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. 2010 Apr 23;285(17):12587-94.
doi: 10.1074/jbc.M109.056259. Epub 2010 Mar 1.

Ribosomal protein L11 associates with c-Myc at 5 S rRNA and tRNA genes and regulates their expression

Mu-Shui Dai  1 , Xiao-Xin Sun, Hua Lu
Affiliations

Ribosomal protein L11 associates with c-Myc at 5 S rRNA and tRNA genes and regulates their expression

Mu-Shui Dai et al. J Biol Chem. .

Erratum in

  • J Biol Chem. 2010 Dec 10;285(50):39574

Abstract

The c-Myc oncoprotein promotes cell growth by enhancing ribosomal biogenesis. Overexpression of c-Myc and aberrant ribosomal biogenesis lead to deregulated cell growth and tumorigenesis. Hence, c-Myc activity and ribosomal biogenesis must be tightly coordinated during normal homeostasis. We previously found that ribosomal protein L11 inhibits c-Myc activity by blocking the recruitment of its co-activator transformation/transcription domain-associated protein (TRRAP) to the promoter regions of c-Myc target genes that are transcribed by RNA polymerases I and II. In this study, we extended the role of L11 to the regulation of c-Myc-driven transcription of the 5 S rRNA and tRNA genes by RNA polymerase III. L11 co-resided with c-Myc at the 5 S rRNA and tRNA genes and significantly inhibited the binding of TRRAP to these genes. Knocking down endogenous L11 enhanced c-Myc-dependent transcription of these genes. Interestingly, in response to ribosomal stress induced by the treatment of cells with a low dose of actinomycin D or serum starvation, L11 binding to these genes was increased, and inversely TRRAP binding to these genes was decreased. Consistently, knockdown of L11 rescued the reduction of the expression 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.

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Figures

FIGURE 1.
FIGURE 1.
Endogenous L11 regulates c-Myc-mediated transcription of the 5 S rRNA and tRNA genes. A, knockdown of endogenous L11 increases the level of endogenous c-Myc. U2OS cells were transfected with siRNAs as indicted followed by IB analysis using antibodies as indicated. B, knockdown of endogenous L11 enhances c-Myc dependent transcription of the 5 S rRNA, tRNATyr, and tRNALeu genes. U2OS cells were transfected with siRNAs as indicated, followed by RT-qPCR assays to determine the relative expression of the 5 S rRNA, tRNATyr, and tRNALeu genes compared with that of glyceraldehyde-3-phosphate dehydrogenase mRNA. Error bars indicate mean ± S.D.
FIGURE 2.
FIGURE 2.
L11 binds to the 5 S rRNA and tRNA genes. ChIP-qPCR assays were conducted to detect the binding of endogenous c-Myc and L11 in the 5 S rRNA and tRNA genes in H1299 cells using anti-c-Myc (N262) or anti-L11 antibodies. Error bars indicate mean ± S.D.
FIGURE 3.
FIGURE 3.
L11 co-resides with c-Myc in the 5 S rRNA and tRNA genes. A, co-IP between ectopic L11 and c-Myc. H1299 cells were transfected with FLAG-L11 with or without V5-c-Myc. The cell lysates were first immunoprecipitated with anti-FLAG antibodies (lanes 3 and 4). The FLAG-L11-associated proteins were eluted with FLAG peptide, and the elution was then immunoprecipitated with anti-V5 antibodies (lanes 5 and 6), followed by an IB assay. B and C, sequential ChIP-qPCR assays were conducted to detect the binding of L11 to c-Myc at the 5 S rRNA and tRNA genes. H1299 cells were transfected with FLAG-L11 with or without V5-c-Myc. The cells were cross-linked, and the cell lysates were immunoprecipitated with anti-FLAG antibody. The FLAG-L11 protein-DNA complexes were eluted with FLAG peptide, and 10% of eluates were used for DNA purification and qPCR amplification (B). The rest of the eluates were subjected to a second IP using anti-V5 antibody, followed by qPCR amplification (C) to detect the 5 S rRNA and tRNA genes. D, overexpression of c-Myc enhances L11 binding to the 5 S rRNA, tRNATyr, and tRNALeu genes. H1299 cells were transfected with FLAG-L11 alone or together with c-Myc. The cells were subjected to ChIP-qPCR assays to detect the binding of L11 to the aforementioned genes. Protein expression of the transfected genes is shown on the right. E, knockdown of endogenous c-Myc decreases L11 binding to the 5 S rRNA, tRNATyr, and tRNALeu genes. U2OS cells were transfected with scrambled (scr) or L11 siRNA, followed by ChIP-qPCR assays to detect the binding of L11 to the aforementioned genes. Protein expression of the transfected genes is shown on the right. The expression of c-Myc and L11 is also shown on the right. Error bars indicate mean ± S.D.
FIGURE 4.
FIGURE 4.
L11 reduces the binding of TRRAP to the 5 S rRNA and tRNA genes. A, H1299 cells transfected with control or FLAG-L11 plasmid were subjected to IB with anti-c-Myc or anti-FLAG antibodies. B and C, L11 reduces the binding of TRRAP to the 5 S rRNA and tRNALeu genes. The transfected cells in A were subjected to ChIP-qPCR assays using goat anti-TRRAP, control goat IgG, rabbit polyclonal anti-c-Myc (N262), or control rabbit IgG, followed by detection of the 5 S rRNA, tRNALeu, and tRNATyr genes. D and E, L11 competes with TRRAP for binding to the 5 S rRNA and tRNALeu genes. H1299 cells transfected with FLAG-TRRAP in the presence of increasing amounts of Myc-tagged L11 were immunoblotted with antibodies as indicated (D). The transfected cells were also subjected to ChIP assays using anti-FLAG antibody, followed by detection of the TRRAP binding to the 5 S rRNA, tRNALeu, and tRNATyr using qPCR assays (E). Error bars indicate mean ± S.D.
FIGURE 5.
FIGURE 5.
c-Myc transcription of the 5 S rRNA and tRNA genes is not affected by L29. A, knockdown of L29 does not affect the expression of the 5 S rRNA, tRNALeu, and tRNATyr genes. U2OS cells were transfected with scrambled or L29 siRNA. The levels of the 5 S rRNA, tRNALeu, and tRNATyr in the cells were analyzed by using RT-qPCR. The knockdown efficiency of L29 mRNA is shown on the right. B, overexpression of L29 does not affect the binding of TRRAP to the 5 S rRNA, tRNALeu, and tRNATyr genes. H1299 cells transfected with control or FLAG-L29 plasmid were subjected to ChIP-qPCR assays using goat anti-TRRAP or control goat IgG, followed by detection of the 5 S rRNA, tRNALeu, and tRNATyr genes. The expression of FLAG-L29 is shown on the right using IB with anti-FLAG antibodies. Error bars indicate mean ± S.D.
FIGURE 6.
FIGURE 6.
L11 is required for the regulation of the 5 S rRNA and tRNA genes in response to ribosomal stress. A, L11 is required for the reduction of the 5 S rRNA, tRNALeu, and tRNATyr genes in response to the treatment of cells with Act D or serum starvation. U2OS cells were transfected with scrambled or L11 siRNA followed by treatment with Act D (5 nm) for 12 h or serum starvation (SS) for 24 h before harvesting. The cells were subjected to RT-qPCR assays to detect the relative expression of the 5 S rRNA and tRNATyr genes. B, the binding of L11 to the 5 S rRNA, tRNALeu, and tRNATyr genes inversely correlates with that of TRRAP in response to Act D treatment or serum starvation. U2OS cells were cultured in 0.2% fetal calf serum-containing medium for 24 h or treated with 5 nm Act D for 12 h. The cells were subjected to ChIP assays using anti-L11, anti-c-Myc, or anti-TRRAP antibodies followed by qPCR detection of the 5 S rRNA, tRNALeu, and tRNATyr genes. Error bars indicate mean ± S.D.
FIGURE 7.
FIGURE 7.
A schematic diagram for the action of L11 in coordinating c-Myc activity with ribosomal biogenesis. In normal growth conditions, c-Myc enhances ribosomal biogenesis through up-regulating Pol I, II, and III activity. In response to ribosomal stress, L11 binds to c-Myc and inhibits TRRAP binding to target gene promoters. Meanwhile, L11 also negatively regulates the level of c-Myc, leading to the inhibition of c-Myc-mediated transcription and ribosomal biogenesis.
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