In vitro 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 [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 trans-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.
Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond 23298.
T A
TA
Zwierzynski
G A
GA
Buck
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012315:RNA Caps / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics* / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000235:genetics*
D012315:RNA Caps; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate