In cycling cells, transcription of ribosomal RNA genes by RNA polymerase I (Pol I) is tightly coordinated with cell growth. Here, we show that the mammalian target of rapamycin (mTOR) regulates Pol I transcription by modulating the activity of TIF-IA, a regulatory factor that senses nutrient and growth-factor availability. Inhibition of mTOR signaling by rapamycin inactivates TIF-IA and impairs transcription-initiation complex formation. Moreover, rapamycin treatment leads to translocation of TIF-IA into the cytoplasm. Rapamycin-mediated inactivation of TIF-IA is caused by hypophosphorylation of Ser 44 (S44) and hyperphosphorylation of Ser 199 (S199). Phosphorylation at these sites affects TIF-IA activity in opposite ways, for example, phosphorylation of S44 activates and S199 inactivates TIF-IA. The results identify a new target for mTOR-signaling pathways and elucidate the molecular mechanism underlying mTOR-dependent regulation of rRNA synthesis.
Noncoding RNAs are important components of regulatory networks controlling the epigenetic state of chromatin. We analyzed the role of pRNA (promoterassociated RNA), a noncoding RNA that is complementary to the rDNA promoter, in mediating de novo CpG methylation of rRNA genes (rDNA). We show that pRNA interacts with the target site of the transcription factor TTF-I, forming a DNA:RNA triplex that is specifically recognized by the DNA methyltransferase DNMT3b. The results reveal a compelling new mechanism of RNA-dependent DNA methylation, suggesting that recruitment of DNMT3b by DNA:RNA triplexes may be a common and generally used pathway in epigenetic regulation.Supplemental material is available at http://www.genesdev.org.Received April 28, 2010; revised version accepted August 26, 2010. Noncoding RNAs (ncRNAs) play a profound and complex role in regulating gene expression (Goodrich and Kugel 2009). While the mechanistic details of how RNA and chromatin are connected remain unclear, there is increasing evidence that epigenetic regulation likely represents a balanced interplay of both RNA and chromatin fields (Bernstein and Allis 2005;Mattick 2009). Previous studies have established that ncRNA plays a key role in epigenetic silencing of rRNA genes. Mammalian genomes contain several clusters of tandemly arrayed rRNA genes (rDNA), of which only a subset is transcribed. Silent rDNA repeats are marked by heterochromatic histone modifications and CpG methylation of the rDNA promoter . The establishment and maintenance of the heterochromatic state is mediated by NoRC (Strohner et al. 2001), a chromatin remodeling complex comprising the ATPase SNF2h and a large subunit, termed TIP5 (TTF-Iinteracting protein #5). NoRC silences rRNA genes by recruiting enzymes that mediate heterochromatin formation and silencing (Zhou et al. 2002;Zhou and Grummt 2005). Transcriptional silencing involves DNA methylation at a critical CpG residue (CpG-133) within the upstream control element (UCE) of the rDNA promoter, thereby impairing binding of the transcription factor UBF and abrogating transcription complex formation . Importantly, NoRC function requires the association of TIP5 with RNA that originates from an RNA polymerase I (Pol I) promoter located in the intergenic spacer ;2 kb upstream of the pre-rRNA transcription start site (Mayer et al. 2006). These intergenic transcripts are of low abundance and usually do not accumulate in vivo because they are rapidly degraded or processed into 150-to 250-nucleotide (nt) RNAs that are shielded from degradation by binding to NoRC. These NoRC-associated transcripts are termed pRNA (for promoter-associated RNA), as their sequence matches the rDNA promoter. TIP5 recognizes the secondary structure of pRNA, and the interaction of TIP5 with pRNA changes the structure of both pRNA and NoRC in an induced fit mechanism (Mayer et al. 2008). Antisensemediated depletion of pRNA leads to nucleoplasmic distribution of NoRC, hypomethylation of rDNA, and enhanced Pol I transcription. ''RNA refeeding'' a...
Transcripts originating from the intergenic spacer (IGS) that separates rRNA genes (rDNA) have been known for two decades; their biological role, however, is largely unknown. Here we show that IGS transcripts are required for establishing and maintaining a specific heterochromatic configuration at the promoter of a subset of rDNA arrays. The mechanism of action appears to be mediated through the interaction of TIP5, the large subunit of the chromatin remodeling complex NoRC, with 150-300 nucleotide RNAs that are complementary in sequence to the rDNA promoter. Mutations that abrogate RNA binding of TIP5 impair the association of NoRC with rDNA and fail to promote H3K9&H4K20 methylation and HP1 recruitment. Knockdown of IGS transcripts abolishes the nucleolar localization of NoRC, decreases DNA methylation, and enhances rDNA transcription. The results reveal an important contribution of processed IGS transcripts in chromatin structure and epigenetic control of the rDNA locus.
Proximal spinal muscular atrophy (SMA) is a motoneuron disease for which there is currently no effective treatment. In animal models of SMA, spinal motoneurons exhibit reduced axon elongation and growth cone size. These defects correlate with reduced β-actin messenger RNA and protein levels in distal axons. We show that survival motoneuron gene (Smn)–deficient motoneurons exhibit severe defects in clustering Cav2.2 channels in axonal growth cones. These defects also correlate with a reduced frequency of local Ca2+ transients. In contrast, global spontaneous excitability measured in cell bodies and proximal axons is not reduced. Stimulation of Smn production from the transgenic SMN2 gene by cyclic adenosine monophosphate restores Cav2.2 accumulation and excitability. This may lead to the development of new therapies for SMA that are not focused on enhancing motoneuron survival but instead investigate restoration of growth cone excitability and function.
All organisms sense and respond to conditions that stress their homeostatic mechanisms. Here we review current studies showing that the nucleolus, long regarded as a mere ribosome producing factory, plays a key role in monitoring and responding to cellular stress. After exposure to extra- or intracellular stress, cells rapidly down-regulate the synthesis of ribosomal RNA. Impairment of nucleolar function in response to stress is accompanied by perturbation of nucleolar structure, cell cycle arrest and stabilization of p53. The nucleolar target for down-regulation of rDNA transcription is TIF-IA, an essential transcription factor that modulates the activity of RNA polymerase I (Pol I). Upon stress, TIF-IA is phosphorylated by c-Jun N-terminal kinase 2 (JNK2). Phosphorylation prevents TIF-IA from interaction with Pol I, thereby impairing transcription complex formation and rRNA synthesis. Furthermore, stress-induced inactivation of TIF-IA is accompanied by translocation of TIF-IA from the nucleolus to the nucleoplasm. These findings, together with other data showing stress-induced release of nucleolar proteins to carry out other regulatory functions, reinforce the growing realization that nucleoli orchestrate the chain of events the cell uses to properly respond to stress signals.
Silencing of ribosomal RNA genes (rDNA) requires binding of the chromatin remodelling complex NoRC to RNA that is complementary to the rDNA promoter. NoRC-associated RNA (pRNA) folds into a conserved stem-loop structure that is required for nucleolar localization and rDNA silencing. Mutations that disrupt the stem-loop structure impair binding of TIP5, the large subunit of NoRC, to pRNA and abolish targeting of NoRC to nucleoli. Binding to pRNA results in a conformational change of TIP5, as shown by enhanced sensitivity of TIP5 towards trypsin digestion. Our results indicate an RNA-dependent mechanism that targets NoRC to chromatin and facilitates the interaction with co-repressors that promote heterochromatin formation and rDNA silencing.
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