The sequential co-IP of the L11-c-Myc complex using anti-FLAG antibodies followed by anti-V5 antibodies is shown in Fig. to ribosomal stress induced by the treatment of cells with Mouse monoclonal to CHK1 a low dose of actinomycin D or serum starvation, L11 binding to these genes was improved, and inversely TRRAP binding to these genes was decreased. Consistently, knockdown of L11 rescued the reduction of the manifestation of these genes by the two treatments. These results demonstrate that L11 suppresses c-Myc-dependent and RNA polymerase III-catalyzed transcription of 5 S rRNA and tRNA genes in response to ribosomal stress, ensuring a tight coordination between c-Myc activity and ribosomal biogenesis. transgene prospects to neoplastic and malignant phenotypes in mice (4,C7). Thus, it is essential to maintain the proper physiological level and activity of c-Myc during normal cell homeostasis. One of the important functions of c-Myc is definitely to regulate ribosomal biogenesis (8). Ribosomal biogenesis is definitely a PHA-665752 tightly controlled cellular process that requires coordinated transcription mediated by all three RNA polymerases (Pols)3 in order to guarantee efficient and accurate production of ribosomes. c-Myc enhances the transcription of many ribosomal biogenesis-related genes catalyzed by RNA Pol II, such as those encoding ribosome assembly proteins, translation initiation and elongation factors, and ribosomal proteins (9,C12). c-Myc also enhances RNA Pol I-catalyzed rRNA synthesis (13,C15) and RNA Pol III-mediated 5 S and tRNA transcription (16). Although these are essential functions for c-Myc in regulating PHA-665752 cell growth and proliferation, overuse of them would convert this transcriptional element into an oncogenic element and favor tumor growth. Indeed, deregulation of c-Myc activity as mentioned above and/or of ribosomal biogenesis has been highly associated with tumorigenesis (17). In our previous attempt to understand the coordination between ribosomal biogenesis and c-Myc activity, we exposed that ribosomal protein L11 functions as a novel c-Myc inhibitor via a opinions mechanism (18). L11 binds to c-Myc at Myc package II, a critical region required for all known c-Myc functions. This binding prospects to inhibition PHA-665752 of the recruitment of the TRRAP co-activator and subsequent reduction of histone acetylation at promoters of c-Myc target genes, including RNA Pol II-transcribed nucleolin, E2F2, eIF4E, and RNA Pol I-transcribed pre-rRNA genes (18). As a result, L11 suppresses the manifestation of these genes. However, it remains unclear whether the inhibitory effect of L11 on c-Myc activity is definitely ubiquitously true to c-Myc-dependent gene transcription catalyzed by all three RNA Pols, because the promoter companies for RNA Pol I and II target genes are different from those for RNA Pol III target genes, such as 5 S rRNA and tRNA genes. Interestingly, regardless of the difference in their target promoter architectures, mechanisms underlying c-Myc-mediated transcription by all RNA Pols look like similar. For instance, similar to the case of c-Myc-induced transcription by RNA Pol I and II, the TRRAP co-activator is also required for c-Myc-mediated transcription by RNA Pol III of the 5 S rRNA and tRNA genes, and c-Myc can bind to these genes and promote the association of TRRAP with them, although no standard E-box elements specific for c-Myc-binding were recognized in the promoters of these genes (19). Because the 5 S rRNA and tRNA genes are essential for ribosomal biogenesis and translation, the manifestation of these genes must also become tightly controlled for normal cell growth. Normally, overexpression of RNA Pol III-specific transcription element, Brf1, results in highly specific elevation of tRNA and 5 S rRNA manifestation and oncogenic transformation in cells (20). Strikingly, overexpression of the RNA Pol III-transcribed tRNAiMet only is sufficient to drive cell proliferation and induce tumors in mice (20). This study suggests that deregulated overexpression of Pol III activity may contribute to tumorigenesis too. To further consolidate the part of L11 in regulating c-Myc-specific transcriptional activity, we have carried out a series of experiments to test whether L11 also regulates c-Myc-dependent transcription of class III genes by RNA Pol III. Indeed, our study as presented here demonstrates L11 can inhibit c-Myc-dependent transcription of the 5 S rRNA and tRNA PHA-665752 genes. L11 bound to c-Myc in the 5 S rRNA and tRNA genes, and this binding prospects to reduction of TRRAP association with these genes. Knockdown of endogenous L11 significantly enhanced c-Myc-mediated transcription of these genes..
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