RNP and find that it contains core Sm proteins as well as four specific proteins (175, 40, 30, and 28 kDa). Using in vitro assembly assays; we show that association of the 175- and 30-kDa SL-specific proteins correlates with SL RNP function in trans-splicing. Binding of these proteins depends upon the sequence of the core Sm binding site; SL RNAs containing the U1 snRNA Sm binding site assemble into Sm RNPs that contain core, but not SL-specific proteins. Furthermore, mutational and thiophosphate interference approaches reveal that both the primary nucleotide sequence and a specific phosphate oxygen within a segment of Stemloop II of the SL RNA are required for function. Finally, mutational activation of an unusual cryptic 5' splice site within the SL sequence itself suggests that U5 snRNA may play a primary role in selecting and specifying the 5' splice site in SL addition trans-splicing.
Assuntos
Ascaris/genética , Splicing de RNA , RNA de Helmintos/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Oxigênio/metabolismo , RNA de Helmintos/química , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
To facilitate biochemical studies of protective filarial antigens, a lambda gt11 cDNA library was constructed from Brugia malayi adult mRNA and screened with rabbit sera that recognizes a limited set of filarial antigens of approximately 25, 42, 60, and 112 kDa. Antigens of approximately equal to 25 and approximately equal to 60 kDa have been shown previously to induce enhanced clearance of microfilaremia in mice. A 154-base pair clone detected by immunological reactivity was used to isolate by hybridization a nearly full-length cDNA clone of 1.8 kilobases. Nucleotide-sequence analysis indicated that this clone was derived from a mRNA encoding a 63-kDa antigen. A fusion polypeptide containing 37 kDa of the Escherichia coli TrpE protein (anthranilate synthase) and 55 kDa of the cloned protein was recognized in immunoblot experiments with antisera raised against a partially purified preparation of the approximately equal to 60-kDa protective filarial antigen. These data relate the cloned antigen to a potentially protective antigen in lymphatic filariasis.