Two separate regions essential for nuclear import of the hnRNP D nucleocytoplasmic shuttling sequence

Authors:

Maiko Suzuki, Megumi Iijima, Akira Nishimura, Yusuke Tomozoe, Daisuke Kamei, and Michiyuki Yamada

Field:

Molecular Biology / Cell Biology

Document Content:

This document investigates the nuclear import mechanism of the heterogeneous nuclear ribonucleoprotein (hnRNP) D/AUF1, a protein involved in mRNA genesis and decay that shuttles between the nucleus and cytoplasm. The study identifies a nucleocytoplasmic shuttling sequence (DNS) within hnRNP D, specifically a 19-amino acid sequence at the C-terminus. Experimental results reveal that two distinct regions within this DNS, namely the N-terminal seven residues and the C-terminal two residues, are critical for both in vivo and in vitro nuclear import, as well as for interaction with the nuclear transport receptor, transportin-1 (Trn-1). The research further elucidates that the nuclear import mediated by Trn-1 is energy-dependent and requires Ran GTPase. The identified N- and C-terminal motifs show conservation in other shuttling sequences, suggesting a broader biological relevance.

Detailed Table of Contents:

• Two separate regions essential for nuclear import of the hnRNP D nucleocytoplasmic shuttling sequence
• Authors and Affiliations
• Keywords
• Correspondence
• Abstract
• Introduction
• Results
• Determination of hnRNP D NLS
• Role of amino acid residues of a D02 NLS in nuclear import
• Identification of an hnRNP D NLS as a nucleocytoplasmic shuttling sequence
• Trn-1-dependent import of hnRNP D
• Direct interaction of DNS NLS with Trn-1
• Affinities of D02, DNS and DNS mutants for Trn-1
• Discussion
• Experimental procedures
• Clonings of cDNAs for hnRNP D02, Trn-1, RanGAP and Ran
• Plasmid constructs for hnRNP D NLS mutants
• Preparation of GST-fusion proteins
• In vivo nuclear import assay and heterokaryon assay
• In vitro nuclear import assay
• Interaction between hnRNP D NLS and Trn-1
• Acknowledgements
• References