GW bodies (or P-bodies) are cytoplasmic granules containing proteins involved in both mRNA degradation and storage, including the RNA interference machinery. Their mechanism of assembly and function are still poorly known although their number depends upon the flux of mRNA to be stored or degraded. We show here that silencing of the translational regulator CPEB1 leads to their disappearance, as reported for other GW body components. Surprisingly, the same results were obtained with several siRNAs targeting genes encoding proteins unrelated to mRNA metabolism. The disappearance of GW bodies did not correlate with the silencing activity of the siRNA and did not inhibit further silencing by siRNA. Importantly, in most cases, GW bodies were rapidly reinduced by arsenite, indicating that their assembly was not prevented by the inhibition of the targeted or off-target genes. We therefore propose that some siRNA sequences affect mRNA metabolism so as to diminish the amount of mRNA directed to the GW bodies. As an exception, GW bodies were not reinduced following Rck/p54 depletion by interference, indicating that this component is truly required for the GW body assembly. Noteworthy, Rck/p54 was dispensable for the assembly of stress granules, in spite of their close relationship with the GW bodies.

The 2-5 oligoadenylate (2-5A)/RNase L pathway is one of the enzymatic pathways induced by interferon. RNase L is a latent endoribonuclease which is activated by 2-5A and inhibited by a specific protein known as RLI (RNase L inhibitor). This system has an important role in regulating viral infection. Additionally, variations in RNase L activity have been observed during cell growth and differentiation but the significance of the 2-5A/RNase L/RLI pathway in these latter processes is not known. To determine the roles of RNase L and RLI in muscle differentiation, C2 mouse myoblasts were transfected with sense and antisense RLI cDNA constructs. Importantly, the overexpression of RLI in C2 cells was associated with diminished RNase L activity, an increased level of MyoD mRNA, and accelerated kinetics of muscle differentiation. Inversely, transfection of the RLI antisense construct was associated with increased RNase L activity, a diminished level of MyoD mRNA, and delayed differentiation. In agreement with these data, MyoD mRNA levels were also decreased in C2 cells transfected with an inducible RNase L construct. The effect of RNase L activity on MyoD mRNA levels was relatively specific because expression of several other mRNAs was not altered in C2 transfectants. Therefore, RNase L is directly involved in myoblast differentiation, probably through its role in regulating MyoD stability. This is the first identification of a potential mRNA target for RNase L.The 2Ј-5Ј oligoadenylate (2-5A)/RNase L system is an interferon (IFN)-inducible RNA degradation pathway which is responsible for many of the antiviral and antiproliferative effects of IFNs (37, 41).The 2-5A pathway is composed of at least three types of enzymatic activities: 2-5A-synthetase, 2-5A-degrading enzymes, and RNase L. 2-5A, an oligoadenylate with 2Ј-5Ј phosphodiester bonds, activates RNase L (53), a latent endoribonuclease. Upon activation, RNase L cleaves mRNAs 3Ј of UpNp sequences, thus leading to the inhibition of protein synthesis (14,21).The activity of RNase L was originally thought to be modulated solely by the concentration of the 2-5A activator (11,21). Moreover, we have previously established that RNase L activity can also be regulated by RLI (RNase L inhibitor), a protein inhibitor (5). Overexpression of the RLI cDNA in HeLa cells results in the inhibition of the IFN-activated 2-5A pathway. RLI is induced by viruses such as encephalomyocarditis virus (EMCV) and human immunodeficiency virus (HIV), causing an inhibition of the 2-5A/RNase L system (27, 28). The role of the 2-5A/RNase L pathway in the selective reduction of viral mRNA during EMCV and HIV infection has been demonstrated elsewhere (16,25,27).Variations in intracellular 2-5A and 2-5A-synthetase levels have been observed during cell growth and differentiation even in the absence of exogenous IFN treatment. Indeed, expression of IFN-inducible proteins, such as 2-5A-synthetase, doublestanded RNA-activated protein kinase (PKR), and p202 (a member of the "200 family" of murine proteins) h...

P-bodies are cytoplasmic granules that are linked to mRNA decay, mRNA storage, and RNA interference (RNAi). They are known to interact with stress granules in stressed cells, and with late endosomes. Here, we report that P-bodies also interact with mitochondria, as previously described for P-body-related granules in germ cells. The interaction is dynamic, as a large majority of P-bodies contacts mitochondria at least once within a 3-min interval, and for about 18 s. This association requires an intact microtubule network. The depletion of P-bodies does not seem to affect mitochondria, nor the mitochondrial activity to be required for their contacts with P-bodies. However, inactivation of mitochondria leads to a strong decrease of miRNAmediated RNAi efficiency, and to a lesser extent of siRNA-mediated RNAi. The defect occurs during the assembly of active RISC and is associated with a specific delocalization of endogeneous Ago2 from P-bodies. Our study reveals the possible involvement of RNAi defect in pathologies involving mitochondrial deficiencies. P-bodies are ribonucleoprotein granules present in the cytoplasm of eukaryotic cells. They contain all proteins involved in the 5Ј to 3Ј mRNA degradation pathway, such as the decapping enzyme Dcp2, its enhancers Dcp1, Lsm1-7, Edc3, Hedls/Ge1, and the exonuclease Xrn1. This list extends to factors involved in specific degradation pathways, such as RNAi, NMD, and NGD (1, 2). They also contain proteins involved in translational repression, such as eIF4ET, Rck/p54/Dhh1, CPEB1, and the RISC complex. Some of the latter proteins also play a role in mRNA degradation, in particular Rck/p54/Dhh1, which is known as an enhancer of decapping, and the RISC complex when it is guided by a siRNA. Such catalogue of components indicates that P-bodies participate in these two aspects of mRNA metabolism. In addition, P-bodies increase in number and size when free untranslated mRNA accumulates. In mammals, this is observed when degradation is compromised by XrnI silencing (3) or when polysomes are disrupted with puromycin or arsenite (4). Taken together, these data support a role of P-bodies in mRNA degradation, mRNA storage, and RNA interference. Yet, their exact participation is unclear, as none of these functions is markedly affected in cells where P-bodies have been depleted (5-9).Live cell observations show that the number of P-bodies is quite stable over hours, although occasional formation of new P-bodies or fusions of pre-existing ones are observed (10).

All vectors were constructed by classical molecular biology techniques (see online-only Data Supplement Methods). Plasmid DNA was prepared by using the Maxiprep 500 EF kit (Macherey Nagel), Background-Genetic defects in calpain3 (CAPN3) lead to limb-girdle muscular dystrophy type 2A, a disease of the skeletal muscle that affects predominantly the proximal limb muscles. We previously demonstrated the potential of adeno-associated virus-mediated transfer of the CAPN3 gene to correct the pathological signs in a murine model for limb-girdle muscular dystrophy type 2A after intramuscular and locoregional administrations. Methods and Results-Here, we showed that intravenous injection of calpain3-expressing vector in mice can induce mortality in a dose-dependent manner. An anatomopathological investigation revealed large areas of fibrosis in the heart that we related to unregulated proteolytic activity of calpain3. To circumvent this toxicity, we developed new adeno-associated virus vectors with skeletal muscle-restricted expression by using new muscle-specific promoters that include the CAPN3 promoter itself and by introducing a target sequence of the cardiac-specific microRNA-208a in the cassette. Our results show that CAPN3 transgene expression can be successfully suppressed in the cardiac tissue, preventing the cardiac toxicity, whereas expression of the transgene in skeletal muscle reverts the pathological signs of calpain3 deficiency. Conclusions-The molecular strategies used in this study may be useful for any gene transfer strategy with potential toxicity in the heart.