Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP.
trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.
Cross
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Max-Planck-Institut für Molekulare Genetik, Otto-Warburg-Laboratorium, Berlin, Dahlem, Germany.
Günzl
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Palfi
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Bindereif
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D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000235:genetics*; D016914:Ribonuclease H; D012261:Ribonucleoproteins / Q000235:genetics* / Q000302:isolation & purification; D017411:Ribonucleoproteins, Small Nuclear; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000235:genetics*
D009841:Oligonucleotides; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D016914:Ribonuclease H