INVITRO CAPPING IN TRYPANOSOMA-CRUZI IDENTIFIES AND SHOWS SPECIFICITY FOR THE SPLICED LEADER RNA AND U-RNAS

Messenger RNA maturation in trypanosomes requires a trans-splicing event in which a capped 39 nucleotide leader sequence, the spliced leader (SL), from the 5' terminus of a small RNA (SL-RNA) is joined to the 5' termini of protein coding gene transcripts. We have developed nuclear extracts from Trypanosoma cruzi that label three small endogenous RNAs in the presence of [α-32P]GTP. Herein, we have characterized this labelling as 5' capping and shown that the capping activity exhibits an unusual ATP dependence. Moreover, partial sequence analysis identified the three cap-labelled RNAs as the T. cruzi SL-RNA, and two U-RNAs previously uncharacterized in T. cruzi, U2 and Ux. Finally, the capping reaction in the T. cruzi extracts showed apparent specificity for these RNAs-other endogenous or exogenous transcripts were not capped. The apparent specificity of this in vitro capping activity closely reflects the in vivo requirements; i.e., only the SL- and U-RNAs need to be capped since mature mRNAs are capped via frans-splicing. These observations are consistent with the hypothesis that one of the functions of trans-splicing is to supply 5' caps to mature trypanosome mRNAs. © 1990 Oxford University Press.