Concurrent versus individual binding of HuR and AUF1 to common labile target mRNAs

Abstract

RNA-binding proteins HuR and AUF1 bind to many common AU-rich target mRNAs and exert opposing influence on target mRNA stability, but the functional interactions between HuR and AUF1 have not been systematically studied. Here, using common target RNAs encoding p21 and cyclin D1, we provide evidence that HuR and AUF1 can bind target transcripts on both distinct, nonoverlapping sites, and on common sites in a competitive fashion. In the nucleus, both proteins were found together within stable ribonucleoprotein complexes; in the cytoplasm, HuR and AUF1 were found to bind to target mRNAs individually, HuR colocalizing with the translational apparatus and AUF1 with the exosome. Our results indicate that the composition and fate (stability, translation) of HuR- and/or AUF1-containing ribonucleoprotein complexes depend on the target mRNA of interest, RNA-binding protein abundance, stress condition, and subcellular compartment

Clean western blot signals from immunoprecipitated samples

“…HRP-conjugated secondary antibodies also bind the intact Fc portion of the primary antibody but, unlike Protein A and Protein G, they also bind the denatured individual HC and LC polypeptides on WB filters. The use of Protein A-or protein G-HRP as substitute for secondary antibody in WBs of IP samples is principally indicated for the detection of these and many other proteins whose molecular weights are close to those of the HC or LC, or for low abundance proteins of any size that are obscured by IP antibodies [7]. The main limitation of this approach is that neither Protein A-nor Protein G-HRP bind adequately to mouse IgG1 or rat antibodies (not shown).…”

“…Ten million RKO cells were rinsed with PBS and lysed in 500 μl RIPA buffer to obtain wholecell lysates [7]. Whole-cell lysates (∼5 μg/μl) were stored at -20 °C or used directly for IP.…”

Mechanisms of regulatory diversity within the p53 transcriptional network

“…Reports from outside the p53 field reveal the importance of post-transcriptional regulation of the p21 waf1 gene in several biological processes such as tissue differentiation. A number of cis-regulatory elements present in the 3 0 -UTR of the p21 waf1 mRNA have been identified, as well as RNA-binding proteins recognizing these sequences (Johannessen et al, 1999;Figueroa et al, 2003;Lal et al, 2004;Yang et al, 2004;Shi et al, 2005). Recently, the Chen group identified a novel p53 target gene, named RNPC1, which codes for an RNA-binding protein capable of stabilizing p21 waf1 mRNA (Shu et al, 2006).…”

AUF1, the regulator of tumor necrosis factor α messenger RNA decay, is targeted by autoantibodies of patients with systemic rheumatic diseases

“…The AUF1 proteins are multifunctional proteins that show a predominant nuclear localization. They partially colocalize with snRNP, but are also part of cytoplasmic mRNA decay complexes, where they mediate mRNA degradation via the exosome (25). The high expression and partial cytoplasmic localization of AUF1 in HEp-2 and HeLa cells as well as in synovial cells from patients with RA leads us to suggest that non-spliceosomal structures in the cytoplasm such as mRNA transport or mRNA decay complexes may form autoimmune targets.…”