RNA-protein complexes mediate in vitro capping of the spliced-leader primary transcript and U-RNAs in Trypanosoma cruzi.
A 39-nucleotide spliced leader (SL) is joined to the 5' ends of trypanosome mRNAs in a bimolecular or trans-splicing process. The SL in Trypanosoma cruzi is transcribed as an approximately 110-nucleotide RNA (SL-RNA or SL primary transcript) bearing the 39-nucleotide SL at the 5' end. The SL-RNA is 5' capped by a guanylyltransferase activity prior to trans-splicing and trypanosome mRNAs thus obtain their mature caps from the SL by trans-splicing. We have previously characterized a guanylyltransferase activity from T. cruzi nuclear extracts and shown that this capping activity has an unusual ATP dependence and an apparent specificity for the SL-RNA and U-RNAs. Herein, we show that the capping activity sediments as a 12-15S particle during velocity sedimentation in glycerol gradients and fractionates as a greater than 150-kDa particle during large-pore gel filtration chromatography. Moreover, the endogenous substrate RNAs--the SL-RNA and U-RNAs--consistently copurify with the capping activity, suggesting that the activity and the substrates form a ribonucleoprotein particle. The capping activity and substrate RNAs are not dissociated in isopycnic Cs2SO4 gradients and band at a density expected for an RNA-protein complex, confirming the existence of ribonucleoprotein particles bearing both the activity and its substrate RNAs. Finally, we partially purified these ribonucleoprotein particles and showed that the capping activity remains ATP dependent and highly specific for the SL-RNA and the U-RNAs. These observations are consistent with the hypothesis that one of the functions of trans-splicing is for mRNA capping.
Zwierzynski
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TA
Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond 23298.
Buck
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GA
D000818:Animals; D002499:Centrifugation, Density Gradient; D002586:Cesium; D015800:Protozoan Proteins / Q000378:metabolism; D012315:RNA Caps; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000378:metabolism
D015800:Protozoan Proteins; D012315:RNA Caps; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D002586:Cesium; C025407:cesium sulfate