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(Received for publication, July 2, 1997)
From the 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.
Volume 272, Number 45,
Issue of November 7, 1997
pp. 28732-28741
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Modulation of AUUUA Response Element Binding by Heterogeneous
Nuclear Ribonucleoprotein A1 in Human T Lymphocytes
THE ROLES OF CYTOPLASMIC LOCATION, TRANSCRIPTION, AND
PHOSPHORYLATION
,
,§¶
Section of Connective Tissue Diseases,
Departments of Medicine and ¶ Microbiology, Dartmouth Medical
School, Lebanon, New Hampshire 03756 and the § Veterans
Administration Medical Center,
White River Junction, Vermont 05009
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