Abstract
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.
Highlights
Protein-coding genes can be expressed from an rDNA promoter [3, 4] and that the major surface proteins of the organisms appear to be transcribed by a polymerase I-like polymerase [5, 6]
RGG motifs are usually found in conjunction with other types of RNA binding domains, RGG boxes are capable of binding nucleic acids independently, at least in some cases
We report here the subcellular localization, cDNA cloning, sequencing, and nucleic acid binding activity of a set of tyrosine-phosphorylated proteins from T. brucei
Summary
Pp44/46 Is a Nucleolar Protein—In earlier studies we identified a set of phosphorylated proteins, pp44/46, whose abundance and tyrosine phosphorylation are regulated in the life cycle of T. brucei. To further investigate the subnuclear localization, we carried out immunogold labeling of procyclic form cells using antibody ID2. Structure of the Nopp44/46 cDNA—Using antibody ID2, we screened a T. brucei Zap expression library and isolated four cDNA clones. The cDNAs were incomplete at the 5Ј end as no spliced leader (the 39nucleotide sequence trans-spliced to form the 5Ј end of all nuclearly encoded mRNAs) was found. The 5Ј end of the Nopp44/46 mRNA was obtained by cloning a fragment obtained by reverse transcription and PCR amplification, using a Nopp44/46-specific primer (primer 486; see Fig. 3 for primer locations) for cDNA synthesis, and a spliced leader primer plus a Nopp44/46-specific primer for amplification (primer 380). The repeat motifs fit the consensus (F/N/D)-R-G-G, with this particular cDNA containing 22 repeats. The aberrant migration appears to be a property of Nopp44/46 protein (see below)
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