RAM function is dependent on Kapβ2-mediated nuclear entry

Abstract

Eukaryotic gene expression is dependent on the modification of the first transcribed nucleotide of pre-mRNA by the addition of the 7-methylguanosine cap. The cap protects transcripts from exonucleases and recruits complexes which mediate transcription elongation, processing and translation initiation. The cap is synthesized by a series of reactions which link 7-methylguanosine to the first transcribed nucleotide via a 5' to 5' triphosphate bridge. In mammals, cap synthesis is catalysed by the sequential action of RNGTT (RNA guanylyltransferase and 5'-phosphatase) and RNMT (RNA guanine-7 methyltransferase), enzymes recruited to RNA pol II (polymerase II) during the early stages of transcription. We recently discovered that the mammalian cap methyltransferase is a heterodimer consisting of RNMT and the RNMT-activating subunit RAM (RNMT-activating mini-protein). RAM activates and stabilizes RNMT and thus is critical for cellular cap methylation and cell viability. In the present study we report that RNMT interacts with the N-terminal 45 amino acids of RAM, a domain necessary and sufficient for maximal RNMT activation. In contrast, smaller components of this RAM domain are sufficient to stabilize RNMT. RAM functions in the nucleus and we report that nuclear import of RAM is dependent on PY nuclear localization signals and Kapβ2 (karyopherin β2) nuclear transport protein.

Figure 1. The RAM RAD, NR and QYP domains are required for RAM function

(A) Depiction of human RAM domains investigated in this study. RAD, amino acids 1–55, interacts with RNMT and stimulates its activity. NR domain, amino acids 56–90, is an RNA-binding domain. QYP domain, amino acids 91–118, is uncharacterized. Two PY NLSs at amino acids 98 and 114 are indicated. (B) HeLa cells were transfected with pcDNA5 RAM-GFP, RAM-GFP mutants or GFP alone. At 3 days following transfection, Western blots were performed to detect the antigens indicated in cell extracts. Molecular masses are indicated in kDa. (C) HeLa cells were co-transfected with RAM or control siRNA (c), and pcDNA5 RAM-GFP, RAM-GFP mutants or GFP alone. At 3 days after transfection the number of cells relative to siRNA control/pcDNA5 GFP transfection were calculated. The histogram depicts the average for three independent experiments and the error bars indicate±S.D. ***P<0.001 using Student's t test in comparison with RAM siRNA/pcDNA5 GFP transfection.

Figure 2. RAM 1–45 activates RNMT

(A) Recombinant GST–RAM WT, truncation mutants or GST alone were incubated with recombinant RNMT. GST–RAM complexes were purified on glutathione–Sepharose, resolved by SDS/PAGE and co-purified RNMT was visualised by Coomassie Blue staining and Western blotting (WB). Molecular masses are indicated in kDa. (B) HeLa cells were transfected with pcDNA5 HA-RNMT or pcDNA5 (c), and pcDNA4 RAM-GFP (RAM WT), RAM-GFP mutants or GFP. Immunoprecipitations (IP) were performed with anti-HA and anti-GFP antibodies. Western blots were performed to detect GFP, RAM, HA and RNMT in inputs and immunoprecipitates. (* indicates cross-reacting antibody heavy or light chain). (C) Cap methyltransferase assay was performed using 15 nM RNMT plus 15 nM GST–RAM, truncation mutants or GST control. Protein complexes were incubated with [³²P]GpppG transcript and S-adenosylmethionine for 10 mins. Following the reaction, transcripts were digested and GpppG and m7GpppG were resolved by TLC and visualized by phosphoimaging. A representative image is shown. The average fold change in cap methyltransferase activity compared with that generated by RNMT alone for six independent experiments is depicted. Error bars indicate±S.D. ***P<0.0001 using Student's t test for results in comparison with RNMT plus GST. endo, endogenous.

Figure 3. RAM stabilizes RNMT against proteosomal degradation

(A) HeLa cells were transfected with RAM or control (c) siRNA, and pcDNA4 RAM-GFP or RAM-GFP mutants. Western blots were performed to detect GFP, RAM, RNMT and β-tubulin. (B) HeLa cells were transfected with pcDNA5 Fg-RAM and pcDNA5 HA-RNMT alone or in combination with the relevant vector controls. Two days following transfection, cells were incubated with 10 μM MG132, 20 μM clasto-lactacystin β-lactone (c-Lactacys) or vehicle control at 8 h before lysis. Western blots were performed to detect RAM, HA, RNMT and β-tubulin in cell extracts. Molecular masses are indicated in kDa. endo, endogenous.

Figure 4. RAM nuclear localization is dependent on the C-terminus

(A) HeLa cells were transfected with pcDNA5 Fg-RAM WT, truncation mutants or vector control. Immunoprecipitations (IP) were performed on normalized cell extracts using anti-Fg antibody–agarose conjugates. Western blots were performed to detect Fg-tagged proteins, RNMT and β-tubulin in immunoprecipitates and extracts. Molecular masses are indicated in kDa. (B) HeLa cells were transfected with control or RAM siRNA and with pcDNA5 Fg-RAM WT, truncation mutants or vector control. IF analysis was used to detect RAM localization and DAPI staining was used to detect nuclei. The overlay of RAM IF, DAPI staining and bright field is also presented.

Figure 5. RAM nuclear localization is dependent on the C-terminus

(A) HeLa cells were transfected with pcDNA5 RAM-GFP, RAM-GFP mutants or GFP. Fluorescence microscopy was used to detect GFP localization in HeLa cells and DAPI staining was used to detect nuclei. An overlay of GFP fluorescence, DAPI staining and bright field is presented. (B) The ratio of nuclear to cytoplasmic GFP fluorescence was quantified. The average for 12 images is presented and error bars indicate±S.D. ***P<0.0001 using Student's t test for average ratio in comparison with GFP control.

Figure 6. RAM nuclear localization is dependent on the PY domains

(A) HeLa cells were transfected with pcDNA5 RAM-GFP, RAM-GFP PY/AA mutants or GFP control. Fluorescence microscopy was used to detect GFP localization and DAPI staining was used to detect nuclei. An overlay of GFP fluorescence, DAPI staining and bright field is presented. (B) Western blots were performed to detect GFP, RNMT, Kapβ2 and β-tubulin in cell extracts. Molecular masses are indicated in kDa. (C) The ratio of nuclear to cytoplasmic GFP fluorescence was quantified. The average of 12 images is presented and error bars indicate±S.D. ***P<0.0001 and *P<0.01 using Student's t test for average ratio in comparison with the GFP control. (D) HeLa cells were co-transfected with RAM or control siRNA, and pcDNA5 RAM-GFP, RAM-GFP PY/AA mutants or GFP. At 3 days after transfection cells were counted and the number expressed as relative to control/pcDNA5 GFP transfection. The histogram depicts the average for three independent experiments and the error bars indicate±S.D. **P<0.001 using Student's t test relative to GFP control.

Figure 7. Kapβ2 mediates RAM import

(A) Recombinant GST–RAM or GST was incubated with recombinant Kapβ2. GST–RAM and Kapβ2 complexes were purified on glutathione–Sepharose, resolved by SDS/PAGE and visualized by Western blotting. Molecular masses are indicated in kDa. (B) HeLa cells were transfected with pcDNA5 Myc-Kapβ2 or vector control and pcDNA5 RAM-GFP or GFP. Immunoprecipitations (IP) were performed on cell extracts using anti-Myc antibodies. Western blots were performed to detect Myc–Kapβ2, RAM and β-tubulin in inputs and immunoprecipitates. (C) HeLa cells were transfected with pcDNA5 Myc-Kapβ2 or vector control (c) and pcDNA5 RAM-GFP, RAM-GFP PY/AA mutants or GFP. Immunoprecipitations were performed on cell extracts using anti-Myc antibodies. Western blots were performed to detect Myc–Kapβ2, RAM and β-tubulin in inputs and immunoprecipitates. (D) HeLa cells were transfected with two independent Kapβ2 siRNAs or control siRNA. After 2 days RNA was extracted and real-time PCR performed to detect expression of Kapβ2, RNMT and RAM. The average result for three independent experiments is presented and the error bars indicate±S.D. (E) Western blots were performed to detect Kapβ2, RNMT, RAM and β-tubulin in cell extracts. (F) IF was used to detect RAM localization and DAPI staining was used to detect nuclei. The overlay of RAM IF, DAPI staining and bright field is also presented. si, siRNA. (G) HeLa cells were transfected with pcDNA3.1 Myc-M9M or vector control. IF was used to detect RAM and Myc-M9M. DAPI staining was used to detect nuclei. The overlay of RAM or RNMT, DAPI staining and bright field is presented.

Figure 8. Summary of RAM functional domains

Depictions of the functionality of the RAM mutants used in the present study and previously [12].