RESUMO
The sarcin-ricin loop (SRL) of 23S rRNA in the large ribosomal subunit is a factor-binding site that is essential for GTP-catalyzed steps in translation, but its precise functional role is thus far unknown. Here, we replaced the 15-nucleotide SRL with a GAAA tetraloop and affinity purified the mutant 50S subunits for functional and structural analysis in vitro. The SRL deletion caused defects in elongation-factor-dependent steps of translation and, unexpectedly, loss of EF-Tu-independent A-site tRNA binding. Detailed chemical probing analysis showed disruption of a network of rRNA tertiary interactions that hold together the 23S rRNA elements of the functional core of the 50S subunit, accompanied by loss of ribosomal protein L16. Our results reveal an influence of the SRL on the higher-order structure of the 50S subunit, with implications for its role in translation.
Assuntos
Conformação de Ácido Nucleico , RNA Ribossômico 23S/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Endorribonucleases/química , Escherichia coli/metabolismo , Proteínas Fúngicas/química , RNA Ribossômico 23S/genética , Subunidades Ribossômicas Maiores de Bactérias/genética , Ricina/química , Deleção de SequênciaRESUMO
Fusion of undifferentiated myoblasts into multinucleated myotubes is a prerequisite for developmental myogenesis and postnatal muscle growth. We report that deacetylase inhibitors favor the recruitment and fusion of myoblasts into preformed myotubes. Muscle-restricted expression of follistatin is induced by deacetylase inhibitors and mediates myoblast recruitment and fusion into myotubes through a pathway distinct from those utilized by either IGF-1 or IL-4. Blockade of follistatin expression by RNAi-mediated knockdown, functional inactivation with either neutralizing antibodies or the antagonist protein myostatin, render myoblasts refractory to HDAC inhibitors. Muscles from animals treated with the HDAC inhibitor trichostatin A display increased production of follistatin and enhanced expression of markers of regeneration following muscle injury. These data identify follistatin as a central mediator of the fusigenic effects exerted by deacetylase inhibitors on skeletal muscles and establish a rationale for their use to manipulate skeletal myogenesis and promote muscle regeneration.