Trypanosoma brucei Encodes a Bifunctional Capping Enzyme Essential for Cap 4 Formation on the Spliced Leader RNA

Yuko Takagi,Shalaka Sindkar,Dimitra Ekonomidis,Megan P Hall,C Kiong Ho

doi:10.1074/jbc.m701569200

Abstract

The 5' end of kinetoplastid mRNA possesses a hypermethylated cap 4 structure, which is derived from standard m7GpppN (cap 0) with additional methylations at seven sites within the first four nucleosides on the spliced leader RNA. In addition to TbCe1 guanylyltransferase and TbCmt1 (guanine N-7) methyltransferase, Trypanosoma brucei encodes a second cap 0 forming enzyme. TbCgm1 (T. brucei cap guanylyltransferase-methyltransferase) is a novel bifunctional capping enzyme consisting of an amino-terminal guanylyltransferase domain and a carboxyl-terminal methyltransferase domain. Recombinant TbCgm1 transfers the GMP to spliced leader RNA (SL RNA) via a covalent enzyme-GMP intermediate, and methylates the guanine N-7 position of the GpppN-terminated RNA to form cap 0 structure. The two domains can function autonomously in vitro. TbCGM1 is essential for parasite growth. Silencing of TbCGM1 by RNA interference increased the abundance of uncapped SL RNA and lead to accumulation of hypomethylated SL RNA. In contrast, silencing of TbCE1 and TbCMT1 did not affect parasite growth or SL RNA capping. We conclude that TbCgm1 specifically cap SL RNA, and cap 0 is a prerequisite for subsequent methylation events leading to the formation of mature SL RNA.

Highlights

Phatase-guanylyltransferase protein and a separate methyltransferase protein (4 –9)
The cap 4 structure is formed exclusively on the spliced leader RNA (SL RNA) synthesized by pol II, and is transferred via trans-splicing to the 5Ј end of individual pre-mRNAs derived from a polycistronic transcript to form mature mRNAs
TbCgm1 Is a Bifunctional Capping Enzyme with Guanylyltransferase and Methyltransferase Activities—To determine whether TbCgm1 has an intrinsic capping activity, we expressed the recombinant protein in E. coli as an NH2-terminal His-Smt3-tagged fusion protein to facilitate solubility and purification

Summary

Introduction

Phatase-guanylyltransferase protein and a separate (guanine N-7) methyltransferase protein (4 –9). The methyltransferase activity of TbCgm1 was assayed by conversion of 32P cap-labeled poly(A) to methylated caplabeled poly(A) in the presence of AdoMet. Digestion by nuclease P1 liberated a labeled species that co-migrated with m7GpppA, generated in a parallel reaction mixture containing purified yeast Abd1 (Fig. 1D, lanes 2 and 5, respectively).

Results

Conclusion