The role of intron structures in trans-splicing and Cap 4 formation for the Leishmania spliced leader RNA

A 39-nucleotide leader is trans-spliced onto all trypanosome nuclear mRNAs. The precursor spliced leader RNA was tested for trans-splicing function in vivo by mutating the intron. We report that in Leishmania tarentolae splic ed leader RNA 5' modification is influenced by the primary sequence of stem -loop II, the Sm-binding site, and the secondary structure of stem-loop III . The sequence of stem-loop II was found to be important for cap 4 formatio n and splicing. As in Ascaris, mutagenesis of the bulge nucleotide in stem- loop II was detrimental to trans-splicing. Because restoration of the L. ta rentolae stem-loop II structure was not sufficient to restore splicing, thi s result contrasts the findings in the kinetoplastid Leptomonas, where muta tions that restored stem-loop II structure supported splicing. Methylation of the cap 4 structure and splicing was also dependent on both the Sm-bindi ng site and the structure of stem-loop III and was inhibited by incomplete 3' end processing. The critical nature of the L. tarentolae Sm-binding site is consistent with its essential role in the Ascaris spliced leader RNA, w hereas in Leptomonas mutation of the Sm-binding site and deletion of stem-l oop III did not affect trans-splicing. A pathway for Leishmania spliced lea der RNA processing and maturation is proposed.