A Major Tyrosine-phosphorylated Protein of Trypanosoma brucei Is a Nucleolar RNA-binding Protein

doi:10.1074/jbc.271.26.15675

A Major Tyrosine-phosphorylated Protein of Trypanosoma brucei Is a Nucleolar RNA-binding Protein*

From the ^‡ Seattle Biomedical Research Institute, Seattle, Washington 98109
^§ the Department of Pathobiology, University of Washington, Seattle, Washington 98195
^| Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, and the
^” Department of Medical and Molecular Parasitology, New York University Medical Center, New York, New York 10010

‴ To whom correspondence should be addressed:
Seattle Biomedical Research Institute, 4 Nickerson St., Seattle, WA 98109.
Tel.: 206-284-8846 (ext. 315); Fax: 206-284-0313.

↵^¶ Present address: Dept. of Laboratory Medicine, University of Washington, Seattle, WA 98195.

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.

Footnotes

↵* This work was supported in part by National Institutes of Health Grant AI R01-31077. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U53863[GenBank].
↵¹ The abbreviations used are:

hnRNP

heterogeneous nuclear ribonucleoprotein

Nopp44/46

nucleolar phosphorylated protein 44/46

rRNA

ribosomal RNA

PCR

polymerase chain reaction

RNase H

ribonuclease H

PAGE

polyacrylamide gel electrophoresis

dsDNA

double-stranded DNA

ssDNA

single-stranded DNA.
- Received November 20, 1995.
- Revision received March 19, 1996.