Identification and characterization of nuclear and nucleolar localization signals in 58-kDa microspherule protein (MSP58).

Chuan-Pin Yang,Chang-Han Chen,Yi-Chao Lee,Mei-Hsiang Wu,Ding-Yen Lin,Yi-Huan Tsou,Wen-Chang Chang,Yu-San Yang,Chi-Wu Chiang

doi:10.1186/s12929-015-0136-0

Abstract

BackgroundMSP58 is a nucleolar protein associated with rRNA transcription and cell proliferation. Its mechanism of translocation into the nucleus or the nucleolus, however, is not entirely known. In order to address this lack, the present study aims to determine a crucial part of this mechanism: the nuclear localization signal (NLS) and the nucleolar localization signal (NoLS) associated with the MSP58 protein.ResultsWe have identified and characterized two NLSs in MSP58. The first is located between residues 32 and 56 (NLS1) and constitutes three clusters of basic amino acids (KRASSQALGTIPKRRSSSRFIKRKK); the second is situated between residues 113 and 123 (NLS2) and harbors a monopartite signal (PGLTKRVKKSK). Both NLS1 and NLS2 are highly conserved among different vertebrate species. Notably, one bipartite motif within the NLS1 (residues 44–56) appears to be absolutely necessary for MSP58 nucleolar localization. By yeast two-hybrid, pull-down, and coimmunoprecipitation analysis, we show that MSP58 binds to importin α1 and α6, suggesting that nuclear targeting of MSP58 utilizes a receptor-mediated and energy-dependent import mechanism. Functionally, our data show that both nuclear and nucleolar localization of MSP58 are crucial for transcriptional regulation on p21 and ribosomal RNA genes, and context-dependent effects on cell proliferation.ConclusionsResults suggest that MSP58 subnuclear localization is regulated by two nuclear import signals, and that proper subcellular localization of MSP58 is critical for its role in transcriptional regulation. Our study reveals a molecular mechanism that controls nuclear and nucleolar localization of MSP58, a finding that might help future researchers understand the MSP58 biological signaling pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-015-0136-0) contains supplementary material, which is available to authorized users.

Highlights

58-kDa microspherule protein (MSP58) is a nucleolar protein associated with ribosomal RNA (rRNA) transcription and cell proliferation
The three clusters of basic residues triple mutant (10A) display a similar phenotype as the 8A mutant (Figure 2C), a result were shown in a number of different cells, including HeLa and MCF7. Together these results suggest that both NLS1 and NLS2 can contribute to the nuclear import of MSP58 and that NLS1 functioned as a nucleolar localization signal (NoLS)
The 10A/NLS1 mutant failed to interact with the rDNA promoter. These results suggest that the NLS1/NoLS motif or nucleolar localization is required for MSP58 to play its role in regulating the rDNA gene promoter activity

Summary

Introduction

MSP58 is a nucleolar protein associated with rRNA transcription and cell proliferation. The active transport of macromolecules into the nucleus occurs through the nuclear pore complex (NPC) in the nuclear envelope This process is mediated by specific signals called nuclear localization sequences (NLSs). The nucleolus, as accumulated studies suggest, plays critical roles in regulating cell proliferation, senescence, stress sensing, signal recognition particle (SRP) assembly, the modulating telomerase function, and tumor suppression and oncogenic activities [13,14,15]. Some of these functions are mediated through the sequestration or release of transcription factors that control the cell cycle [15,16,17]. Many NoLSs are usually rich in arginine and lysine residues, which overlap with NLSs (reviewed in [12])

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