Assuntos
Ascaris lumbricoides/metabolismo , RNA de Helmintos/metabolismo , RNA Nuclear Pequeno/metabolismo , Spliceossomos/metabolismo , Animais , Ascaris lumbricoides/genética , Sequência de Bases , Genes de Helmintos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Splicing de RNA , RNA de Helmintos/química , RNA de Helmintos/genética , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genéticaRESUMO
The spliced leader RNAs of both trypanosomes and nematodes can form similar secondary structures where the trans-splice donor site is involved in intramolecular base pairing with the spliced leader sequence. It has been proposed that this base pairing could serve to activate autonomously the SL RNA splice donor site. Here, we have examined exon requirements for trans-splicing in a nematode cell free system. Complete disruption of secondary structure interactions at and around the trans-splice donor site did not affect the ability of the SL RNA to function in trans-splicing. In addition, the highly conserved 22 nt sequence could be productively replaced by artificial exons ranging in size from 2 to 246 nucleotides. These results reinforce the view that the 'intron' portion of the SL RNA functions as an independent Sm snRNP whose role is to deliver exon sequences to the trans-spliceosome.
Assuntos
Composição de Bases , Nematoides/genética , Sinais Direcionadores de Proteínas/metabolismo , Splicing de RNA , Animais , Sequência de Bases , Éxons , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismoRESUMO
We have used block substitution mutagenesis and in vitro transcription to define sequence elements important for efficient initiation and 3' end-formation of the trans-spliced leader RNA (SL RNA) of the parasitic nematode Ascaris lumbricoides. These experiments indicate that the SL RNA has an unusual promoter structure containing elements which include the 22 nt trans-spliced leader exon itself. Efficient transcription is correlated with the binding of a factor to the 22 nt (SL) sequence; mutations within the SL which abolish transcription lead to a loss in binding of this factor. In addition to internal sequences, synthesis of SL RNA in vitro requires an element centered 50 bases upstream of the cap site. Mutations within this element dramatically affect the level of SL RNA synthesis but do not affect accuracy of initiation. Finally, all of the information required for accurate 3' end-formation of SL RNA lies within the transcribed region. Thus, the arrangement of sequences necessary for the synthesis of SL RNAs does not resemble that of sequences important for the synthesis of vertebrate U snRNAs despite the similarities between SL RNAs and U snRNAs.
