RNA-protein complexes mediate in vitro capping of the spliced-leader primary transcript and U-RNAs in Trypanosoma cruzi.

Abstract

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 lO-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 >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. mRNA maturation in the kinetoplastid protozoa requires an unusual trans-splicing process in which a 39-nucleotide leader, the spliced leader (SL), from a small abundant transcript, the SL primary transcript (SL-RNA), is ligated to the 5' ends of structural gene primary transcripts (1). The SL-RNA obtains an unusual 5' cap that is transferred to the mature mRNA with the SL (2-5). In other eukaryotes, mRNA primary transcripts are capped shortly after transcription initiation by guanylyltransferases (GTases) that are probably associated with the RNA polymerase II (Pol II) initiation complex in a reaction requiring a 5'-tri-or -diphosphate substrate RNA (6). Although capping is thought to be mediated by the Pol II transcription initiation complex (6), soluble partially purified GTases lack substrate specificity and require only the energy from cleavage of the GTP substrate for activity. In the kinetoplastid protozoa, there is no evidence that mRNA primary transcripts are capped, many genes are transcribed as "multicistronic" RNAs that are processed into monocistronic mRNAs (cf. refs. 7 and 8), and some protein coding genes may not be transcribed by a normal a-amanitin-sensitive Pol 11 (9-11). Thus, it has been proposed that trans-splicing evolved in the kinetoplastid protozoa as a means of capping protein coding mRNAs (12).We have established (13) nuclear extracts from Trypanosoma cruzi that cap the SL-RNA and at least two U-RNAs...

Genome-wide analysis of trans-splicing in the nematode Pristionchus pacificus unravels conserved gene functions for germline and dauer development in divergent operons

“…This short noncoding RNA is called a spliced leader (SL) and varies in length and sequence in different species, ranging from 16-51 nucleotides (nt). The requirement of trans-splicing in such diverse lineages is not completely understood, but existing models propose their roles in various processes such as resolution of individual cistrons from poly-cistronic transcripts (Blumenthal 1995;Blaxter and Liu 1996;Clayton 2002), stabilization of mRNA by the Tri-Methyl-Guanosine (TMG) cap at the 5 ′ end of the spliced leader (Zwierzynski and Buck 1991), and increased translational efficiency (Maroney et al 1995;Lall et al 2004). Trypanosomes have essentially polycistronic transcription, and hence all genes are trans-spliced to a spliced leader.…”