NF90 selectively represses the translation of target mRNAs bearing an AU-rich signature motif

The RNA-binding protein nuclear factor 90 (NF90) has been implicated in the stabilization, transport and translational control of several target mRNAs. However, a systematic analysis of NF90 target mRNAs has not been performed. Here, we use ribonucleoprotein immunoprecipitation analysis to identify a large subset of NF90-associated mRNAs. Comparison of the 3′-untranslated regions (UTRs) of these mRNAs led to the elucidation of a 25- to 30-nucleotide, RNA signature motif rich in adenines and uracils. Insertion of the AU-rich NF90 motif (‘NF90m’) in the 3′UTR of an EGFP heterologous reporter did not affect the steady-state level of the chimeric EGFP-NF90m mRNA or its cytosolic abundance. Instead, the translation of EGFP-NF90m mRNA was specifically repressed in an NF90-dependent manner, as determined by analysing nascent EGFP translation, the distribution of chimeric mRNAs on polysome gradients and the steady-state levels of expressed EGFP protein. The interaction of endogenous NF90 with target mRNAs was validated after testing both endogenous mRNAs and recombinant biotinylated transcripts containing NF90 motif hits. Further analysis showed that the stability of endogenous NF90 target mRNAs was not significantly influenced by NF90 abundance, while their translation increased when NF90 levels were reduced. In summary, we have identified an AU-rich RNA motif present in NF90 target mRNAs and have obtained evidence that NF90 represses the translation of this subset of mRNAs

Paradoxical microRNAs: Individual gene repressors, global translation enhancers

In mammalian cells, microRNAs regulate the expression of target mRNAs generally by reducing their stability and/or translation, and thereby control diverse cellular processes such as senescence. We recently reported the differential abundance of microRNAs in young (early-passage, proliferating) relative to senescent (late-passage, non-proliferating) WI-38 human diploid fibroblasts. Here we report that the levels of the vast majority of mRNAs were unaltered in senescent compared to young WI-38 cells, while overall mRNA translation was potently reduced in senescent cells. Downregulation of Dicer or Drosha, two major enzymes in microRNA biogenesis, lowered microRNA levels, but, unexpectedly, it also reduced global translation. While a reduction in Dicer levels markedly enhanced cellular senescence, reduction of Drosha levels did not, suggesting that the Drosha/Dicer effects on translation may be independent of senescence and further suggesting that microRNAs may directly or indirectly enhance mRNA translation in WI-38 cells. We discuss possible scenarios through which Dicer/Drosha/microRNAs could enhance translation

Polyamine depletion induces nucleophosmin modulating stability and transcriptional activity of p53 in intestinal epithelial cells

Our previous studies have shown that polyamines are required for normal intestinal mucosal growth and that decreased levels of polyamines inhibit intestinal epithelial cell (IEC) proliferation by stabilizing p53 and other growth-inhibiting proteins. Nucleophosmin (NPM) is a multifunctional protein that recently has been shown to regulate p53 activity. In the present study, we sought to determine whether polyamine depletion increases NPM modulating, the stability and transcriptional activity of p53 in a normal IEC-6 intestinal epithelial cell line. Depletion of cellular polyamines by α- difluoromethylornithine, the specific inhibitor of polyamine biosynthesis, stimulated expression of the NPM gene and induced nuclear translocation of NPM protein. Polyamine depletion stimulated NPM expression primarily by increasing NPM gene transcription and its mRNA stability, and it induced NPM nuclear translocation through activation of phosphorylation of mitogen-activated protein kinase kinase. Increased NPM interacted with p53 and formed a NPM/p53 complex in polyamine-deficient cells. Inhibition of NPM expression by small interfering RNA targeting NPM (siNPM) not only destabilized p53 as indicated by a decrease in its protein half-life but also prevented the increased p53-dependent transactivation as shown by suppression of the p21 promoter activity. Decreased expression of NPM by siNPM also promoted cell growth in polyamine-deficient cells. These results indicate that 1) polyamine depletion increases expression of the NPM gene and enhances NPM nuclear translocation and 2) increased NPM interacts with and stabilizes p53, leading to inhibition of IEC-6 cell proliferation

miR-375 Inhibits Differentiation of Neurites by Lowering HuD Levels▿

Neuronal development and plasticity are maintained by tightly regulated gene expression programs. Here, we report that the developmentally regulated microRNA miR-375 affects dendrite formation and maintenance. miR-375 overexpression in mouse hippocampus potently reduced dendrite density. We identified the predominantly neuronal RNA-binding protein HuD as a key effector of miR-375 influence on dendrite maintenance. Heterologous reporter analysis verified that miR-375 repressed HuD expression through a specific, evolutionarily conserved site on the HuD 3′ untranslated region. miR-375 overexpression lowered both HuD mRNA stability and translation and recapitulated the effects of HuD silencing, which reduced the levels of target proteins with key functions in neuronal signaling and cytoskeleton organization (N-cadherin, PSD-95, RhoA, NCAM1, and integrin α1). Moreover, the increase in neurite outgrowth after brain-derived neurotrophic factor (BDNF) treatment was diminished by miR-375 overexpression; this effect was rescued by reexpression of miR-375-refractory HuD. Our findings indicate that miR-375 modulates neuronal HuD expression and function, in turn affecting dendrite abundance