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Nucleic Acids Research, 1990, Vol. 18, No. 14 4197-4206
© 1990

MOLECULAR BIOLOGY

In vitro capping in Trypanosoma cruzi identifies and shows specificity for the spliced leader RNA and U-RNAs

Tadeusz A. Zwierzynski and Gregory A. Buck*

Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University Richmond, VA 23298, USA

* To whom correspondence should be addressed

Received March 21, 1990. Revised June 8, 1990. Accepted June 8, 1990.

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 [{alpha}-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.


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E. Silva, E. Ullu, R. Kobayashi, and C. Tschudi
Trypanosome Capping Enzymes Display a Novel Two-Domain Structure
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[Abstract] [Full Text]


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