Functional analyses of positions across the 5 ' splice site of the trypanosomatid spliced leader RNA - Implications for base-pair Interaction with U5and U6 snRNAs

In this study, we have used a genetic compensatory approach to examine the functional significance of the previously proposed interaction of spliced l eader (SL) RNA with U5 small nuclear RNA (snRNA) (Dungan, J. D., Watkins, K . P., and Agabian, N. (1996) EMBO J. 15, 4016-4029; Xu, Y.-X., Ben Shlomo, H., and Michaeli, S. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 8473-8478) an d the interaction of the SL RNA intron with U6 snRNA analogous to cis-splic ing. Mutations were introduced at positions -4, -1, +1, +4, +5, and +7/+8 r elative to the SL RNA 5' splice site that were proposed to interact with U5 and U6 snRNAs, All mutants exhibited altered splicing phenotypes compared with the parental strain, showing the importance of these intron and exon p ositions for transsplicing. Surprisingly, mutation at invariant +1 position did not abolish splicing completely, unlike cia-splicing, but position +2 had the most severe effect on transsplicing. Compensatory mutations were in troduced in U5 and U6 snRNAs to examine whether the defects resulted from f ailure to interact with these snRNAs by base pairing, Suppression was obser ved only for positions +5 and +7/+8 with U5 compensatory mutations and for position +5 with a U6 compensatory mutation, supporting the existence of a base pair interaction of U5 and U6 with the SL RNA intron region. The failu re to suppress the other SL RNA mutants by the U5 compensatory mutations su ggests that another factor(s) interacts with these key SL RNA positions.