RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.

SUMMARY BIK protein is an initiator of mitochondrial apoptosis and BIK expression is induced by pro-apoptotic signals including DNA damage. Here we demonstrate that 3′-end processing and expression of BIK mRNA are controlled by the nuclear PI4,5P2-regulated poly(A) polymerase Star-PAP downstream of DNA damage. Nuclear PKCδ is a key mediator of apoptosis and DNA damage stimulates PKCδ association with the Star-PAP complex where PKCδ is required for Star-PAP-dependent BIK expression. PKCδ binds the PI4,5P2-generating enzyme PIPKIα, which is essential for PKCδ interaction with the Star-PAP complex and PKCδ activity is directly stimulated by PI4,5P2. Features in the BIK 3′-UTR uniquely define Star-PAP specificity and may block canonical PAP activity toward BIK mRNA. This reveals a nuclear phosphoinositide signaling nexus where PIPKIα, PI4,5P2 and PKCδ regulate Star-PAP control of BIK expression and induction of apoptosis. This pathway is distinct from the Star-PAP-mediated oxidative stress pathway indicating signal-specific regulation of mRNA 3′-end processing.

Star-PAP is a poly (A) polymerase (PAP) that is putatively required for 3 0 -end cleavage and polyadenylation of a select set of pre-messenger RNAs (mRNAs), including heme oxygenase (HO-1) mRNA. To investigate the underlying mechanism, the cleavage and polyadenylation of premRNA was reconstituted with nuclear lysates. siRNA knockdown of Star-PAP abolished cleavage of HO-1, and this phenotype could be rescued by recombinant Star-PAP but not PAPa. Star-PAP directly associated with cleavage and polyadenylation specificity factor (CPSF) 160 and 73 subunits and also the targeted pre-mRNA. In vitro and in vivo Star-PAP was required for the stable association of CPSF complex to pre-mRNA and then CPSF 73 specifically cleaved the mRNA at the 3 0 -cleavage site. This mechanism is distinct from canonical PAPa, which is recruited to the cleavage complex by interacting with CPSF 160. The data support a model where Star-PAP binds to the RNA, recruits the CPSF complex to the 3 0 -end of pre-mRNA and then defines cleavage by CPSF 73 and subsequent polyadenylation of its target mRNAs.

While the presence of phosphoinositides in the nuclei of eukaryotes and the identity of the enzymes responsible for their metabolism have been known for some time, their functions in the nucleus are only now emerging. This is illustrated by the recent identification of effectors for nuclear phosphoinositides. Like the cytosolic phosphoinositide signaling pathway, nuclear phosphatidylinositol 4,5 bisphosphate (PI4,5P2) is at the center of the pathway and acts both as a messenger and as a precursor for many additional messengers. Here, recent advances in the understanding of nuclear phosphoinositide signaling and its functions are reviewed with an emphasis on PI4,5P2 and its role in gene expression. The compartmentalization of nuclear phosphoinositide phosphates (PIPn) remains a mystery, but emerging evidence suggests that phosphoinositides occupy several functionally distinct compartments.

In vivo transcription of the Escherichia coli argO gene, which encodes an arginine (Arg) exporter, requires the LysR-family regulator protein ArgP (previously called IciA) and is induced in the presence of Arg or its naturally occurring antimetabolite analog canavanine. Lysine (Lys) addition, on the other hand, phenocopies an argP mutation to result in the shutoff of argO expression. We now report that the ArgP dimer by itself is able to bind the argO promoter-operator region to form a binary complex, but that the formation of a ternary complex with RNA polymerase is greatly stimulated only in presence of a coeffector. Both Arg and Lys were proficient as coeffectors for ArgP-mediated recruitment of RNA polymerase to, and open complex formation at, the argO promoter, although only Arg (but not Lys) was competent to activate transcription. The two coeffectors competed for binding to ArgP, and the ternary complex that had been assembled on the argO template in the presence of Lys could be chased into a transcriptionally active state upon Arg addition. Our results support a novel mechanism of argO regulation in which Lys-bound ArgP reversibly restrains RNA polymerase at the promoter, at a step (following open complex formation) that precedes, and is common to, both abortive and productive transcription. This represents, therefore, the first example of an environmental signal regulating the final step of promoter clearance by RNA polymerase in bacterial transcription. We propose that, in E. coli cells, the ternary complex remains assembled and poised at the argO promoter at all times to respond, positively or negatively, to instantaneous changes in the ratio of intracellular Arg to Lys concentrations.[Keywords: ArgP; arginine export; bacterial transcription; gene regulation; promoter clearance] Supplemental material is available at http://www.genesdev.org.