Widespread Regulation of miRNA Biogenesis at the Dicer Step by the Cold-Inducible RNA-Binding Protein, RBM3

MicroRNAs (miRNAs) play critical roles in diverse cellular events through their effects on translation. Emerging data suggest that modulation of miRNA biogenesis at post-transcriptional steps by RNA-binding proteins is a key point of regulatory control over the expression of some miRNAs and the cellular processes they influence. However, the extent and conditions under which the miRNA pathway is amenable to regulation at posttranscriptional steps are poorly understood. Here we show that RBM3, a cold-inducible, developmentally regulated RNA-binding protein and putative protooncogene, is an essential regulator of miRNA biogenesis. Utilizing miRNA array, Northern blot, and PCR methods, we observed that over 60% of miRNAs detectable in a neuronal cell line were significantly downregulated by knockdown of RBM3. Conversely, for select miRNAs assayed by Northern blot, induction of RBM3 by overexpression or mild hypothermia increased their levels. Changes in miRNA expression were accompanied by changes in the levels of their ∼70 nt precursors, whereas primary transcript levels were unaffected. Mechanistic studies revealed that knockdown of RBM3 does not reduce Dicer activity or impede transport of pre-miRNAs into the cytoplasm. Rather, we find that RBM3 binds directly to ∼70 nt pre-miRNA intermediates and promotes / de-represses their ability as larger ribonucleoproteins (pre-miRNPs) to associate with active Dicer complexes. Our findings suggest that the processing of a majority of pre-miRNPs by Dicer is subject to an intrinsic inhibitory influence that is overcome by RBM3 expression. RBM3 may thus orchestrate changes in miRNA expression during hypothermia and other cellular stresses, and in the euthermic contexts of early development, differentiation, and oncogenesis where RBM3 expression is highly elevated. Additionally, our data suggest that temperature-dependent changes in miRNA expression mediated by RBM3 may contribute to the therapeutic effects of hypothermia, and are an important variable to consider in in vitro studies of translation-dependent cellular events

Charting the NF-κB Pathway Interactome Map

Inflammation is part of a complex physiological response to harmful stimuli and pathogenic stress. The five components of the Nuclear Factor κB (NF-κB) family are prominent mediators of inflammation, acting as key transcriptional regulators of hundreds of genes. Several signaling pathways activated by diverse stimuli converge on NF-κB activation, resulting in a regulatory system characterized by high complexity. It is increasingly recognized that the number of components that impinges upon phenotypic outcomes of signal transduction pathways may be higher than those taken into consideration from canonical pathway representations. Scope of the present analysis is to provide a wider, systemic picture of the NF-κB signaling system. Data from different sources such as literature, functional enrichment web resources, protein-protein interaction and pathway databases have been gathered, curated, integrated and analyzed in order to reconstruct a single, comprehensive picture of the proteins that interact with, and participate to the NF-κB activation system. Such a reconstruction shows that the NF-κB interactome is substantially different in quantity and quality of components with respect to canonical representations. The analysis highlights that several neglected but topologically central proteins may play a role in the activation of NF-κB mediated responses. Moreover the interactome structure fits with the characteristics of a bow tie architecture. This interactome is intended as an open network resource available for further development, refinement and analysis

Regulation of MicroRNA Biogenesis: A miRiad of mechanisms

microRNAs are small, non-coding RNAs that influence diverse biological functions through the repression of target genes during normal development and pathological responses. Widespread use of microRNA arrays to profile microRNA expression has indicated that the levels of many microRNAs are altered during development and disease. These findings have prompted a great deal of investigation into the mechanism and function of microRNA-mediated repression. However, the mechanisms which govern the regulation of microRNA biogenesis and activity are just beginning to be uncovered. Following transcription, mature microRNA are generated through a series of coordinated processing events mediated by large protein complexes. It is increasingly clear that microRNA biogenesis does not proceed in a 'one-size-fits-all' manner. Rather, individual classes of microRNAs are differentially regulated through the association of regulatory factors with the core microRNA biogenesis machinery. Here, we review the regulation of microRNA biogenesis and activity, with particular focus on mechanisms of post-transcriptional control. Further understanding of the regulation of microRNA biogenesis and activity will undoubtedly provide important insights into normal development as well as pathological conditions such as cardiovascular disease and cancer

Advancing a Conceptual Model of Evidence-Based Practice Implementation in Public Service Sectors

Implementation science is a quickly growing discipline. Lessons learned from business and medical settings are being applied but it is unclear how well they translate to settings with different historical origins and customs (e.g., public mental health, social service, alcohol/drug sectors). The purpose of this paper is to propose a multi-level, four phase model of the implementation process (i.e., Exploration, Adoption/Preparation, Implementation, Sustainment), derived from extant literature, and apply it to public sector services. We highlight features of the model likely to be particularly important in each phase, while considering the outer and inner contexts (i.e., levels) of public sector service systems

The NF-kappa B precursor p105 and the proto-oncogene product Bcl-3 are I kappa B molecules and control nuclear translocation of NF-kappa B.

We have examined the interaction of the NF-kappa B precursor p105 with NF-kappa B subunits. Similar to an I kappa B molecule, p105 associates in the cytoplasm with p50 or p65. Through this assembly, p105 efficiently blocks nuclear transfer of either subunit. Moreover, the p105 protein inhibits DNA binding of dimeric NF-kappa B subunits in a similar, but not identical, manner to its isolated C-terminal domain, which contains an ankyrin-like repeat domain (ARD). The proto-oncogene product Bcl-3 also controls nuclear translocation of p50, but not of p65. Hence, p50 can be retained in the cytoplasm via at least three distinct interactions: through direct interactions either with its own precursor, with Bcl-3 or indirectly through I kappa B alpha or -beta when attached to p65. We discuss a function of p105 as a cytoplasmic assembly unit for homo- and heteromeric NF-kappa B complexes and of Bcl-3 as an I kappa B with novel subunit specificity

Different mechanisms control signal-induced degradation and basal turnover of the NF-kappaB inhibitor IkappaB alpha in vivo.

The transcription factor NF-kappaB is sequestered in the cytoplasm by a family of IkappaB molecules. Upon cellular stimulation with diverse agents, one of these molecules, IkappaB alpha, is rapidly phosphorylated and subsequently degraded. This process triggers nuclear translocation of NF-kappaB and the successive activation of target genes. Independent of its rapid stimulation-induced breakdown, IkappaB alpha is inherently unstable and undergoes a continuous turnover. To compare the mechanisms and protein domains involved in inducible and basal degradation of IkappaB alpha in intact cells we employed a transfection strategy using tagged IkappaB alpha and ubiquitin molecules. We show that tumor necrosis factor alpha (TNFalpha) induced breakdown of IkappaB alpha but not its basal turnover coincides with ubiquitination in the amino-terminal signal response domain (SRD) of IkappaB alpha. Neither the SRD nor the carboxy-terminal PEST sequence is needed for basal turnover, which instead depends only on the core ankyrin repeat domain. Despite the differences in the requirements of protein domains and ubiquitin-conjugation for both degradation pathways, each one is mediated by the proteasome. This finding is important for understanding alternative modes of controlling NF-kappaB activity

Signal-dependent degradation of IkappaBalpha is mediated by an inducible destruction box that can be transferred to NF-kappaB, bcl-3 or p53.

Activation of the transcription factor NF-kappaB in response to a variety of stimuli is governed by the signal-induced proteolytic degradation of NF-kappaB inhibitor proteins, the IkappaBs. We have investigated the sequence requirements for signal-induced IkappaBalpha phosphorylation and proteolysis by generating chimeric proteins containing discrete sub-regions of IkappaBalpha fused to the IkappaBalpha homologue Bcl-3, the transcription factor NF-kappaB1/p50 and the tumour suppressor protein p53. Using this approach we show that the N-terminal signal response domain (SRD) of IkappaBalpha directs their signal-dependent phosphorylation and degradation when transferred to heterologous proteins. The C-terminal PEST sequence from IkappaBalpha was not essential for induced proteolysis of the chimeric proteins. A deletion analysis conducted on the SRD identified a 25 amino acid sub-domain of IkappaBalpha that is necessary and sufficient for the degradative response in vivo and for recognition by TNFalpha-dependent IkappaBalpha kinase in vitro . The results obtained should prove instrumental in the further characterization of IkappaB-specific kinases, as well as the E2 and E3 enzymes responsible for IkappaBalpha ubiquitination. Furthermore, they suggest a novel strategy for generating conditional mutants, by targetting heterologous proteins for transient elimination by the IkappaBalpha pathway