Domain structure of U2 and U4/U6 small nuclear ribonucleoprotein particles from Trypanosoma brucei: identification of trans-spliceosomal specific RNA-protein interactions.
Maturation of mRNAs in trypanosomes involves trans splicing of the 5' end of the spliced leader RNA and the exons of polycistronic pre-mRNAs, requiring small nuclear ribonucleoproteins (snRNPs) as cofactors. We have mapped protein-binding sites in the U2 and U4/U6 snRNPs by a combination of RNase H protection analysis, native gel electrophoresis, and CsCl density gradient centrifugation. In the U2 snRNP, protein binding occurs primarily in the 3'-terminal domain; through U2 snRNP reconstitution and chemical modification-interference assays, we have identified discrete positions within stem-loop IV of Trypanosoma brucei U2 RNA that are essential for protein binding; significantly, some of these positions differ from the consensus sequence derived from cis-spliceosomal U2 RNAs. In the U4/U6 snRNP, the major protein-binding region is contained within the 3'-terminal half of U4 RNA. In sum, while the overall domain structure of the U2 and U4/U6 snRNPs is conserved between cis- and trans-splicing systems, our data suggest that there are also trans-spliceosomal specific determinants of RNA-protein binding.
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Max-Planck-Institut für Molekulare Genetik, Otto-Warburg-Laboratorium, Berlin Dahlem, Germany.
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D002498:Centrifugation; D016384:Consensus Sequence; D004586:Electrophoresis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D011485:Protein Binding; D012326:RNA Splicing; D016053:RNA, Protozoan / Q000737:chemistry / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear; D014346:Trypanosoma brucei brucei / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D016053:RNA, Protozoan; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D016914:Ribonuclease H