Caspase-mediated cleavage of HuR in the cytoplasm contributes to pp32/PHAP-I regulation of apoptosis.

The RNA-binding protein HuR affects cell fate by regulating the stability and/or the translation of messenger RNAs that encode cell stress response proteins. In this study, we delineate a novel regulatory mechanism by which HuR contributes to stress-induced cell death. Upon lethal stress, HuR translocates into the cytoplasm by a mechanism involving its association with the apoptosome activator pp32/PHAP-I. Depleting the expression of pp32/PHAP-I by RNA interference reduces both HuR cytoplasmic accumulation and the efficiency of caspase activation. In the cytoplasm, HuR undergoes caspase-mediated cleavage at aspartate 226. This cleavage activity is significantly reduced in the absence of pp32/PHAP-I. Substituting aspartate 226 with an alanine creates a noncleavable isoform of HuR that, when overexpressed, maintains its association with pp32/PHAP-I and delays the apoptotic response. Thus, we propose a model in which HuR association with pp32/PHAP-I and its caspase-mediated cleavage constitutes a regulatory step that contributes to an amplified apoptotic response.

The RNA-binding protein HuR promotes cell migration and cell invasion by stabilizing the beta-actin mRNA in a U-rich-element-dependent manner.

A high expression level of the beta-actin protein is required for important biological mechanisms, such as maintaining cell shape, growth, and motility. Although the elevated cellular level of the beta-actin protein is directly linked to the long half-life of its mRNA, the molecular mechanisms responsible for this effect are unknown. Here we show that the RNA-binding protein HuR stabilizes the beta-actin mRNA by associating with a uridine-rich element within its 3' untranslated region. Using RNA interference to knock down the expression of HuR in HeLa cells, we demonstrate that HuR plays an important role in the stabilization but not in the nuclear/cytoplasmic distribution of the beta-actin mRNA. HuR depletion in HeLa cells alters key beta-actin-based cytoskeleton functions, such as cell adhesion, migration, and invasion, and these defects correlate with a loss of the actin stress fiber network. Together our data establish that the posttranscriptional event involving HuR-mediated beta-actin mRNA stabilization could be a part of the regulatory mechanisms responsible for maintaining cell integrity, which is a prerequisite for avoiding transformation and tumor formation.

Humanized telomeres and an attempt to express a functional human telomerase in yeast.

The maintenance of telomeric repeat DNA depends on an evolutionarily conserved reverse trans criptase called telomerase. In vitro, only the catalytic subunit and a telomerase-associated RNA are required for the synthesis of species-specific repeat DNA. In an attempt to establish a heterologous system for the study of the human telomerase enzyme, we expressed the two core components and predicted regulatory subunits in the yeast Saccharomyces cerevisiae. We show that adequate substrates for human telomerase can be generated; the expressed enzyme was localized in the nucleus and it had the capacity to synthesize human-specific repeats in vitro. However, there was no evidence for human telomerase activity at yeast telomeres in vivo. Therefore functional replacement of the yeast telomerase by the human enzyme may require additional human-specific components. We also replaced the template region of the yeast telomerase RNA with one that dictates the synthesis of vertebrate repeats and performed a detailed molecular analysis of the composition of the telomeres upon outgrowth of such strains. The results suggest that vertebrate repeats on yeast telomeres are subject to a very high degree of repeat turnover and show that an innermost tract of 50 bp of yeast repeats are resistant to replacement.

RNAi-mediated HuR depletion leads to the inhibition of muscle cell differentiation.

The formation of muscle fibers involves the sequential expression of many proteins that regulate key steps during myoblast-to-myotube transition. MyoD, myogenin, and the cyclin-dependent kinase inhibitor p21cip1 are major players in the initiation and maintenance of the differentiated state of mouse embryonic muscle cells (C2C12). The messenger RNAs encoding these three proteins contain typical AU-rich elements (AREs) in their 3'-untranslated regions (3'-UTRs), which are known to affect the half-life of many short-lived mRNAs. HuR, an RNA-binding protein that regulates both the stability and cellular movement of ARE-containing mRNAs, interacts and stabilizes the p21cip1 message under UV stress in human RKO colorectal carcinoma cells. Here, by the use of gel shift experiments and immunoprecipitation followed by reverse transcription-PCR analysis, we show that HuR interacts with MyoD, myogenin, and p21cip1 mRNAs through specific sequences in their 3'-UTRs. To demonstrate the implication of endogenous HuR in myogenesis, we knocked down its expression in myoblasts using RNA interference and observed a significant reduction of HuR expression, associated with complete inhibition of myogenesis. Moreover, the expression of MyoD and myogenin mRNAs, as well as proteins, is significantly reduced in the HuR knockdown C2C12 cells. We were able to completely re-establish the myogenic process of these defective cells by introducing back HuR protein conjugated to a cell-permeable peptide. Finally, HuR accumulates in the cytoplasm during myogenesis. Thus, our results clearly demonstrated that endogenous HuR plays a crucial role in muscle differentiation by regulating the expression and/or the nuclear export of ARE-containing mRNAs that are essential for this process.