Identification of a novel nucleocytoplasmic shuttling RNA helicase of trypanosomes.

Alexandre Haruo Inoue,Maria Cristina Machado Motta,Sueli Fumie Yamada-Ogatta,Newton Medeiros Vidal,João Renato Carvalho Muniz,Mariana Serpeloni,Andréa Rodrigues Ávila,Priscila Mazzocchi Hiraiwa,Samuel Goldenberg,Alexander F Palazzo

doi:10.1371/journal.pone.0109521

Abstract

Gene expression in trypanosomes is controlled mostly by post-transcriptional pathways. Little is known about the components of mRNA nucleocytoplasmic export routes in these parasites. Comparative genomics has shown that the mRNA transport pathway is the least conserved pathway among eukaryotes. Nonetheless, we identified a RNA helicase (Hel45) that is conserved across eukaryotes and similar to shuttling proteins involved in mRNA export. We used in silico analysis to predict the structure of Trypanosoma cruzi Hel45, including the N-terminal domain and the C-terminal domain, and our findings suggest that this RNA helicase can form complexes with mRNA. Hel45 was present in both nucleus and cytoplasm. Electron microscopy showed that Hel45 is clustered close to the cytoplasmic side of nuclear pore complexes, and is also present in the nucleus where it is associated with peripheral compact chromatin. Deletion of a predicted Nuclear Export Signal motif led to the accumulation of Hel45ΔNES in the nucleus, indicating that Hel45 shuttles between the nucleus and the cytoplasm. This transport was dependent on active transcription but did not depend on the exportin Crm1. Knockdown of Mex67 in T. brucei caused the nuclear accumulation of the T. brucei ortholog of Hel45. Indeed, Hel45 is present in mRNA ribonucleoprotein complexes that are not associated with polysomes. It is still necessary to confirm the precise function of Hel45. However, this RNA helicase is associated with mRNA metabolism and its nucleocytoplasmic shuttling is dependent on an mRNA export route involving Mex67 receptor.

Highlights

Chagas disease is a neglected disease endemic to Latin America, where about eight million people are affected [1]
We show that Hel45 has a nuclear export signal and its shuttling is dependent on an messenger RNA (mRNA) export pathway, involving a homolog of the Mex67 nuclear receptor
Our findings suggest that this protein is involved in mRNA metabolism and its nucleocytoplasmic shuttling is dependent on an mRNA export route involving Mex67 receptor

Summary

Introduction

Chagas disease is a neglected disease endemic to Latin America, where about eight million people are affected [1]. This disease is caused by infection with Trypanosoma cruzi (T. cruzi). Several genes are grouped together under the control of a single promoter region [2,3] and give rise to long polycistronic transcripts. These transcripts are processed by trans-splicing and polyadenylation to form monocistronic messenger RNA (mRNA) [4,5,6]. Little is known about the mechanisms of mRNA nucleocytoplasmic transport in these parasites, and the identity of factors that determine the fate of mRNA in the cytoplasm remains to be unveiled

Methods

Results

Conclusion