Localization of Nucleoporin Tpr to the Nuclear Pore Complex Is Essential for Tpr Mediated Regulation of the Export of Unspliced RNA

Kalpana Rajanala,Vinay Kumar Nandicoori,Juan Mata

doi:10.1371/journal.pone.0029921

Kalpana Rajanala, Vinay Kumar Nandicoori + Show 1 more

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https://doi.org/10.1371/journal.pone.0029921

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Journal: PLoS ONE	Publication Date: Jan 13, 2012
Citations: 129	License type: CC BY 4.0

Affiliation: National Institute of Immunology

Abstract

Nucleoporin Tpr is a component of the nuclear pore complex (NPC) that localizes exclusively to intranuclear filaments. Tpr functions as a scaffolding element in the nuclear phase of the NPC and plays a role in mitotic spindle checkpoint signalling. Export of intron-containing mRNA in Mason Pfizer Monkey Virus is regulated by direct interaction of cellular proteins with the cis-acting Constitutive Transport Element (CTE). In mammalian cells, the transport of Gag/Pol-CTE reporter construct is not very efficient, suggesting a regulatory mechanism to retain this unspliced RNA. Here we report that the knockdown of Tpr in mammalian cells leads to a drastic enhancement in the levels of Gag proteins (p24) in the cytoplasm, which is rescued by siRNA resistant Tpr. Tpr's role in the retention of unspliced RNA is independent of the functions of Sam68 and Tap/Nxf1 proteins, which are reported to promote CTE dependent export. Further, we investigated the possible role for nucleoporins that are known to function in nucleocytoplasmic transport in modulating unspliced RNA export. Results show that depletion of Nup153, a nucleoporin required for NPC anchoring of Tpr, plays a role in regulating the export, while depletion of other FG repeat-containing nucleoporins did not alter the unspliced RNA export. Results suggest that Tpr and Nup153 both regulate the export of unspliced RNA and they are most likely functioning through the same pathway. Importantly, we find that localization of Tpr to the NPC is necessary for Tpr mediated regulation of unspliced RNA export. Collectively, the data indicates that perinuclear localization of Tpr at the nucleopore complex is crucial for regulating intron containing mRNA export by directly or indirectly participating in the processing and degradation of aberrant mRNA transcripts.

Highlights

Nucleoporin Tpr was originally identified as the oncogenic activator of the met, raf, and trk protooncogenes [1,2,3]
Tpr does not play a significant role in cellular protein transport or in mRNA export Nucleoporin Tpr has been reported to play a role in nuclear export of proteins containing leucine rich nuclear export signal, [12] and the ectopic expression of mammalian Tpr has been reported to result in the accumulation of poly (A)+ RNA in the nucleus [28]
In order to examine the function of Tpr in cellular transport of proteins and nuclear export of mRNA, we depleted Tpr protein in HEK293T cells using three independent siRNA oligonucleotides

Summary

Introduction

Nucleoporin Tpr was originally identified as the oncogenic activator of the met, raf, and trk protooncogenes [1,2,3]. Cellular transformations and human tumors have been shown to occur due to the fusion of N-terminal residues of Tpr (residues 140–230) with the protein kinase domains of the protooncogenes met, raf, and trk [1,2,3]. Tpr has been shown to be localized exclusively to intranuclear filaments associated with the nucleoplasmic side of the NPC, by directly binding to Nup153 [5,6,7]. Tpr has been shown to play a role in nuclear export of proteins containing leucine rich nuclear export signal (NES) and it aids in the export of proteins with no apparent NES, as in the Huntington protein [12,15]. The association of Mad and Mad proteins with Tpr has been shown to affect mitotic spindle checkpoint signalling [16]

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