An Investigation of the Regulation of RNA Polymerase II Transcription Termination by SMN, FUS, and TDP-43 in a pathway involving Symmetric Arginine Dimethylation (R1810) of the RNAPII CTD

The C-terminal domain (CTD) of the largest RNA polymerase II (RNAPII) subunit, POLR2A, is a platform for modifications specifying the recruitment of factors that regulate transcription, messenger RNA (mRNA) processing, and chromatin remodeling. I found that a CTD arginine residue (R1810) is symmetrically dimethylated (me2s), allowing it to recruit the Tudor domain of the SMN protein, which is mutated in spinal muscular atrophy (SMA). SMN can oligomerize and interact with Senataxin (SETX), which is sometimes mutated in ALS4 (Amyotrophic Lateral Sclerosis). I showed that CTD R1810me2s and SMN, like SETX, are required for resolving R-loops created by RNAPII in transcription termination regions. A defect in this process is recapitulated in the SMA disease state. FUS and TDP-43 (TARDBP) are DNA/RNA binding proteins that are involved in numerous aspects of gene regulation, and they are known to interact with each other and with SMN. As well, they are sometimes mutated in neurodegenerative diseases such as ALS and FTD (Frontal Temporal Dementia). I was able to show that FUS and TDP-43 act downstream of the CTD R1810me2s-SMN pathway, and that defects in the recruitment of these proteins can influence RNAPII transcription termination through the accumulation of R-loops and also cause DNA damage. I propose that mis-regulation of RNAPII termination could lead to DNA damage, which may contribute to neurodegenerative disorders like SMA and ALS/FTD. iii Acknowledgements I feel tremendously grateful for having Dr. Jack Greenblatt as my supervisor. My graduate training with Jack, one of the best scientific thinkers that I know of, has been the most enriching and enlightening learning experience in my life. I also feel truly thankful for having Dr. Tony Pawson as my co-supervisor, for his extreme kindness in giving me a beginning in the graduate program at the University of Toronto. I feel extremely lucky for having Dr. Ben Blencowe and Dr. Dan Durocher in my thesis committee, for their support and guidance through the years, and for their amazing insights and tastes in science. I am also very grateful for my external thesis member, for taking parts in my defense, in spite of the short notice. As well, I am indebted to many people who helped me during my studies in the Greenblatt and Pawson labs. I am especially grateful for Dr. Jerry Gish for technical support and scientific discussions, Guoqing Zhong for countless help with cell culture, Shuye Pu for help with sequencing analysis, Dr. Zuyao Ni for discussions, Dr. Karen Colwill, Monika Tucholska, Lorne Taylor, and Hongbo Guo for help with Mass Spectrometry, Dr. Sarang Kulkarni for training in microscopy, the lab managers Joyce Li, Edyta Marcon, Geraldine Mbamalu and the rest of the lab for being wonderfully supportive. I am also thankful for the collaborations from the Blencowe lab especially with help from Dr. Ulrich Braunschweig and Kevin Ha, and the assistance from Dr. Dax Torti and Danica Leung of the CCBR sequencing center. I am also thankful for the helps from several labs at the Structural and Genomic Consortium (Toronto), Dr. Frank Schmitges from the Hughes lab, and Dr. Yue Li from the Zhang lab at U of T. And finally, I’d like to acknowledge Dr. Danny Reinberg, Dr. James Manley, Dr. Dirk Eick, Dr.