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.All protein-coding mRNAs analyzed in trypanosomes to date arise by trans splicing of the 140-nucleotide spliced leader RNA (SL RNA), which contributes the common, 40-nucleotide spliced leader, and a precursor RNA with the protein-coding exon. The chemistry of cleavage-ligation reactions follows the same principles in both cis and trans splicing, with small nuclear ribonucleoproteins (snRNPs) functioning as essential cofactors (51). Trypanosomatids appear to process mRNAs exclusively through trans splicing, whereas in nematodes both cis and trans splicing can occur within the same precursor RNA (reviewed in references 1, 6, 25, and 37). Of the spliceosomal snRNAs, only U2, U4, and U6 homologs have been identified in trypanosomes (35,49,50). From extensive phylogenetic sequence comparisons, it is evident that many sequence elements and parts of the proposed secondary structures of U2, U4, and U6, such as the U4-U6 base-pairing interaction, are conserved between cis-and trans-spliceosomal systems (18). These common elements may reflect pathways of snRNA maturation, snRNP assembly, and snRNP transport or mechanistic principles that are shared by cis-and transsplicing systems. However, phylogenetic analysis has also revealed that trypanosomal snRNAs principally deviate from their counterparts of other eucaryotes functioning in cis splicing. It will be important to relate these differences in RNA sequence and secondary structure to the organization of the RNA-protein complexes and ultimately to functional, mechanistic differences between cis and trans splicing.In trypanosomes, no homologs of the cis-spliceosomal Ul and U5 snRNPs are known; the SL RNA, in the form of a ribonucleoprotein, may perform the function of the Ul snRNP, catalyzing trans splicing of its own 5'-terminal spliced leader portion (9). In contrast to the trans-spliceosomal U2 and U4/U6 snRNPs, detailed structural information is available on the cis-spliceo...