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 d erivatives and human-trypanosome hybrid RNAs. Stable binding of both t he U2-specific 40-kDa and the common proteins requires only the 3'-ter minal 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 p roteins; surprisingly, the sequence of the ''Sm-analogous'' single-str anded region between stem-loops IIb and IV is not essential for protei n binding. Our mutational analysis further indicates that interactions between common and specific proteins play an important role in the as sembly of a stable core complex. Finally, a partially assembled U2 RNP complex could be identified as a kinetic intermediate of U2 snRNP ass embly. We propose a model of the domain structure and assembly of the trans-spliceosomal U2 snRNP, which deviates in several aspects from th at of the cis-spliceosomal U2 snRNP; these differences may be related to the trans-splicing-specific functions of the trypanosomal U2 snRNP.