Human cap methyltransferase (RNMT) N-terminal non-catalytic domain mediates recruitment to transcription initiation sites

Abstract

Gene expression in eukaryotes is dependent on the mRNA methyl cap which mediates mRNA processing and translation initiation. Synthesis of the methyl cap initiates with the addition of 7-methylguanosine to the initiating nucleotide of RNA pol II (polymerase II) transcripts, which occurs predominantly during transcription and in mammals is catalysed by RNGTT (RNA guanylyltransferase and 5' phosphatase) and RNMT (RNA guanine-7 methyltransferase). RNMT has a methyltransferase domain and an N-terminal domain whose function is unclear; it is conserved in mammals, but not required for cap methyltransferase activity. In the present study we report that the N-terminal domain is necessary and sufficient for RNMT recruitment to transcription initiation sites and that recruitment occurs in a DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole)-dependent manner. The RNMT-activating subunit, RAM (RNMT-activating miniprotein), is also recruited to transcription initiation sites via an interaction with RNMT. The RNMT N-terminal domain is required for transcript expression, translation and cell proliferation.

Figure 1. RNMT amino acids 1–120 are not required for catalytic activity

(A) Human RNMT mutants used in the present study: RNMTcat, amino acids 121–476; and RNMT-N, amino acids 1–120. (B) Western blots were performed with anti-HA antibodies to detect expression of HA–RNMT and HA–RNMTcat immunoprecipitated from 50 μg of HEK-293 cell extracts using anti-HA antibodies (HA IP), and in 10 μg cell extracts (extracts). Cap methyltransferase assay was performed on RNMT proteins immunoprecipitated from 10 μg cell extracts or negative control for the time course indicated (C) and on RNMT proteins immunoprecipitated from the quantity of cell extracts indicated for 10 min (D). The plots depict the means±S.D. for three independent experiments (P<0.001 for both plots, Student's t test).

Figure 2. RNMT nuclear localization is mediated independently by the N-terminal and catalytic domains

(A) Immunofluoresence analysis was performed on HeLa cells transfected with control or RNMT-directed siRNA for 48 h. The subcellular localization of RNMT was detected with anti-RNMT antibodies. (B) Immunofluoresence analysis was performed on HeLa cells transfected with pcDNA4 (control) or pcDNA4 HA–RNMT, HA–RNMTcat and HA–RNMT-N plus pcDNA4 FLAG–RAM. The subcellular localization of RNMT was detected via the HA tag. In (A) and (B) DAPI stain was used to detect nuclei and a bright field view to detect the cell membrane.

Figure 3. RNMT is recruited to chromatin via the N-terminus

ChIP was performed on HeLa cells transfected with pcDNA4 HA–RNMT, HA–RNMTcat or pcDNA4 (control), plus pcDNA4 FLAG–RAM. Immunoprecipitations were performed with antibodies raised against HA to detect RNMT (A) and FLAG to detect RAM (B). PCR was performed against the promoter-proximal region of the genes indicated. PCR signal relative to input, subtracted from negative control immunoprecipitation is depicted as means±S.D. for three independent experiments (**P<0.01, Student's t test). (C) For the same immunoprecipitations, binding to the c-Myc gene TSS and 2000 bases upstream of the start site (−2000) was compared. Results are an average of three independent experiments and the error bars indicate S.D. (D) HeLa cells were transfected with pcDNA4 HA–RNMT, HA–RNMT-N or vector control plus pcDNA4 FLAG–RAM and Western blotting was performed to detect RNMT proteins, RAM and tubulin. Molecular masses are shown on the left-hand side in kDa. (E) ChIPs were performed using anti-HA antibodies as above. Results are an average of three independent experiments and the error bars indicate S.D.

Figure 4. RNMT recruitment to chromatin is inhibited by DRB

(A) ChIPs were performed on HeLa cells transfected with pcDNA4 HA–RNMT, HA–RNMT-N or pcDNA4 (control), plus pcDNA4 FLAG–RAM. Cells were treated with 50 μM DRB or vehicle control for 1 h prior to lysis. Immunoprecipitations were performed with anti-HA antibodies. PCR signal relative to input, subtracted from negative control immunoprecipitation is depicted as means±S.D. for three independent experiments (comparison with and without DRB resulted in P values<0.05 for the HA–RNMT and HA–RNMT-N transfections, Student's t test). (B) Purified recombinant GST, GST–RNMT and GST–RNGTT were incubated with HeLa nuclear cell extracts and glutathione pull downs were performed. Western blots were performed to detected RNA pol II Ser⁵ CTD phosphorylation and GST. Molecular masses are shown on the left-hand side in kDa.

Figure 5. The RNMT N-terminus is required for gene expression

HeLa cells were transfected with pcDNA4 HA–RNMT, pcDNA4 HA–RNMTcat and vector control plus pcDNA4 FLAG–RAM WBL (siRNA-resistant DNA). (A) At 24 h after transfection, RNA was harvested and real-time PCR was performed to detect expression of the c-Myc transcripts. Results are an average of three independent experiments and the error bars indicate the S.D. (B) Western blots were performed to detect c-Myc, RNMT and tubulin. Molecular masses are shown on the left-hand side in kDa. (C) As in (A) except performed with the genes indicated. (D) [³⁵S]Methionine in vivo labelling was performed to estimate amino acid incorporation rates in the same experimental system. Results are means±S.D. for three independent experiments (C) and a representative of three independent experiments performed in triplicate (D). *P<0.05 and **P<0.01 when comparing HA–RNMT with the vector control or HA-RNMTcat, Student's t test.

Figure 6. RNMT amino acids 1–120 are required for cell proliferation

RNMT–RAM was knocked down in HeLa cells using RAM-directed siRNA or a control, and cells were transfected with pcDNA4 HA–RNMT, pcDNA4 HA–RNMTcat or a vector control, and pcDNA4 FLAG–RAM WBL (siRNA-resistant cDNA). After 3 days Western blots were performed to detect the antigens indicated (A) and cells were counted (B). RNMT–RAM was knocked down using RNMT-directed siRNA in IMEC expressing HA–RNMT and HA–RNMTcat and control lines. After 3 days Western blots were performed to detect the antigens indicated (C) and cells were counted (D). (E) IMECs (5×10³) expressing RNMT–GFP and RNMTcat–GFP or control lines were plated in soft agar. (F) At 10 days later the number of established colonies (over 50 μm) were counted. Histograms depict mean±S.D. for at least three independent experiments. **P<0.01 and ***P<0.001, Student's t test. Molecular masses are shown on the left-hand side of the blots in kDa.