Characterization of GFP-Gdown1 interactors by IP and LC-MS/MS

ABSTRACT: Approximately half of purified mammalian RNA polymerase II (Pol II) is associated with a tightly interacting sub-stoichiometric subunit, Gdown1. Previous studies have established that Gdown1 inhibits transcription initiation through competitive interactions with general transcription factors and blocks the Pol II termination activity of transcription termination factor 2 (TTF2). However, the biological functions of Gdown1 remain poorly understood. Here, we utilized genetic, microscopic, and multi-omics approaches to functionally characterize Gdown1 in three human cell lines. Acute depletion of Gdown1 caused minimal direct effects on transcription. We show that Gdown1 resides predominantly in the cytoplasm of interphase cells, shuttles between the cytoplasm and nucleus, and is regulated by nuclear export. Gdown1 enters the nucleus at the onset of mitosis. Consistently, genetic ablation of Gdown1 is associated with partial de-repression of mitotic transcription, and Gdown1 KO cells present with evidence of aberrant mitoses coupled to p53 pathway activation. Evidence is presented demonstrating that Gdown1 modulates the combined functions of purified productive elongation factors PAF1C, RTF1, SPT6, DSIF, and P-TEFb in vitro. Collectively, our findings support a model wherein the Pol II-regulatory function of Gdown1 occurs during mitosis and is required for stably maintaining a pattern of gene expression across cell divisions. To characterize potential interactors of Gdown1 in the cytoplasm, an eGFP-TEV-Gdown1 HAP1 cell line was developed. Cytosolic extracts were prepared, GFP-Trap Agarose (Chromotek) was utilized to pull down Gdown1 and associated factors, and associated proteins were characterized by LC-MS/MS.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Christopher Ball  

LAB HEAD: David Price

PROVIDER: PXD029425 | Pride | 2022-02-14

REPOSITORIES: Pride