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.

an orderly manner on the pre-mRNA, thereby forming the Jü rgen Lauber 1 , William S.Lane 2 and catalytic splicing machinery known as the spliceosome. Reinhard Lü hrmann 3Despite the fact that exogenous phosphates are not Keller, 1985) and has been shown to be involved in Cambridge, MA 02138, USA several steps from spliceosome assembly to product release 1 Present address: Qiagen GmbH, Max-Volmer-Strasse 4, 40724 Hilden, (reviewed in Guthrie, 1991;Moore et al., 1993). GermanyTwo classes of splicing factors are distinguished cur-3 Corresponding author rently. The first class comprises four evolutionarily cone-mail: luehrmann@imt.uni-marburg.de served small nuclear ribonucleoprotein (snRNP) particles, U1, U2, U4/U6 and U5, that contain either one (U1, U2, The driving forces behind the many RNA conform-U5) or two (U4/U6) snRNA components (for review, see ational changes occurring in the spliceosome are not Green, 1991;Guthrie, 1991;Rymond and Rosbash, 1992; well understood. Here we characterize an evolu- Moore et al., 1993); the second class consists of an as yet tionarily conserved human U5 small nuclear ribounknown number of proteins that are not tightly bound to nucleoprotein (snRNP) protein (U5-116kD) that is snRNPs and are therefore termed non-snRNP splicing strikingly homologous to the ribosomal elongation factors (see Lamm and Lamond, 1993;Beggs, 1995 ; factor EF-2 (ribosomal translocase). A 114 kDa proteinKrämer, 1995). (Snu114p) homologous to U5-116kD was identified inThe composition of the U snRNPs has been studied Saccharomyces cerevisiae and was shown to be essential most extensively in HeLa cells . At low for yeast cell viability. Genetic depletion of Snu114p salt concentrations (up to 100 mM), where HeLa nuclear results in accumulation of unspliced pre-mRNA, extracts support pre-mRNA splicing in vitro, a 12S U1 indicating that Snu114p is essential for splicing in vivo.snRNP, 17S U2 snRNP and a 25S [U4/U6·U5] tri-snRNP Antibodies specific for U5-116kD inhibit pre-mRNA complex are found. At high salt concentrations (350-splicing in a HeLa nuclear extract in vitro. In HeLa 450 mM), the tri-snRNP complex dissociates into a 20S cells, U5-116kD is located in the nucleus and co-U5 and a 12S U4/U6 particle. In the U4/U6 snRNP, the localizes with snRNP-containing subnuclear structures U4 and U6 snRNAs interact through extensive sequence referred to as speckles. The G domain of U5-116kD/ complementarity (Bringmann et al., 1984; Hashimoto and Snu114p contains the consensus sequence elements Steitz, 1984;Rinke et al., 1985;Brow and Guthrie, 1988). G1-G5 important for binding and hydrolyzing GTP.The proteins of the snRNPs fall into two groups, the Consistent with this, U5-116kD can be cross-linked common proteins (B/BЈ, D1, D2, D3, E, F and G), specifically to GTP by UV irradiation of U5 snRNPs.which are present in each snRNP, and the particle-specific Moreover, a single amino acid substitution in the G1proteins. While U1 and U2 snRNPs contain three (70K, sequence motif of Snu114p, expected to abolish GTP-A and C...

Spinal muscular atrophy (SMA) is a motoneuron disease caused by reduced levels of survival motoneuron (SMN) protein. Previous studies have assigned SMN to uridine-rich small nuclear ribonucleoprotein particle (U snRNP) assembly, splicing, transcription, and RNA localization. Here, we have used gene silencing to assess the effect of SMN protein deficiency on U snRNP metabolism in living cells and organisms. In HeLa cells, we show that reduction of SMN to levels found in SMA patients impairs U snRNP assembly. In line with this, induced silencing of SMN expression in Xenopus laevis or zebrafish arrested embryonic development. Under less severe knock-down conditions, zebrafish embryos proceeded through development yet exhibited dramatic SMA-like motor axon degeneration. The same was observed after silencing two other essential factors in the U snRNP assembly pathway, Gemin2 and pICln. Importantly, the injection of purified U snRNPs into either SMN-or Gemin2-deficient embryos of Xenopus and zebrafish prevented developmental arrest and motoneuron degeneration, respectively. These findings suggest that motoneuron degeneration in SMA patients is a direct consequence of impaired production of U snRNPs.[Keywords: Survival motor neurons (SMN); U snRNP assembly; motoneuron; spinal muscular atrophy; zebrafish] Supplemental material is available at http://www.genesdev.org.

Chronic cardiac stress induces pathologic hypertrophy and fibrosis of the myocardium. The microRNA-29 (miR-29) family has been found to prevent excess collagen expression in various organs, particularly through its function in fibroblasts. Here, we show that miR-29 promotes pathologic hypertrophy of cardiac myocytes and overall cardiac dysfunction. In a mouse model of cardiac pressure overload, global genetic deletion of miR-29 or antimiR-29 infusion prevents cardiac hypertrophy and fibrosis and improves cardiac function. Targeted deletion of miR-29 in cardiac myocytes in vivo also prevents cardiac hypertrophy and fibrosis, indicating that the function of miR-29 in cardiac myocytes dominates over that in non-myocyte cell types. Mechanistically, we found cardiac myocyte miR-29 to de-repress Wnt signaling by directly targeting four pathway factors. Our data suggests that, cell- or tissue-specific antimiR-29 delivery may have therapeutic value for pathological cardiac remodeling and fibrosis.

Background— Several microRNAs (miRs) have been shown to regulate gene expression in the heart, and dysregulation of their expression has been linked to cardiac disease. miR-378 is strongly expressed in the mammalian heart but so far has been studied predominantly in cancer, in which it regulates cell survival and tumor growth. Methods and Results— Here, we report tight control of cardiomyocyte hypertrophy through miR-378. In isolated primary cardiomyocytes, miR-378 was found to be both necessary and sufficient to repress cardiomyocyte hypertrophy. Bioinformatic prediction suggested that factors of the mitogen-activated protein kinase (MAPK) pathway are enriched among miR-378 targets. Using mRNA and protein expression analysis along with luciferase assays, we validated 4 key components of the MAPK pathway as targets of miR-378: MAPK1 itself, insulin-like growth factor receptor 1, growth factor receptor-bound protein 2, and kinase suppressor of ras 1. RNA interference with these targets prevented the prohypertrophic effect of antimiR-378, suggesting their functional relation with miR-378. Because miR-378 significantly decreases in cardiac disease, we sought to compensate for its loss through adeno-associated virus–mediated, cardiomyocyte-targeted expression of miR-378 in an in vivo model of cardiac hypertrophy (pressure overload by thoracic aortic constriction). Restoration of miR-378 levels significantly attenuated thoracic aortic constriction–induced cardiac hypertrophy and improved cardiac function. Conclusions— Our data identify miR-378 as a regulator of cardiomyocyte hypertrophy, which exerts its activity by suppressing the MAPK signaling pathway on several distinct levels. Restoration of disease-associated loss of miR-378 through cardiomyocyte-targeted adeno-associated virus–miR-378 may prove to be an effective therapeutic strategy in myocardial disease.

Distal spinal muscular atrophy type 1 (DSMA1) is an autosomal recessive disease that is clinically characterized by distal limb weakness and respiratory distress. In this disease, the degeneration of alpha-motoneurons is caused by mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2). This protein has been implicated in DNA replication, pre-mRNA splicing and transcription, but its precise function in all these processes has remained elusive. We have purified catalytically active recombinant IGHMBP2, which has enabled us to assess its enzymatic properties and to identify its cellular targets. Our data reveal that IGHMBP2 is an ATP-dependent 5' --> 3' helicase, which unwinds RNA and DNA duplices in vitro. Importantly, this helicase localizes predominantly to the cytoplasm of neuronal and non-neuronal cells and associates with ribosomes. DSMA1-causing amino acid substitutions in IGHMBP2 do not affect ribosome binding yet severely impair ATPase and helicase activity. We propose that IGHMBP2 is functionally linked to translation, and that mutations in its helicase domain interfere with this function in DSMA1 patients.