An RNA Recognition Motif Mediates the Nucleocytoplasmic Transport of a Trypanosome RNA-binding Protein

Alejandro Cassola,Alberto C Frasch

doi:10.1074/jbc.m109.031633

Abstract

RNA-binding proteins (RBPs) and RNA metabolism are considered to be important for modulating gene expression in trypanosomes, because these protozoan parasites mainly rely on post-transcriptional mechanisms to regulate protein levels. Previously, we have identified TcUBP1, a single RNA recognition motif (RRM)-type RBP from Trypanosoma cruzi. TcUBP1 is a cytoplasmic protein with roles in stabilization/degradation of mRNAs and in the protection of transcripts through their recruitment into cytoplasmic granules. We now show that TcUBP1, and the closely related protein TcUBP2, can be found in small amounts in the nucleus under normal conditions, and are able to accumulate in the nucleus under arsenite stress. The kinetics of nuclear accumulation, and export to the cytoplasm, are consistent with the shuttling of TcUBP1 between the nucleus and the cytoplasm. The sequence required for TcUBP1 nuclear accumulation was narrowed to the RRM, and point mutations affecting RNA binding abolished nuclear import. This RRM was also shown to be efficiently exported from the nucleus in unstressed parasites, a property that relied on the binding to RNA. TcUBP1 nuclear accumulation was dependent on active transcription, and colocalized with transcripts in the nucleus, suggesting nuclear binding of the mRNA. We propose that TcUBP1 could be linking the mRNA metabolism at both sides of the nuclear pore complex, using the RRM as a nuclear localization signal, and being exported as a cargo on mRNA.

Highlights

DECEMBER 11, 2009 VOLUME 284 NUMBER 50 transcriptional mechanisms [2, 3]
Stress-mediated Nuclear Accumulation of TcUBP1—Cytoplasmic messenger RNAs (mRNAs) granules in T. cruzi and T. brucei insect forms contain proteins involved in mRNA metabolism, including RNA recognition motif (RRM)-type RNA-binding proteins (RBPs) such as TcUBP1 and TcPABP1
We found that arsenite treatment did not induce cytoplasmic granules in culture epimastigotes, but after 4 h of treatment made TcUBP1 and TcUBP2 GFP fusion proteins accumulate in the nucleus (Fig. 1A)

Summary

Introduction

DECEMBER 11, 2009 VOLUME 284 NUMBER 50 transcriptional mechanisms [2, 3]. Polycistronic pre-messenger RNAs (mRNAs) are processed into monocistronic mRNAs by 5Ј trans-splicing coupled to 3Ј polyadenylation [4]. We found that arsenite treatment did not induce cytoplasmic granules in culture epimastigotes, but after 4 h of treatment made TcUBP1 and TcUBP2 GFP fusion proteins accumulate in the nucleus (Fig. 1A). The cytoplasmic localization and arsenite-induced nuclear accumulation of TcUBP1-GFP was evidenced throughout the culture of parasites (supplemental Fig. S1B).

Results

Conclusion