RESUMO
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.
Assuntos
Capuzes de RNA , Splicing de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Trypanosoma cruzi/genética , Animais , Centrifugação com Gradiente de Concentração , Césio , Proteínas de Protozoários/metabolismo , Ribonucleoproteínas Nucleares Pequenas , Trypanosoma cruzi/metabolismoRESUMO
The nuclear extracts from HeLa cells subjected to heat shock at 43 or 46 degrees C for 2 h were unable to splice pre-mRNA in vitro. Analysis of snRNPs in the extracts revealed that the U4.U5.U6 small nuclear ribonucleoprotein particle (snRNP) complex was disrupted at both temperatures while U1 and U2 snRNPs remained unaffected at 43 degrees C but were disrupted to certain extent during heat shock at 46 degrees C. During splicing reaction, the extract from cells heat shocked at 43 degrees C formed intermediate splicing complexes alpha and beta but was unable to form a functional spliceosome, complex gamma. Addition of fractions from a normal nuclear extract restored splicing activity only in the extract from cells subjected to heat shock at 43 degrees C. Using this complementation assay, we have partially purified the factor(s) inactivated at this temperature. The purified factor(s) was essentially devoid of snRNAs and snRNPs and resistant to micrococcal nuclease, indicating that the factor(s) inactivated by in vivo heat shock at 43 degrees C is a protein. We have also subjected the nuclear extracts from normal HeLa cells to in vitro heat treatment at 43 or 46 degrees C. The results indicate that during in vitro heat treatment of the extracts the damage to splicing machinery is more extensive than that during in vivo heat shock. These experiments also suggest that the factor(s) inactivated by heat shock at 43 degrees C is different from previously identified thermolabile splicing factors.
Assuntos
Precursores de RNA/genética , Splicing de RNA , Ribonucleoproteínas/metabolismo , Núcleo Celular/metabolismo , Células HeLa/metabolismo , Temperatura Alta , Humanos , Cinética , Nuclease do Micrococo , Precursores de RNA/metabolismo , Ribonucleoproteínas/isolamento & purificação , Ribonucleoproteínas Nucleares Pequenas , Moldes Genéticos , Transcrição GênicaRESUMO
Messenger RNA maturation in trypanosomes requires a trans-splicing event in which a capped 39 nucleotide leader sequence, the spliced leader (SL), from the 5' terminus of a small RNA (SL-RNA) is joined to the 5' termini of protein coding gene transcripts. We have developed nuclear extracts from Trypanosoma cruzi that label three small endogenous RNAs in the presence of [alpha-32P]GTP. Herein, we have characterized this labelling as 5' capping and shown that the capping activity exhibits an unusual ATP dependence. Moreover, partial sequence analysis identified the three cap-labelled RNAs as the T. cruzi SL-RNA, and two U-RNAs previously uncharacterized in T. cruzi, U2 and Ux. Finally, the capping reaction in the T. cruzi extracts showed apparent specificity for these RNAs--other endogenous or exogenous transcripts were not capped. The apparent specificity of this in vitro capping activity closely reflects the in vivo requirements; i.e., only the SL- and U-RNAs need to be capped since mature mRNAs are capped via trans-splicing. These observations are consistent with the hypothesis that one of the functions of trans-splicing is to supply 5' caps to mature trypanosome mRNAs.
Assuntos
Capuzes de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , Trypanosoma cruzi/genética , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Guanosina Trifosfato/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismoRESUMO
Pre-mRNA in kinetoplastids is processed to maturity following unique pathways requiring a transplicing event that links a common 39 nucleotide leader to the 5' termini of the mature mRNAs. The mechanisms of this reaction and other steps of mRNA processing; i.e., 5' capping and 3' cleavage and polyadenylation, have not been resolved. Herein, we describe a 3' polyadenylation activity in cell-free extracts prepared from nuclei isolated from Trypanosoma cruzi, the kinetoplastid agent of Chagas' Disease. Synthetic RNA transcripts incubated in these extracts in the presence of ATP are 3' polyadenylated. This polyadenylation activity is sensitive to heat or pre-treatment of the extract with Micrococcal nuclease, suggesting that an RNA-protein complex is required. As these are characteristics of polyadenylation activities in other eukaryotes, we believe that this activity may participate in the in vivo trypanosome mRNA polyadenylation system. Several other modification activities specific for RNA 3' termini, including terminal nucleotide transferases, a tRNA CCA maturation activity, and a 3' exonuclease were also identified in these T. cruzi nuclear extracts.
Assuntos
Poli A/genética , Processamento Pós-Transcricional do RNA , RNA/genética , Trypanosoma cruzi/genética , Animais , Ligação Competitiva , Núcleo Celular/enzimologia , Núcleo Celular/metabolismo , Citidina Desaminase/metabolismo , DNA Nucleotidilexotransferase/metabolismo , Exonucleases/metabolismo , Cinética , Poli A/antagonistas & inibidores , Poli A/metabolismo , RNA/antagonistas & inibidores , RNA/metabolismo , RNA Mensageiro , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/metabolismoRESUMO
The effect of the alkaloid sparteine on arginyl-tRNA formation was studied. It was demonstrated that sparteine sulfate in the concentration range 10-60 mM inhibits the charging reaction when amino acid, ATP and tRNA are used as variable substrates. The mode of action is different for all pattern of inhibition for all varied substrates is generally uncompetitive. A pattern of inhibition for all varied substrates is generally uncompetitive. A non-competitive mechanism for amino acid and tRNA was observed at low sparteine concentration, but in the case of ATP it is also uncompetitive.
Assuntos
RNA de Transferência/metabolismo , Esparteína/farmacologia , Alcaloides/farmacologia , Arginina-tRNA Ligase/metabolismo , Aminoacil-RNA de Transferência/antagonistas & inibidoresRESUMO
A method for detection of enzymatic activities which use aminoacyl-tRNA is described. It is based on the synthesis of aminoacyl-tRNA in the reaction mixture (in situ) without additional purification. The results are the same as when using purified AA-tRNA.
