Evidence for a capping enzyme with specificity for the trypanosome spliced leader RNA.
Capping of the pre-mRNA 5' end by addition a monomethylated guanosine cap (m(7)G) is an essential and the earliest modification in the biogenesis of mRNA. The reaction is catalyzed by three enzymes: triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase. Whereas this modification occurs co-transcriptionally in most eukaryotic organisms, trypanosomatid protozoa mRNAs acquire the m(7)G cap by trans-splicing, which entails the transfer of the capped spliced leader (SL) from the SL RNA to the mRNA. Intriguingly, the genomes of all trypanosomatid protozoa sequenced to date possess two distinct proteins with the signature motifs of guanylyltransferases: TbCGM1 and the previously characterized TbCE1. Here we provide biochemical evidence that TbCgm1 is a capping enzyme. Whereas RNAi-induced downregulation of TbCe1 had no phenotypic consequences, we found that TbCGM1 is essential for trypanosome viability and is required for SL RNA capping. Furthermore, consistent with co-transcriptional addition of the m(7)G cap, chromatin immunoprecipitation revealed recruitment of TbCgm1 to the SL RNA genes.
Ruan
Jia-Peng
JP
Department of Epidemiology & Public Health, Yale University Medical School, 295 Congress Avenue, New Haven, CT 06536-0812, USA.
Shen
Shuiyuan
S
Ullu
Elisabetta
E
Tschudi
Christian
C
D000818:Animals; D002470:Cell Survival; D047369:Chromatin Immunoprecipitation; D020868:Gene Silencing; D020043:Genes, Essential; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011485:Protein Binding; D015800:Protozoan Proteins / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism*; D034622:RNA Interference; D020038:RNA, Spliced Leader / Q000378:metabolism*; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
D015800:Protozoan Proteins; D012315:RNA Caps; D020038:RNA, Spliced Leader; C526771:CGM1 protein, Trypanosoma brucei; D009713:Nucleotidyltransferases