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. 2009 Dec;29(23):6182-91.
doi: 10.1128/MCB.00973-09. Epub 2009 Oct 5.

S-adenosyl homocysteine hydrolase is required for Myc-induced mRNA cap methylation, protein synthesis, and cell proliferation

Maria Elena Fernandez-Sanchez  1 , Thomas Gonatopoulos-Pournatzis, Gavin Preston, Margaret A Lawlor, Victoria H Cowling
Affiliations

S-adenosyl homocysteine hydrolase is required for Myc-induced mRNA cap methylation, protein synthesis, and cell proliferation

Maria Elena Fernandez-Sanchez et al. Mol Cell Biol. 2009 Dec.

"VSports app下载" Abstract

The c-Myc proto-oncogene promotes mRNA cap methylation, which is essential for almost all mRNA translation. The mRNA cap methylation reaction produces an inhibitory byproduct, S-adenosyl homocysteine. Here we report that Myc promotes upregulation of S-adenosyl homocysteine hydrolase (SAHH), an enzyme which hydrolyzes S-adenosyl homocysteine, thus neutralizing its inhibitory effects, and this is required for c-Myc-induced mRNA cap methylation. c-Myc-induced mRNA cap methylation was repressed by inhibiting the expression or activity of SAHH, whereas the same treatments did not have a significant effect on c-Myc-induced transcription or other c-Myc-dependent methylation events. The selective inhibition of mRNA cap methylation afforded by SAHH repression revealed that c-Myc-induced cap methylation could be correlated with the core c-Myc functions of protein synthesis, cell proliferation, and cell transformation VSports手机版. .

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"V体育官网" Figures

FIG. 1.
FIG. 1.
The SAHH gene is a Myc target gene. (A) P493-6 cells were incubated with doxycycline (Dox) to inhibit exogenous Myc expression or vehicle control (veh). Rat1A fibroblasts, primary MEFs, and IMECs were infected with retroviruses to express vector control, MycWT, or MycΔMBII, and pools of cells were drug selected. Primary murine T cells were incubated with IL-2, IL-7, and IL-15 for 24 h. Rat1A cells were incubated with a control siRNA (con si; cyclophilin B siRNA) or an siRNA directed against Myc (Myc si) for 24 h. Western blotting was performed to detect SAHH, Myc, β-tubulin, or POLR2A expression, as loading controls. (B) RNA was extracted from the cell lines indicated, and RT-PCR was performed to detect SAHH mRNA levels relative to GAPDH (or 18S rRNA for P493-6). Prim., primary. (C) In the IMEC lines indicated, SAHH mRNA was detected by RT-PCR (black bars) and m7G (cap-methylated) SAHH mRNA was detected by anti-m7G IP followed by RT-PCR (gray bars). (D) Rat fibroblasts expressing the vector control or MycER were incubated with 4-hydroxytamoxifen (OHT) for 2 h to activate MycER and with cycloheximide (CHX) 30 min prior to OHT to inhibit protein synthesis. RT-PCR was used to detect SAHH mRNA levels relative to GAPDH levels. (E) Chromatin IP was performed in P493-6 cells incubated with Dox (low Myc; −) or the vehicle control (high Myc; +) and IMECs expressing the vector control (−) or c-Myc (+). Polyclonal control antibodies (cont) or anti-c-Myc antibodies (Myc) were used for immunoprecipitation, as indicated. Myc was found to bind significantly to the SAHH TSS but not to the E-box (CACGTG) found in intron 1 at +7645 or to Hbb.
FIG. 2.
FIG. 2.
Myc-induced SAHH expression is necessary for Myc-induced mRNA cap methylation. In Rat1A cells (A) and IMECs (B), SAHH expression was reduced in cells expressing exogenous Myc to a level equivalent to that found in vector control cells by transfection of two independent siRNAs directed against SAHH (SAHH si1 and SAHH si2). Vector control (vec) and exogenous c-Myc-expressing cells (Myc) were incubated with control siRNA (cont. si). Western blotting was performed on cell extracts to detect SAHH, Myc, and β-tubulin. (C) In the Rat1A cells, homocysteine levels in the extracellular medium were measured (n = 3). In Rat1A cells (D) and IMECs (E), mRNA and m7G (cap-methylated) mRNA levels were measured as described for Fig. 1C, for the Myc target genes encoding Nol5a, NPM, NUCL, and eIF4E (n = 4). For each gene examined, the values for m7G mRNA in exogenous c-Myc-expressing cells were significantly higher (t test; P < 0.01) in cells transfected with control siRNA than in cells transfected with SAHH siRNA.
FIG. 3.
FIG. 3.
Myc-induced SAHH expression does not regulate total RNA, DNA, and protein methylation. SAHH expression was reduced in Rat1A cells expressing exogenous Myc to a level equivalent to that found in vector control cells by transfection of two independent siRNAs directed against SAHH (SAHH si1 and SAHH si2). Vector control (vec) and exogenous c-Myc-expressing cells (Myc) were incubated with control siRNA (cont. si). Methylation of (A) RNA, (B) DNA, and (C) protein was determined by measuring [methyl-3H]methionine incorporation (n = 3). For all three panels, the values for cells transfected with vector control siRNA were significantly lower than the values for cells expressing c-Myc and transfected with control siRNA (t test; P < 0.0001). The values for c-Myc/control siRNA and c-Myc/SAHH siRNAs were not significantly different (t test; P > 0.01).
FIG. 4.
FIG. 4.
Myc-induced SAHH expression is necessary for Myc-induced protein synthesis. SAHH expression was reduced in (A) Rat1A cells and (B) IMECs expressing exogenous Myc to a level equivalent to that found in vector control cells by transfection of two independent siRNAs directed against SAHH (SAHH si1 and SAHH si2). Vector control (vec) and exogenous c-Myc-expressing (Myc) cells were incubated with control siRNA (cont. si). The protein synthesis rate was analyzed by measuring incorporation of [35S]methionine and [35S]cysteine into TCA-precipitable material (n = 5). For both panels, the values for cells expressing exogenous c-Myc and transfected with siRNA were significantly higher than the values for cells expressing vector control and transfected with control siRNA and for cells expressing exogenous c-Myc and transfected with SAHH siRNAs (t test; P < 0.01).
FIG. 5.
FIG. 5.
Myc-induced SAHH expression is required for Myc-induced cell proliferation and cell transformation. SAHH expression was reduced in (A and C) Rat1A cells and (B and D) IMECs expressing exogenous Myc to a level equivalent to that found in vector control cells by transfection of two independent siRNAs directed against SAHH (SAHH si1 and SAHH si2). Vector control (vec) and exogenous c-Myc-expressing cells (Myc) were incubated with control siRNA (cont. si). (A and B) Cell proliferation was analyzed by counting cells each day (n = 4). (C and D) Cell transformation was investigated using the soft agar transformation assay. At day 7, representative micrographs were taken and number of plated cells which form colonies over 50 or 100 μm was determined (n = 2).
FIG. 6.
FIG. 6.
Myc-induced cap methylation correlates with Myc-induced protein synthesis and cell proliferation. (A) Rat1A cells were incubated with 100 nM tubercidin (Tub) or vehicle control (Veh) for 3 h, and RNA was extracted. mRNA and m7G mRNA levels for three Myc target genes were measured as described for Fig. 1 (n = 3). (B) The protein synthesis rate was analyzed by measuring incorporation of [35S]methionine and [35S]cysteine into TCA-precipitable material (n = 3). (C) Cell proliferation was analyzed by counting cells each day (n = 3). Methylation of (D) RNA, (E) DNA, and (F) protein was determined by measuring [methyl-3H]methionine incorporation (n = 3).
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