Fragile X syndrome is a common form of inherited mental retardation. Most fragile X patients exhibit mutations in the fragile X mental retardation gene 1 (FMR1) that lead to transcriptional silencing and hence to the absence of the fragile X mental retardation protein (FMRP). Since FMRP is an RNA-binding protein which associates with polyribosomes, it had been proposed to function as a regulator of gene expression at the post-transcriptional level. In the present study, we show that FMRP strongly inhibits translation of various mRNAs at nanomolar concentrations in both rabbit reticulocyte lysate and microinjected Xenopus laevis oocytes. This effect is specific for FMRP, since other proteins with similar RNA-binding domains, including the autosomal homologues of FMRP, FXR1 and FXR2, failed to suppress translation in the same concentration range. Strikingly, a disease-causing Ile-->Asn substitution at amino acid position 304 (I304N) renders FMRP incapable of interfering with translation in both test systems. Initial studies addressing the underlying mechanism of inhibition suggest that FMRP inhibits the assembly of 80S ribosomes on the target mRNAs. The failure of FMRP I304N to suppress translation is not due to its reduced affinity for mRNA or its interacting proteins FXR1 and FXR2. Instead, the I304N point mutation severely impairs homo-oligomerization of FMRP. Our data support the notion that inhibition of translation may be a function of FMRP in vivo. We further suggest that the failure of FMRP to oligomerize, caused by the I304N mutation, may contribute to the pathophysiological events leading to fragile X syndrome.
Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA-RNA interactions, such as the unwinding of the U4͞U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5-200kD and U5-100kD, which share homology with the DEAD͞DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4͞U6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5-200kD protein but lack U5-100kD, suggesting that the U5-200kD protein could mediate U4͞U6 duplex unwinding. Finally, U5-200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5-200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4͞U6 RNA duplex in the spliceosome.The spliceosome, which catalyzes splicing of nuclear precursor mRNA (pre-mRNA), is formed by an ordered recruitment of the small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4͞U6, and numerous non-snRNP proteins onto the pre-mRNA (1, 2). During spliceosome assembly a complex and highly dynamic network of both small nuclear RNA (snRNA)-snRNA and snRNA-pre-mRNA interactions is formed (1, 3, 4). Initially, U1 snRNA hybridyzes with the 5Ј splice site, and U2 snRNA interacts with the branch site region of a premRNA. Although the latter interaction is likely to persist through all steps of splicing (3), U1 snRNA must be displaced from the intron in an ATP-dependent reaction when the [U4͞U6.U5] tri-snRNP enters the pre-spliceosome (5). Before or concomitant with this step, the phylogenetically conserved duplex between U4 and U6 snRNA, which exists in the [U4͞U6.U5] tri-snRNP, dissociates and U6 snRNA interacts simultaneously with U2 snRNA and the 5Ј splice site (reviewed in refs. 1, 3, and 4). The unwinding of the U4͞U6 RNA duplex thus is essential for establishing an RNA network in the catalytic center of the spliceosome. What triggers this or other rearrangements of RNA is presently unknown. Yet it is generally thought that one or more spliceosomal proteins of the DEAD͞DEXH-box family of ATP-dependent RNA helicases (6, 7) play a major role in these events (4,8,9). In the yeast Saccharomyces cerevisiae, the precursor RNA processing proteins Prp2p, Prp5p, Prp16p, Prp22p, Prp28p, and Prp43p have been identified as putative DEAD͞DEXH-box RNA helicases required for pre-mRNA splicing (10-15), and potential mammalian homologs of some are known (16)(17)(18)(19). Indirect evidence that they may funct...
Background Cardiomyocytes (CM) utilize Ca2+ not only in excitation-contraction coupling (ECC), but also as a signaling molecule promoting for example cardiac hypertrophy. It is largely unclear how Ca2+ triggers signaling in CM in the presence of the rapid and large Ca2+ fluctuations that occur during ECC. A potential route is store-operated Ca2+ entry (SOCE), a drug-inducible mechanism for Ca2+ signaling that requires stromal interaction molecule 1 (STIM1). SOCE can also be induced in cardiomyocytes, which prompted us to study STIM1-dependent Ca2+-entry with respect to cardiac hypertrophy in vitro and in vivo. Methods and Results Consistent with earlier reports, we found drug-inducible SOCE in neonatal rat cardiomyocytes, which was dependent on STIM1. While this STIM1-dependent, drug-inducible SOCE was only marginal in adult cardiomyocytes isolated from control hearts, it significantly increased in cardiomyocytes isolated from adult rats that had developed compensated cardiac hypertrophy after abdominal aortic banding. Moreover, we detected an inwardly rectifying current in hypertrophic cardiomyocytes that occurs under native conditions (i.e. in the absence of drug-induced store depletion) and is dependent on STIM1. By manipulating its expression, STIM1 was found to be both sufficient and necessary for cardiomyocyte hypertrophy both in vitro and in the adult heart in vivo. Stim1 silencing by AAV9-mediated gene transfer protected rats from pressure overload-induced cardiac hypertrophy. Conclusions STIM1 promotes cardiac hypertrophy by controlling a previously unrecognized sarcolemmal current.
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