Hu Proteins Regulate Polyadenylation by Blocking Sites Containing U-rich Sequences

Hua Lou,Hui Zhu,Robert A. Hasman,Hua-Lin Zhou

doi:10.1074/jbc.m609349200

Abstract

A recent genome-wide bioinformatic analysis indicated that 54% of human genes undergo alternative polyadenylation. Although it is clear that differential selection of poly(A) sites can alter gene expression, resulting in significant biological consequences, the mechanisms that regulate polyadenylation are poorly understood. Here we report that the neuron-specific members of a family of RNA-binding proteins, Hu proteins, known to regulate mRNA stability and translation in the cytoplasm, play an important role in polyadenylation regulation. Hu proteins are homologs of the Drosophila embryonic lethal abnormal visual protein and contain three RNA recognition motifs. Using an in vitro polyadenylation assay with HeLa cell nuclear extract and recombinant Hu proteins, we have shown that Hu proteins selectively block both cleavage and poly(A) addition at sites containing U-rich sequences. Hu proteins have no effect on poly(A) sites that do not contain U-rich sequences or sites in which the U-rich sequences are mutated. All three RNA recognition motifs of Hu proteins are required for this activity. Overexpression of HuR in HeLa cells also blocks polyadenylation at a poly(A) signal that contains U-rich sequences. Hu proteins block the interaction between the polyadenylation cleavage stimulation factor 64-kDa subunit and RNA most likely through direct interaction with poly(A) cleavage stimulation factor 64-kDa subunit and cleavage and polyadenylation specificity factor 160-kDa subunit. These studies identify a novel group of mammalian polyadenylation regulators. Furthermore, they define a previously unknown nuclear function of Hu proteins.

Highlights

The systemic therapies currently available for the treatment of various solid tumors of adult life, including those of lung, colon, breast, prostate, and ovary, remain primarily palliative, and there is an urgent need for more effective therapies
Initial reports showed that MLN944 can bind strongly to DNA [2] and that it may interfere with the normal function of topoisomerase I and II in vitro [1], recent studies have indicated that topoisomerase I and II may not be its primary cellular target and that it works by a novel mechanism of action
The N10 of the phenazine ring of MLN944 is protonated (Fig. 1a) in the DNA complex at pH 7, and this HN10 proton is clearly observed in the NMR data

Summary

Introduction

The systemic therapies currently available for the treatment of various solid tumors of adult life, including those of lung, colon, breast, prostate, and ovary, remain primarily palliative, and there is an urgent need for more effective therapies. We present the first NMR structure of MLN944 complexed with d(ATGCAT)2 DNA duplex, demonstrating a novel binding mode in which the two phenazine rings bis-intercalate at the 5؅-TpG site, with the carboxamide amino linker lying in the major groove of DNA.

Results

Conclusion