ARGININE METHYLATION AND m 6 A RNA METHYLATION
A.3 Role of arginine methylation in regulating m 6 A RNA methylation
Multiple proteomic studies have reported that the subunits of RNA-MT complex are methylated on multiple arginine residues. Particularly, Virilizer has a C-terminal tail that is heavily decorated by the all three methylarginine marks – ADMA, SDMA and MMA (81,82).
We confirmed this by methylating recombinant Virilizer protein in vitro with PRMT1, CARM1, PRMT5 or PRMT7 (Fig. A4A, top panel).
The Giallourakis lab (Harvard Medical School), in collaboration with us, recently observed that m6A levels of mRNA are highly reduced in cells lacking PRMT5, CARM1 or PRMT7. We isolated total RNA from WT and PRMT KO MEFs, which was used by the Giallourakis group to enrich for mRNA and then detect m6A levels by dot-blot analyses. They observed a marked loss in m6A mark from MEFs that lacked Prmt5, Carm1 or Prmt7 (data not shown).
Additionally, we observed that in MEFs, upon loss of PRMT5 levels of Virilizer were reduced (Figure A4B). Importantly, the levels of RNA m6A are more drastically reduced upon loss of the scaffolding proteins – WTAP and Virilizer – of RNA-MT complex than upon loss of the methyltransferases METTL3 or METTL14 (220). Taken together, this indicates that PRMT5 likely regulates the deposition of m6A mark via methylation of RNA-MT complex components.
We hypothesized that PRMT5 regulates the subset of m6A marked transcripts that are likely Virilizer-dependent (WTAP-independent, as mentioned in the study by Schwartz et. al
In order to quantitatively assess the changes in m6A, we collaborated with the He group (University of Chicago) and performed a LC-MS/MS experiment. We employed two approaches: 1) Genetic ablation of Prmt5 and 2) chemical inhibition of its enzymatic activity using small molecule inhibitors to PRMT5 (PR-5i). We isolated total RNA from WT and PRMT5 KO MEFs; PR-5i treated Prmt5fl/fl MEFs and from PR-5i treated HeLa cells. We confirmed the loss of PRMT5 and Virilizer from the 4-OHT treated cells (Fig. A4B) by western blot analyses.
Extracts from all the cells – KOs and PR-5i treated – were probed with αSDMA, αH4R3me2s to confirm ablation of PRMT5’s activity; and α β-actin (control) (Fig. A5A). Total RNA isolated from these cells was processed to isolate mRNA (polyA selection) followed by ribo-depletion, to further enrich for mRNA. Upon LC-MS/MS analysis of this RNA, unfortunately, contrary to the dot-blot results obtained by the Giallourakis group, we did not see any change in the levels of mRNA m6A between cells with impaired PRMT5 and corresponding controls (Fig. A5B), indicating that PRMT5 does not regulate m6A levels on mRNA transcripts. We performed similar LC-MS/MS analyses with mRNA isolated from PRMT1 and CARM1 knockout cells.
We did not observe significant difference in the m6A levels in KOs of PRMT5, CARM1 or PRMT1 as compared to WT (Fig. A5).
Figure A4. Virilizer a PRMT substrate. (legend on the next page)
Figure A4. Virilizer is a PRMT substrate. Recombinant Virilizer (1500-1812) was cloned as a GST-tagged protein and purified from bacteria. GST-Virilizer was subject to in vitro methylation using recombinant PRMT1, CARM1, PRMT7 or Myc-PRMT5 (A, top).
Ponceau stained blot serves as loading control (A, middle). The same enzyme was used to methylate recombinant Histones H2B, H3 or H4, as mentioned. This serves as positive control showing that the purified enzymes are active (A, bottom). Prmt5fl/fl MEFs were treated with 4-OHT to induce loss of PRMT5. Virilizer was IPed from PRMT5 knockout and WT MEFs.
Immunoblot shows that upon loss of PRMT5 (B, top) Virilizer levels are markedly reduced (B, bottom).
Figure A5. Mammalian m6A levels are not PRMT5 dependent. RNA was extracted from cells in which PRMT5 was either genetically ablated (A, left) or its activity chemically inhibited (A, center and right). Loss of PRMT5 or its activity was confirmed by western blot analyses using α-SDMA, α-H4R3me2s antibodies. α-PRMT5 shows loss of PRMT5 (A, left) and α-β-actin shows equal loading. Total RNA was extracted from the cells used in A, enriched for poly(A) and subject to LC-MS/MS to detect levels of m6A. No difference was observed in m6A levels between cells with ablated or intact PRMT5 (B). RNA extracted from WT or PRMT1KO, CARM1KO cells subject to LC-MS/MS to detect changes in the levels of m6A also did not show any significant difference.
Figure A5. Mammalian m6A levels are not PRMT5 dependent. (Legend on the
A.4 Discussion
The interplay between arginine methylation and RNA modifications is likely to be important. Many of the substrates of PRMTs are directly involved in RNA biology. PRMT5 plays a major role in spliceosome biology by methylating Sm proteins. CARM1 regulates alternative splicing, promotes exon skipping, methylates spliceosome associated protein SAP49 (SF3B4), SmB, the transcription factor and CA150. (11). Arginine methylation of hnRNPs regulates their cellular localization (236). Importantly, hnRNPA1/B2 are readers of m6A RNA and regulate efficacy of viral infection (237). Modulating PRMT activity can therefore provide a therapeutic opportunity in treatment of such viral infections.
Many other posttranscriptional modifications have been identified on RNA species recently. Owing to the rapid development of technologies, new posttranscriptional modifications have been identified (Figure A1). Transcriptome-wide sequencing studies have now mapped m6A, m1A, m5C, m6Am modified sites (217). In addition, m6A and m1A have recently been identified in DNA (238). Exploring the functional significance of these marks, their dynamic nature, and their regulation will shed new light on regulation of cellular biology.
Crosstalk between seemingly different cellular processes – like RNA methylation and arginine methylation – will likely have interacting nodes which can be exploited in disease settings.
The fact that we do not see gross changes in m6A RNA levels in PRMT KO cells does not rule out the possibility of subtler regulatory role for arginine methylation in this pathway.
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