Modulation of AUUUA Response Element Binding by Heterogeneous Nuclear Ribonucleoprotein A1 in Human T Lymphocytes

Abstract

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) shuttles between the cytoplasm and nucleus and plays important roles in RNA metabolism. Whereas nuclear hnRNP A1 has been shown to bind intronic sequences and modulate splicing, cytoplasmic hnRNP A1 is associated with poly(A) ؉ RNA, indicating different RNA ligand specificity. Previous studies indicated that cytoplasmic hnRNP A1 is capable of high-affinity binding of reiterated AUUUA sequences (ARE) that have been shown to modulate mRNA turnover and translation. Through a combination of two-dimensional gel and proteolysis studies, we establish hnRNP A1 (or structurally related proteins that are post-translationally regulated in an identical manner) as the dominant cytoplasmic protein in human T lymphocytes capable of interacting with the ARE contained within the context of full-length granulocyte-macrophage colony-stimulating factor mRNA. We additionally demonstrate that cytoplasmic hnRNP A1 preferentially binds ARE relative to pre-mRNAs in both cross-linking and mobility shift experiments. RNA polymerase II inhibition increased the binding of ARE (AUBP activity) and poly(U)-Sepharose by cytoplasmic hnRNP A1, while nuclear hnRNP A1 binding was unaffected. Nuclear and cytoplasmic hnRNP A1 could be distinguished by the differential sensitivity of their RNA binding to diamide and N-ethylmaleimide. The increase in AUBP activity of cytoplasmic hnRNP A1 following RNA polymerase II inhibition correlated with serine-threonine dephosphorylation, as determined by inhibitor and metabolic labeling studies. Thus, cytoplasmic and nuclear hnRNP A1 exhibit different RNA binding profiles, perhaps transduced through serine-threonine phosphorylation. These findings are relevant to the specific ability of hnRNP A1 to serve distinct roles in post-transcriptional regulation of gene expression in both the nucleus and cytoplasm.The heterogeneous nuclear ribonucleoproteins (hnRNP) 1 were originally defined as the proteins constituting the 40 S complexes isolated from nuclei following nuclease treatment (reviewed in Refs. 1 and 2). The hnRNP proteins range in mass from 30 to 120 kDa and associate with pre-mRNA as a complex (1, 2). Studies of hnRNP-RNA interactions have demonstrated sequence-specific binding, both in vitro and in vivo (3-5), as well as roles in pre-mRNA splicing and splice-site selection (2, 6, 7). Consistent with this latter observation, native hnRNP A1 and C proteins have been reported to bind a polypyrimidine stretch bordered by AG at the 3Ј end of introns (7,8). Similar findings have been made with recombinant hnRNP A1 (9, 10), which demonstrated equivalent binding to 5Ј-and 3Ј-intronic splice sites (9). These findings are of functional interest because hnRNP A1 has been shown to modulate the effects of splicing factor 2 (SF2/ASF), and promote distal 5Ј-splice site selection (8,(11)(12)(13).The role of hnRNP A1 in mRNA metabolism expanded with demonstration that hnRNP A1 shuttles between the nucleus and the cytoplasm (14). With inhibition of RNA polymerase II t...

Heterogeneous nuclear ribonucleoprotein D0 contains transactivator and DNA-binding domains

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Structure/Function Analysis of Tristetraprolin (TTP): p38 Stress-Activated Protein Kinase and Lipopolysaccharide Stimulation Do Not Alter TTP Function

“…Cytoplasmic preparations were performed as previously described using a method characterized for its lack of contamination by nuclear proteins (22,27). For immunoblotting, cytoplasmic and gradient fractions were boiled in loading buffer (50 mM Tris-HCl (pH 6.8), 100 mM DTT, 2% SDS, 0.1% bromophenol blue, and 10% glycerol) for 3 min, resolved by 12 or 8 -16% SDS-PAGE, and then electrotransferred to nitrocellulose.…”

Human Immunodeficiency Virus Type 1 (HIV-1) Induces the Cytoplasmic Retention of Heterogeneous Nuclear Ribonucleoprotein A1 by Disrupting Nuclear Import

“…What enables hnRNP A1 to have such broad functions in RNA metabolism is its ability to bind both nuclear and cytoplasmic RNAs (10). In addition to its well characterized role in nuclear RNA processing, hnRNP A1 binds to purine-rich sequences of mRNAs for mRNA turnover and translation (13,14).…”

“…The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a predominantly nuclear protein engaged in a number of cellular and viral RNPs for RNA-processing activities, including splicing regulation, nuclear export, microRNA processing, mRNA stability, telomere maintenance, and IRES-mediated translation initiation (7)(8)(9)(10)(11)(12). What enables hnRNP A1 to have such broad functions in RNA metabolism is its ability to bind both nuclear and cytoplasmic RNAs (10).…”