Identification of a Human Cytoplasmic Poly(A) Nuclease Complex Stimulated by Poly(A)-binding Protein

Naoyuki Uchida,Shin-Ichi Hoshino,Toshiaki Katada

doi:10.1074/jbc.m309125200

Naoyuki Uchida, Shin-Ichi Hoshino + Show 1 more

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https://doi.org/10.1074/jbc.m309125200

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Abstract

The poly(A) tail shortening in mRNA, called deadenylation, is the first rate-limiting step in eukaryotic mRNA turnover, and the polyadenylate-binding protein (PABP) appears to be involved in the regulation of this step. However, the precise role of PABP remains largely unknown in higher eukaryotes. Here we identified and characterized a human PABP-dependent poly(A) nuclease (hPAN) complex consisting of catalytic hPan2 and regulatory hPan3 subunits. hPan2 has intrinsically a 3' to 5' exoribonuclease activity and requires Mg2+ for the enzyme activity. On the other hand, hPan3 interacts with PABP to simulate hPan2 nuclease activity. Interestingly, the hPAN nuclease complex has a higher substrate specificity to poly(A) RNA upon its association with PABP. Consistent with the roles of hPan2 and hPan3 in mRNA decay, the two subunits exhibit cytoplasmic co-localization. Thus, the human PAN complex is a poly(A)-specific exoribonuclease that is stimulated by PABP in the cytoplasm.

Highlights

Eukaryotic mRNAs have two major features, a 5Ј-terminal cap and a 3Ј-terminal poly(A) tail
To confirm that the observed RNase activity is derived from the immunopurified human Pan2 (hPan2) rather than from a co-purifying unknown factor, we prepared a hPan2 mutant in which a key conserved catalytic residue (Asp) in the RNase D domain had been replace by Ala (Fig. 2D)
Identification of Human PAN Nuclease—In this study, we have identified novel human cDNAs encoding an human PABP-dependent poly(A) nuclease (hPAN) nuclease complex and analyzed its biochemical properties in terms of its regulation by polyadenylate-binding protein (PABP) and substrate specificity

Summary

Introduction

Eukaryotic mRNAs have two major features, a 5Ј-terminal cap and a 3Ј-terminal poly(A) tail. We identified and characterized a human PABP-dependent poly(A) nuclease (hPAN) complex consisting of catalytic hPan2 and regulatory hPan3 subunits. The hPAN nuclease complex has a higher substrate specificity to poly(A) RNA upon its association with PABP.

Results

Conclusion