Short communication

Cloning and sequence of a Leishmania major homologue to the fibrillarin gene

Giardia lamblia, the ancient eukaryote does not have nucleolus but produces the fibrillarin protein that may be used for pre-rRNA processing. The nucleoli of eukaryotes contain complex population of small nucleolar RNAs, known as snoRNAs, several of which are required for rRNA processing. This report describes the full-length cloning of fibrillarin gene from Giardia lamblia, using RTPCR and the production of recombinant fibrillarin protein in Escherichia coli strain BL21 (DE3) as N-terminal His-tag protein. The condition for production of soluble protein was standardized. The expressed protein was purified by using Ni-chelation chromatography and used for functional studies. The small nuclear RNAs (snRNAs), RNA D, RNA J, and RNA H, containing box C, box D, and box C/D, respectively, of Giardia were also cloned by RTPCR. Antibody raised against the recombinant protein was used to identify the fibrillarin in giardial nuclear extract. The interaction of snRNAs with recombinant fibrillarin was followed using North-Western hybridization. Gel electrophoresis mobility shift assay demonstrated that bacterially expressed protein may participate in the in vitro interaction with RNA J, RNA H, and RNA D. Our results indicate that the recombinant fibrillarin by itself is able to bind and does not require the involvement of any other protein for this binding to the three snRNAs.

We report the identification of 17 box C/D fibrillarin-associated small nucleolar RNAs (snoRNAs) from the ancient eukaryote, Trypanosoma brucei. To systematically isolate and characterize these snoRNAs, the T. brucei cDNA for the box C/D snoRNA common protein, fibrillarin, was cloned and polyclonal antibodies to the recombinant fibrillarin protein were generated in rabbits. Immunoprecipitations from T. bruceiextracts with the anti-fibrillarin antibodies indicated that this trypanosomatid has at least 30 fibrillarin-associated snoRNAs. We have sequenced seventeen of them and designated them TBR for T . b rucei RNA 1–17. All of them bear conserved box C, D, C′, and D′ elements, a hallmark of fibrillarin-associated snoRNAs in eukaryotes. Fourteen of them are novel T. brucei snoRNAs. Fifteen bear potential guide regions to mature rRNAs suggesting that they are involved in 2′-O-ribose methylation. Indeed, eight ribose methylations have been mapped in the rRNA at sites predicted by the snoRNA sequences. Comparative genomics indicates that six of the seventeen are the first trypanosome homologs of known yeast and vertebrate methylation guide snoRNAs. Our results indicate that T. brucei has many fibrillarin-associated box C/D snoRNAs with roles in 2′-O-ribose methylation of rRNA and that the mechanism for targeting the nucleotide to be methylated at the fifth nucleotide upstream of box D or D′ originated in early eukaryotes.

We have previously identified a set of tyrosine-phosphorylated proteins with apparent molecular masses of 44–46 kDa as some of the major tyrosine phosphorylated species in the protozoan parasite Trypanosoma brucei. We now show that these molecules, herein named Nopp44/46, are localized in the nucleolus. Using monoclonal antibodies, we have isolated Nopp44/46 cDNA clones from expression libraries. Sequence analysis reveals that the predicted amino acid sequence of the molecule is composed of an N-terminal unique region, an internal acidic region, and C-terminal repeat region. Analysis of the cDNA clones and genomic Southern analysis indicated that Nopp44/46 belongs to a multigene family in which different gene copies are very similar but vary in the number of repeats. Interestingly, the repetitive amino acid sequence motif contains multiple RGG (Arg-Gly-Gly) boxes characteristic of RNA-binding proteins. In vitro binding experiments demonstrated that Nopp44/46 is indeed capable of binding nucleic acids. Competition experiments with different RNA homopolymers demonstrated that Nopp44/46 preferentially binds to poly(U). These studies suggest that Nopp44/46 may play a role in RNA metabolism in trypanosomes and raise the possibility that tyrosine phosphorylation may regulate the process.