RNA Polymerase II CTD phosphorylation control
Quantitative analysis of gene expression
The Mosely Lab uses a number of quantitative approaches to study how genetic perturbations (such as mutation or deletion) in transcription related proteins impacts gene expression control. Specifically, this team uses peptide mass spectrometry to study protein-protein interactions, protein post-translational modifications, protein expression level, and protein thermal stability. Using high-throughput sequencing, scientists in this lab can determine how the levels of both coding (mRNA) and non-coding transcripts change in response to various perturbations. The laboratory also uses various approaches to study genome-wide protein interactions with DNA using techniques such as chromatin immunoprecipitation. These approaches allow the team to explore hypothesis-driven research questions about the role of specific proteins in gene expression.
RNAPII CTD phosphatases
During the past five years, the Mosley Lab has focused on the RNAPII interacting protein Rtr1 that Amber Mosley, PhD, I characterized during postdoctoral training. Rtr1 is a novel type of protein phosphatase that is conserved throughout most eukaryotes. We, and others, have shown that Rtr1 regulates CTD phosphorylation at the 5th amino acid in the CTD repeats (hence Ser5) during early transcription elongation. Studies led by Mosley indicate that the CTD phosphatases Rtr1 and Ssu72 play unique roles in the regulation of CTD phosphorylation although they can both target Ser5 for dephosphorylation during the transcription cycle. Using approaches such as nascent and steady state RNA-sequencing, chromatin immunoprecipitation followed by sequencing, and dynamic protein-protein interaction network analysis via mass spectrometry, investigators in this lab get a system-level view of the impacts of Rtr1 and/or Ssu72 on the function on RNAPII. The lab team has also discovered that the nuclear exosome-specific subunit Rrp6 plays a role in the regulation of RNAPII transcription termination using high resolution chromatin IP experiments (ChIP-exo) and RNA sequencing (Fox et al, 2015). Rrp6 is required for termination and processing of specific transcripts through the Nrd1-Nab3-Sen1 (NNS) pathway that mainly controls RNAPII termination of noncoding RNA transcripts. The Mosley Lab team is currently exploring the role of Rtr1 in the regulation of Rrp6-dependent transcription termination.
Current Research Funding
NIH R01 GM099714: Regulation of RNA Polymerase II transcription by the phosphatase Rtr1.
NSF 1515748: Quantitative Analysis of Transcription Elongation Perturbation Networks
A complete list of publications from the Mosley Lab is available in PubMed.
Hunter, G.O., Fox, M.J., Smith-Kinnaman, W.R., Gogol, M., Fleharty, B., Mosley, A.L. The phosphatase Rtr1 regulates global levels of serine 5 RNA Polymerase II C-terminal domain phosphorylation and cotranscriptional histone methylation. Mol Cell Biol. 2016 May 31. pii: MCB.00870-15.
Bedard, L.G., Dronamraju, R., Kerschner, J.L., Hunter, G.O., Axley, E.D., Boyd, A.K., Strahl, B.D., Mosley, A.L. Quantitative Analysis of Dynamic Protein Interactions during Transcription Reveals a Role for Casein Kinase II in Polymerase-associated Factor (PAF) Complex Phosphorylation and Regulation of Histone H2B Monoubiquitylation. J Biol Chem. 2016 Jun 24;291(26):13410-20.
Fox, M.J., Gao, H., Smith-Kinnaman, W. R., Liu, Y., Mosley, A.L. The Exosome Component Rrp6 Is Required for RNA Polymerase II Termination at Specific Targets of the Nrd1-Nab3 Pathway. 02/2015. PLoS Genet 10(2): e1004999.
Ni, Z. Xu, C. Guo, X. Hunter, G.O., Kuznetsova, O., Tempel, W., Marcon, E., Zhong, G., Guo, H., Kuo, W., Li, J., Young, P., Olsen, J.B., Wan, C., Loppnau, P., Bakkouri, M.E., Senisterra, G., He, H., Huang, H-M., Sidhu, S., Emili, A., Murphy, S., Mosley, A.L., Arrowsmith, C., Min, J., Greeblatt, J.F. RPRD1A and RPRD1B serve as RNA Polymerase II Carboxy-Terminal Domain Scaffolds to Recruit RPAP2 for Serine 5 Dephosphorylation. Nat Struct Mol Biol. 2014 Aug;21(8):686-95.
Hsu, P.L., Yang, F., Smith-Kinnaman, W.R., Yang, W., Zheng, N., Mosley, A.L., Varani, G. Rtr1 is a dual specificity phosphatase that dephosphorylates Tyr1 and Ser5 on the RNA Polymerase II CTD. J Mol Biol. 2014 Aug 12;426(16):2970-81. doi: 10.1016/j.jmb.2014.06.010.
Additional Lab Team Members
The Mosley Lab team also includes Whitney Smith-Kinnaman (Research Analyst), Sarah Peck (IBMG PhD student), Jose Victorino (IBMG PhD student), Katlyn Hughes (IBMG PhD student), Jason True (Postdoctoral Researcher), and Guihong Qi (Research Analyst – Proteomics Core).