Modulation of AUUUA response element binding by heterogeneous nuclear ribonucleoprotein A1 in human T lymphocytes - The roles of cytoplasmic location, transcription, and phosphorylation

The heterogeneous nuclear ribonucleoprotein Al (hnRNP A1) shuttles between the cytoplasm and nucleus and plays important roles in RNA metabolism, Whereas nuclear hnRNP Al has been shown to bind intronic sequences and modulate splicing, cytoplasmic hnRNP Al is associated with poly(A)(+) RNA, indicating different RNA ligand specificity. Previous studies indicated that cytoplasmic hnRNP Al 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 Al (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 Al 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 Al, while nuclear hnRNP Al binding was unaffected. Nuclear and cytoplasmic hnRNP Al could be distinguished by the differential sensitivity of their RNA binding to diamide and N-ethyhmaleimide. The increase in AUBP activity of cytoplasmic hnRNP Al following RNA polymerase II inhibition correlated with serine-threonine dephosphorylation, as determined by inhibitor and metabolic labeling studies, Thus, cytoplasmic and nuclear hnRNP Al exhibit different RNA binding profiles, perhaps transduced through serine-threonine phosphorylation. These findings are relevant to the specific ability of hnRNP Al to serve distinct roles in post-transcriptional regulation of gene expression in both the nucleus and cytoplasm.