RNA Polymerase II CTD phosphorylation control
RNA transcription in eukaryotes is carried out by multi-subunit molecular machines known as the RNA Polymerases. RNA Polymerase II (RNAPII), responsible for the production of messenger RNA (mRNA) and other noncoding RNAs, is conserved across all eukaryotes including yeast, plant, and human cells. The largest of the RNAPII subunits, Rpb1, has an unstructured C-terminus known as the CTD that has been shown to serve as a platform for the recruitment of various accessory proteins during transcription. Recruitment of many of these factors is controlled through regulation of the phosphorylation status of various residues within the tandem CTD repeats (YPSPTSPS). Observation of the role of CTD phosphorylation in accessory protein recruitment during transcription resulted in the CTD code hypothesis, which states that the CTD acts as a, “contortionist that, upon post-translational modification, adopts different configurations specifically recognized by its partners” (Buratowski, S., Nat Struct Biol, 2003). Many unanswered questions exist regarding the role of the RNAPII CTD in transcription. [Figure: RNAPII ternary complex (RNAPII:DNA:nascent RNA) adapted from PDB 3HOU by Jose Victorino.]
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
Recent Publications
A complete list of publications from the Mosley Lab is available in PubMed.
