Assembly of the U2 small nuclear ribonucleoprotein from Trypanosoma brucei. A mutational analysis.
trans-Splicing in trypanosomes requires the functions of U2 and U4/U6 small nuclear (sn) RNPs. We have analyzed protein binding and assembly of the Trypanosoma brucei U2 snRNP, using specific antibodies against U2 snRNP proteins and in vitro reconstitution assays of U2 deletion derivatives and human-trypanosome hybrid RNAs. Stable binding of both the U2-specific 40-kDa and the common proteins requires only the 3'-terminal domain (stem-loop IIb, single-stranded region, and stem-loop IV), with loop IV providing the critical sequence determinant; stem-loop IV suffices for binding of the 40 kDa-protein, but not of the common proteins; surprisingly, the sequence of the "Sm-analogous" single-stranded region between stem-loops IIb and IV is not essential for protein binding. Our mutational analysis further indicates that interactions between common and specific proteins play an important role in the assembly of a stable core complex. Finally, a partially assembled U2 RNP complex could be identified as a kinetic intermediate of U2 snRNP assembly. We propose a model of the domain structure and assembly of the trans-spliceosomal U2 snRNP, which deviates in several aspects from that of the cis-spliceosomal U2 snRNP; these differences may be related to the trans-splicing-specific functions of the trypanosomal U2 snRNP.
Max-Planck-Institut für Molekulare Genetik, Otto-Warburg-Laboratorium, Berlin, Federal Republic of Germany.
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Günzl
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Cross
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Palfi
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Bindereif
D000818:Animals; D001483:Base Sequence; D016054:DNA, Protozoan; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D016053:RNA, Protozoan / Q000737:chemistry / Q000378:metabolism; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
D016054:DNA, Protozoan; D016053:RNA, Protozoan; D017413:Ribonucleoprotein, U2 Small Nuclear