Subramanya Srikantan

LincRNA-p21 Suppresses Target mRNA Translation

Mammalian long intergenic noncoding RNAs (lincRNAs) are best known for modulating transcription. Here we report a posttranscriptional function for lincRNA-p21 as a modulator of translation. Association of the RNA-binding protein HuR with lincRNA-p21 favored the recruitment of let-7/Ago2 to lincRNA-p21, leading to lower lincRNA-p21 stability. Under reduced HuR levels, lincRNA-p21 accumulated in human cervical carcinoma HeLa cells, increasing its association with JUNB and CTNNB1 mRNAs and selectively lowering their translation. With elevated HuR, lincRNA-p21 levels declined, which in turn derepressed JunB and β-catenin translation and increased the levels of these proteins. We propose that HuR controls translation of a subset of target mRNAs by influencing lincRNA-p21 levels. Our findings uncover a role for lincRNA as a posttranscriptional inhibitor of translation.

p16INK4a translation suppressed by miR-24

Background Expression of the tumor suppressor p16INK4a increases during aging and replicative senescence. Methodology/Principal Findings Here, we report that the microRNA miR-24 suppresses p16 expression in human diploid fibroblasts and cervical carcinoma cells. Increased p16 expression with replicative senescence was associated with decreased levels of miR-24, a microRNA that was predicted to associate with the p16 mRNA coding and 3′-untranslated regions. Ectopic miR-24 overexpression reduced p16 protein but not p16 mRNA levels. Conversely, introduction of antisense (AS)-miR-24 blocked miR-24 expression and markedly enhanced p16 protein levels, p16 translation, and the production of EGFP-p16 reporter bearing the miR-24 target recognition sites. Conclusions/Significance Together, our results suggest that miR-24 represses the initiation and elongation phases of p16 translation.

miR-130 Suppresses Adipogenesis by Inhibiting Peroxisome Proliferator-Activated Receptor Expression

ABSTRACT Adipose tissue development is tightly regulated by altering gene expression. MicroRNAs are strong posttranscriptional regulators of mammalian differentiation. We hypothesized that microRNAs might influence human adipogenesis by targeting specific adipogenic factors. We identified microRNAs that showed varying abundance during the differentiation of human preadipocytes into adipocytes. Among them, miR-130 strongly affected adipocyte differentiation, as overexpressing miR-130 impaired adipogenesis and reducing miR-130 enhanced adipogenesis. A key effector of miR-130 actions was the protein peroxisome proliferator-activated receptor γ (PPARγ), a major regulator of adipogenesis. Interestingly, miR-130 potently repressed PPARγ expression by targeting both the PPARγ mRNA coding and 3′ untranslated regions. Adipose tissue from obese women contained significantly lower miR-130 and higher PPARγ mRNA levels than that from nonobese women. Our findings reveal that miR-130 reduces adipogenesis by repressing PPARγ biosynthesis and suggest that perturbations in this regulation is linked to human obesity.

HuR function in disease.

The cytoplasmic events that control mammalian gene expression, primarily mRNA stability and translation, potently influence the cellular response to internal and external signals. The ubiquitous RNA-binding protein (RBP) HuR is one of the best-studied regulators of cytoplasmic mRNA fate. Through its post-transcriptional influence on specific target mRNAs, HuR can alter the cellular response to proliferative, stress, apoptotic, differentiation, senescence, inflammatory and immune stimuli. In light of its central role in important cellular functions, HuRs role in diseases in which these responses are aberrant is increasingly appreciated. Here, we review the mechanisms that control HuR function, its influence on target mRNAs, and how impairment in HuR-governed gene expression programs impact upon different disease processes. We focus on HuRs well-recognized implication in cancer and chronic inflammation, and discuss emerging studies linking HuR to cardiovascular, neurological, and muscular pathologies. We also discuss the progress, potential, and challenges of targeting HuR therapeutically.

miR-519 reduces cell proliferation by lowering RNA-binding protein HuR levels

Gene expression is potently regulated through the action of RNA-binding proteins (RBPs) and microRNAs (miRNAs). Here, we present evidence of a miRNA regulating an RBP. The RBP HuR can stabilize and modulate the translation of numerous target mRNAs involved in cell proliferation, but little is known about the mechanisms that regulate HuR abundance. We identified two putative sites of miR-519 interaction on the HuR mRNA, one in its coding region (CR), one in its 3′-untranslated region (UTR). In several human carcinoma cell lines tested, HeLa (cervical), HCT116 and RKO (colon), and A2780 (ovarian), overexpression of a miR-519 precursor [(Pre)miR-519] reduced HuR abundance, while inhibiting miR-519 by using an antisense RNA [(AS)miR-519] elevated HuR levels. The influence of miR-519 was recapitulated using heterologous reporter constructs that revealed a greater repressive effect on the HuR CR than the HuR 3′-UTR target sequences. miR-519 did not alter HuR mRNA abundance, but reduced HuR biosynthesis, as determined by measuring nascent HuR translation and HuR mRNA association with polysomes. Modulation of miR-519 leading to altered HuR levels in turn affected the levels of proteins encoded by HuR target mRNAs. In keeping with HuRs proliferative influence, (AS)miR-519 significantly increased cell number and [3H]-thymidine incorporation, while (Pre)miR-519 reduced these parameters. Importantly, the growth-promoting effects of (AS)miR-519 required the presence of HuR, because downregulation of HuR by RNAi dramatically suppressed its proliferative action. In sum, miR-519 represses HuR translation, in turn reducing HuR-regulated gene expression and cell division.

Nuclear HuR accumulation through phosphorylation by Cdk1

A predominantly nuclear RNA-binding protein, HuR translocates to the cytoplasm in response to stress and proliferative signals, where it stabilizes or modulates the translation of target mRNAs. Here, we present evidence that HuR phosphorylation at S202 by the G2-phase kinase Cdk1 influences its subcellular distribution. HuR was specifically phosphorylated in synchronous G2-phase cultures; its cytoplasmic levels increased by Cdk1-inhibitory interventions and declined in response to Cdk1-activating interventions. In keeping with the prominently cytoplasmic location of the nonphosphorylatable point mutant HuR(S202A), phospho-HuR(S202) was shown to be predominantly nuclear using a novel anti-phospho-HuR(S202) antibody. The enhanced cytoplasmic presence of unphosphorylated HuR was linked to its decreased association with 14-3-3 and to its heightened binding to target mRNAs. Our findings suggest that Cdk1 phosphorylates HuR during G2, thereby helping to retain it in the nucleus in association with 14-3-3 and hindering its post-transcriptional function and anti-apoptotic influence.

Functional interplay between RNA-binding protein HuR and microRNAs

The mammalian RNA-binding protein (RBP) HuR associates with numerous mRNAs encoding proteins with roles in cell division, cell survival, immune response, and differentiation. HuR was known to stabilize many of these mRNAs and/or modulated their translation, but the molecular processes by which HuR affected the fate of target mRNAs was largely unknown. Evidence accumulated over the past five years has revealed that the influence of HuR on many bound transcripts depends on HuRs interplay with microRNAs which associate with the same mRNAs. Here, we review the interactions of HuR and microRNAs - both competitive and cooperative - that govern expression of shared target mRNAs. Competition between HuR and microRNAs typically results in enhanced gene expression if the HuR-mRNA interaction prevails, and in repression if the microRNA remains associated. Cooperation between HuR and microRNAs leads to lower expression of the shared mRNA. We also describe the regulation of HuR levels by microRNAs as well as the regulation of microRNA levels by HuR. Finally, we discuss transcriptome-wide analyses of HuR-bound mRNAs with neighboring microRNA sites, and review the emerging mechanisms whereby microRNAs confer versatility and robustness to the post-transcriptional outcomes of HuR targets.

Ubiquitin‐mediated proteolysis of HuR by heat shock

The RNA‐binding protein HuR regulates the stability and translation of numerous mRNAs encoding stress‐response and proliferative proteins. Although its post‐transcriptional influence has been linked primarily to its cytoplasmic translocation, here we report that moderate heat shock (HS) potently reduces HuR levels, thereby altering the expression of HuR target mRNAs. HS did not change HuR mRNA levels or de novo translation, but instead reduced HuR protein stability. Supporting the involvement of the ubiquitin–proteasome system in this process were results showing that (1) HuR was ubiquitinated in vitro and in intact cells, (2) proteasome inhibition increased HuR abundance after HS, and (3) the HuR kinase checkpoint kinase 2 protected against the loss of HuR by HS. Within a central, HS‐labile ∼110‐amino‐acid region, K182 was found to be essential for HuR ubiquitination and proteolysis as mutant HuR(K182R) was left virtually unubiquitinated and was refractory to HS‐triggered degradation. Our findings reveal that HS transiently lowers HuR by proteolysis linked to K182 ubiquitination and that HuR reduction enhances cell survival following HS.

miR-375 Inhibits Differentiation of Neurites by Lowering HuD Levels

ABSTRACT 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.

Post-transcriptional gene regulation by HuR promotes a more tumorigenic phenotype

In a breast tumor xenograft model, the MCT-1 oncogene increases the in vivo tumorgenicity of MCF7 cells by promoting angiogenesis and inhibiting apoptosis. Increases in the tumor microvascular density are accompanied by a strong reduction in the levels of the angiogenesis inhibitor thrombospondin-1 (TSP1), but the mechanisms underlying this process are unknown. We show that TSP1 expression is controlled, at least in part, by post-transcriptional events. Using RNA interference to knock down the expression of the RNA-binding protein HuR in MCF7 cells as well as HuR overexpression, we demonstrate that HuR plays an important role in translation of the TSP1 mRNA. Furthermore, employing the RIP-Chip assay yielded 595 transcripts with significantly altered binding to HuR in the more tumorigenic breast cancer clones compared with the weakly tumorigenic clones. These mRNAs clustered in several pathways implicated in the transformed phenotype, such as the RAS pathway (involved in mitogenesis), the PI3K pathway (evasion of apoptosis) and pathways mediating angiogenesis and the cellular response to hypoxia. These findings demonstrate for the first time that global changes in HuR-bound mRNAs are implicated in the evolution to a more tumorigenic phenotype in an in vivo tumor model and underscore the role of global mRNA-protein interactions toward tumor progression.