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The Escherichia coli protein RhlB is an ATP-dependent motor that unfolds structured RNA for destruction by partner ribonucleases. In E. coli, and probably many other related gamma-proteobacteria, RhlB associates with the essential endoribonuclease RNase E as part of the multi-enzyme RNA degradosome assembly. The interaction with RNase E boosts RhlB's ATPase activity by an order of magnitude. Here, we examine the origins and implications of this effect. The location of the interaction sites on both RNase E and RhlB are refined and analysed using limited protease digestion, domain cross-linking and homology modelling. These data indicate that RhlB's carboxy-terminal RecA-like domain engages a segment of RNase E that is no greater than 64 residues. The interaction between RhlB and RNase E has two important consequences: first, the interaction itself stimulates the unwinding and ATPase activities of RhlB; second, RhlB gains proximity to two RNA-binding sites on RNase E, with which it cooperates to unwind RNA. Our homology model identifies a pattern of residues in RhlB that may be key for recognition of RNase E and which may communicate the activating effects. Our data also suggest that the association with RNase E may partially repress the RNA-binding activity of RhlB. This repression may in fact permit the interplay of the helicase and adjacent RNA binding segments as part of a process that steers substrates to either processing or destruction, depending on context, within the RNA degradosome assembly.

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...

The CD45 protein is a transmembrane tyrosine phosphatase that is required for normal T cell receptor (TCR)-mediated signaling. A chimeric complementary DNA encoding the intracellular enzymatically active portion of murine CD45 preceded by a short amino-terminal sequence from p60 c- src was transfected into CD45- T cells. Expression of this chimeric protein corrected most of the TCR signaling abnormalities observed in the absence of CD45, including TCR-mediated enhancement of tyrosine kinase activity and Ca 2+ flux. Thus, the enzymatically active intracellular portion of CD45 is sufficient to allow TCR transmembrane signaling.