PRMT5 regulates IRES-dependent translation via methylation of hnRNP A1

Abstract

The type II arginine methyltransferase PRMT5 is responsible for the symmetric dimethylation of histone to generate the H3R8me2s and H4R3me2s marks, which correlate with the repression of transcription. However, the protein level of a number of genes (MEP50, CCND1, MYC, HIF1a, MTIF and CDKN1B) are reported to be downregulated by the loss of PRMT5, while their mRNA levels remain unchanged, which is counterintuitive for PRMT5's proposed role as a transcription repressor. We noticed that the majority of the genes regulated by PRMT5, at the posttranscriptional level, express mRNA containing an internal ribosome entry site (IRES). Using an IRES-dependent reporter system, we established that PRMT5 facilitates the translation of a subset of IRES-containing genes. The heterogeneous nuclear ribonucleoprotein, hnRNP A1, is an IRES transacting factor (ITAF) that regulates the IRES-dependent translation of Cyclin D1 and c-Myc. We showed that hnRNP A1 is methylated by PRMT5 on two residues, R218 and R225, and that this methylation facilitates the interaction of hnRNP A1 with IRES RNA to promote IRES-dependent translation. This study defines a new role for PRMT5 regulation of cellular protein levels, which goes beyond the known functions of PRMT5 as a transcription and splicing regulator.

Figure 1.. The protein and mRNA levels of IRES-containing genes in PRMT5 deficient MEFs and MCF-7 cells. (A and B) PRMT5^flox/flox MEFs were treated with or without 2 μM tamoxifen for 10 days for PRMT5 depletion, and cultured in regular DMEM for another 3 days to minimize the effect of tamoxifen (TAM) on transcription. Cells were then harvested and lysed for western blotting for the indicated proteins (A) or subjected to RNA purification and RT-qPCR for the relative levels of indicated genes (B). (C and D) MCF-7 cells were transducted with lentiviruses encoding control shRNA or PRMT5 shRNA, and selected with puromycin to generate stable cell lines (Ctrl and P5-KD lines). MCF-7 cells stably expressing control shRNA or PRMT5 shRNA were then harvested and lysed for Western blotting for the indicated proteins (C) or subjected to RNA purification and RT-qPCR for the relative levels ofindicated genes (D).

Figure 2.

Effect of PRMT5 inhibitor on the protein and mRNA levels of IRES-containing genes in MEFs and MCF-7 cells. (A and B) PRMT5^flox/flox MEFs were treated with DMSO or 5 μM PRMT5 inhibitor (EPZ015666, EPZ for short) for 6 days, and harvested for Western blotting for the indicated proteins (C) or RT-qPCR for the mRNA levels of the indicated genes (D). (C and D) MCF-7 cells were treated with DMSO or PRMT5 inhibitor for 6 days, and harvested for Western blotting (C) or RT-qPCR for the indicated genes (D).

Figure 3.

Effect of PRMT5 on IRES-dependent translation. (A) Schematic representation of bicistronic reporter constructs with different IRESes. (B) MCF-7 Ctrl and MCF-7 P5-KD cells were transfected with indicated IRES-dependent reporters, respectively. The firefly and renilla luciferase activities were measured and the ratios of firefly luciferase activity over renilla luciferase activity were calculated. (C) MCF-7 cells were treated with DMSO or 5 μM EPZ015666 for 5 days and then transfected with indicated IRES-dependent reporters for 24 hours. Cells were then lysed to determine the firefly and renilla luciferase activities. The ratios of firefly luciferase activity over renilla luciferase activity were calculated.

Figure 4.

PRMT5 methylates hnRNP A1 in vitro. (A) Schematic representation of hnRNP A1 domains as well as the reported di-methylated sites. (B) Myc.PRMT5 complex were immunoprecipitated from 293T cells overexpressing Myc-tagged PRMT5, then mixed with ³H-AdoMet and GST, recombinant Histone H4 or indicated GST-fusion proteins, for the in vitro methylation assay by PRMT5. (C) GST-hnRNP A1 wild type or single site mutated proteins were applied to in vitro methylation by PRMT5. (D) GST-hnRNP A1 wild type protein or the double mutant R218/225K was subjected to in vitro methylation by PRMT5. In all cases, the recombinant proteins were subjected to PAGE electrophoresis and stained with Coomassie blue as a loading control.

Figure 5.

hnRNP A1 methylation is required for the translational regulation of IRES-dependent genes by PRMT5. (A) 293T cells were transfected with control siRNA (siCtrl) or siRNA to hnRNP A1 (siA1). After 24 h, indicated IRES-dependent reporters were transfected into each group of cells. The luciferase activities were measured after 24 h and the ratios of firefly luciferase activity over renilla luciferase activity were calculated. A representative western blotting was shown to confirm the knockdown of hnRNP A1. (B and C) 293T cells were transfected with control siRNA or siRNA to hnRNP A1. 24 h later, CCND1 IRES reporter construct was co-transfected with GFP-tagged hnRNP A1 wild type or indicated mutants into siA1-transfected cells. GFP plasmid was used to normalize the total transfected DNA. The luciferase activities were measured the following day, and the ratios of firefly over renilla luciferase activity were calculated (B). The knockdown of hnRNP A1, as well as rescued expression of GFP-tagged hnRNP A1, was shown by Western blots (C). (D and E) Wild type HeLa cells or hnRNP A1 knockout HeLa cells were treated with DMSO or 5 μM EPZ015666 for 4 days. On day 3, cells were transfected with CCND1 IRES reporter construct. At the end of day 4, half of the cells were harvested for luciferase activity assay. The ratios of firefly over renilla luciferase activity were calculated (D). The other half was lysed with RIPA buffer and applied to Western blotting against hnRNP A1 and pan-SDMA substrates (E).

Figure 6.

Methylation of hnRNP A1 by PRMT5 facilitates recognition of IRES-containing RNAs. PRMT5^flox/flox MEFs were treated with 2 μM tamoxifen (TAM) for 8 days. (A) The cell lysates from the treated and untreated cells were incubated overnight with biotin-labeled CCND1 or MYC IRES RNA or their control antisense RNAs. RNA complexes were pulled down by streptavidin-conjugated beads and subjected to Western blotting with anti-hnRNP A1 or anti-PRMT5 antibodies. (B) The relative IRES-bound hnRNP A1 signal intensity from Figure 6A was calculated by densitometry. Signal intensity of hnRNP A1 from lane 1 was set as 1. (C) Cell lysates from tamoxifen treated or untreated cells were incubated with normal mouse IgG or anti-hnRNP A1 antibody in the presence of RNase inhibitors overnight, and immunoprecipitated with protein A/G conjugated beads. Bound RNAs were then eluted, purified and subjected to RT-qPCR for CCND1 and MYC mRNAs. (D) Wild type or double-site mutant of GFP-hnRNP A1 constructs were transfected into hnRNP A1 knockout HeLa cells. The levels of CCND1 and MYC mRNAs bound to wild type or mutated hnRNP A1 were detected by RT-qPCR, following RNA immunoprecipitation with anti-GFP antibody.