Trypanosome Capping Enzymes Display a Novel Two-Domain Structure

ABSTRACT

The ubiquitous m⁷G cap of eukaryotic mRNAs and of precursors to the spliceosomal small nuclear RNAs (snRNAs) is the result of an essential RNA modification acquired during transcript elongation. In trypanosomes, the m⁷G cap is restricted to the spliced leader (SL) RNA and the precursors of U2, U3, and U4 snRNAs. mRNA capping in these organisms occurs posttranscriptionally bytrans splicing, which transfers the capped SL sequence to the 5′ ends of all mRNAs. The SL cap is the most elaborate cap structure known in nature and has been shown to consist of an m⁷G residue followed by four methylated nucleotides. UsingCrithidia fasciculata, we have characterized and purified the guanylyltransferase (capping enzyme), which transfers GMP from GTP to the diphosphate end of RNA. The corresponding gene codes for a protein of 697 amino acids, with the carboxy-terminal half of the C. fasciculata guanylyltransferase containing the six signature motifs previously identified in yeast capping enzymes. The amino-terminal half contains a domain that displays no resemblance to any other domain associated with capping enzymes. Intriguingly, this region harbors a consensus sequence for a phosphate-binding loop which is found in ATP- and GTP-binding proteins. This two-domain structure is also present in the Trypanosoma brucei capping enzyme, which shows 44% overall identity with the C. fasciculatacapping enzyme. Thus, this structure appears to be common to all trypanosomatid protozoa and defines a novel class of capping enzymes.

ACKNOWLEDGMENTS

We thank Diane MacMahon-Pratt for generously providing the C. fasciculata genomic library and Helen Kwon for excellent technical assistance. We thank David Bermudes, Christopher Yoo, and members of our laboratory for valuable criticism on the manuscript.

This investigation received financial support from National Institutes of Health grants AI28798 to E.U. and CA45508 to R.K. and from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) (grant to C.T.).