Identification of a non-canonical nuclear localization signal (NLS) in BRCA1 that could mediate nuclear localization of splice variants lacking the classical NLS

Aruna Korlimarla,Pravin Kumar,Jyothi Prabhu,T Sridhar,Hema Shankar,Jose Remacle,Hari Sankaranarayanan,Lekhana Bhandary,Dipa Natarajan

doi:10.2478/s11658-013-0088-x

Abstract

The breast cancer type 1 susceptibility gene (BRCA1) is a tumor suppressor gene, mutations or loss of which lead to genomic instability and breast cancer. BRCA1 protein is part of a large multi-protein complex involved in a variety of DNA repair and transcription regulatory functions. At least four splice variants have been described and these differ in their function and tissue and spatio-temporal expression patterns. Structural analysis has revealed the presence of two nuclear localization signals (NLS) located in exon 11 of BRCA1. Interestingly, a splice variant of the protein that lacks both of the known NLS still manages to gain entry to the nucleus. While there is experimental proof for the translocation of these proteins by binding to other established nuclear proteins, we examined the possibility of a hitherto unidentified NLS in this particular variant. In this paper, we present evidence for the existence of a previously unreported non-canonical NLS contained within the first 39 amino acids of exon 11. A fusion protein with this 39mer and a reporter green fluorescent protein translocated into the nucleus when it was expressed in breast epithelial cells. We demonstrate the presence of a hitherto unreported noncanonical NLS in exon 11a of BRCA1. This NLS might aid proteins that were encoded by splice variants and lack the canonical NLS to localize to the nucleus.

Highlights

breast cancer type 1 susceptibility gene (BRCA1) is a tumor suppressor gene, mutations of which are associated with hereditary breast cancer [1, 2]
Since the initial report that the fundamental property of nuclear localization signals (NLS) was contained in a stretch of at least four basic amino acids [20], there has been much refinement in defining consensus sequences and the development of methods to predict the presence of an NLS in a protein
It is known from both experimental and modelling studies that canonical nuclear localization signals bind to the major binding pocket of importin alpha, while the nuclear localization signal contained in the 39mer is likely to mediate its effect by binding to the minor binding pocket

Summary

Introduction

BRCA1 is a tumor suppressor gene, mutations of which are associated with hereditary breast cancer [1, 2]. Wang et al showed that the BRCA1, BRCA1a, and BRCA1b proteins are localized in the cytoplasm, nucleus and mitochondria and their nuclear– cytoplasmic shuttling is a regulated process [10]. They demonstrated the growth inhibitory function of these variants and in particular that BRCA1a has antitumor activity in triple negative breast cancer. They have shown that both BRCA1a and BRCA1b associate with the E2F family of transcription factors, cyclins and cyclin-dependant kinases. In 2002, Fabbro et al [17] showed that BRCA1 can bind to the BRCA1associated RING domain (BARD1) through its NH2-terminal RING domain

Methods

Results

Conclusion