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Story about the 5 year journey to a new preprint from our lab: Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs https://www.biorxiv.org/content/10.1101/2020.06.23.167601v1 (Only a few of the contributors are on twitter) /1
Our long interest in PRMTs and the cellular consequences of arginine methylation ("Rme"), with our long mass spec collaboration with Donald Hunt's Lab ( @ChemistryUVA) led us to work with @MillerLehman on a PTMscan approach to ID'ing all the Rme1/Rme2s/Rme2a in the cell /2
At the same time, we were using new (at the time) chemical probes from @thesgconline targeting PRMT5 and type I PRMTs /3
Independently, we developed a new, accurate, proteome hydrolysis and mass spec approach to determine total cellular abundances of methylated Arg and Lys (major credit to Emmanuel Burgos and Eddie Nieves). We showed ~1.1% Rme2a, 0.4% me2s, & 0.1% me1 (all surprisingly high!) /3
We also collaborated with Mark Bedford ( @MDAndersonNews) and @epicypher to use the oriented peptide array library (OPAL) to quantitatively probe the in vitro substrate specificities of the major PRMTs: PRMT1, PRMT4/CARM1, and PRMT5-MEP50. /4
We also performed RNA-Seq on cells treated with PRMT5 and Type I inhibitors (4+ years ago, the first of MANY PRMT-regulated total and nascent transcriptome experiments with at least 3 more manuscripts on the way....) /5
Finally, in the inhibitor-treated cells, we analyzed the total Type I and II PRMT-regulated proteome /6
Now the data we had collected had grown more than we had originally intended, and at this point many of the investigators had moved on in their careers to new labs. We were faced with a challenging path to integrating everything together /7
Luckily for us, we had recruited @SimoneSidoli to our Biochemistry dept at @EinsteinMed. Simone is a master mass spectrometrist and proteomics guru. He worked with @MillerLehman and new data she had collected with the samples @GarciaLabMS. /8
But for the PTMScan, the combo of multiply modified methylarg peptides and trypsin digestion, sequential IPs, and cell treatment created a complicated network of peptides. How to decipher? The Hunt lab's ETD fragmentation and their new computational decision tree to the rescue /9
@SimoneSidoli then characterized the enzyme dependent changes in protein abundances and methylation states. Our bioinformatics collaborator Varun Gupta (Einstein Cell Bio, in Charles Query's group) and my MSTP student Maxim Maron worked on the RNA-Seq and ontology analysis /10
Finally, we weaved this story together into a big picture view of the role of PRMTs in many cellular consequences. Note that these studies were done in lung adenocarcinoma cells, providing basic knowledge and key insights for the use of PRMTi in lung cancer /11
What did we learn? 1) Much more methylarg is in the cell than we previously thought, may not all be due to PRMTs... 2) Even though PRMTs (sometimes) scavenge each others substrates and may complement, their actual substrate preference is more diverse than thought (cont'd) /12
3) each methylarginine protein state may regulate distinct transciptomic and proteomic consequences, but are primarily found in the nucleus, in RNA-binding, intrinsically disordered proteins, and maybe phase separation... (cont'd) /13
4) consistent with the many proteins methylated by PRMTs, the transcriptome and proteome regulated by PRMTs are different, as are the transcriptome and proteomes regulated by Type I and Type II enzymes cont'd) /14
5) Finally, our overall conclusion is that the transcriptome and proteome regulated by PRMTs are distinct from the classes of proteins directly methylated by PRMTs (at least 383 on thousands of sites), suggesting an elaborate cellular regulatory regime /15
Thanks to all the (16!) authors, their hard work, and long time to getting this story in shape. I point out that a number of other studies have been published in the last year on methylarg PTMscan, but ours is unique in the breadth of approaches and depth of results /16
And thanks to the funders of this (and other related work rapidly approaching conclusion)- @NIGMSgenes @AmericanLung. We hope that these studies will both increase our basic knowledge and lead to new understanding of the efficacy and consequences of PRMT drugs on lung cancer /end
And we have opportunities available on related studies: https://jobrxiv.org/job/albert-einstein-college-of-medicine-27778-postdoctoral-or-research-associate/
