Examining the Complexity of Human RNA Polymerase Complexes using HaloTag Technology Coupled to Label Free Quantitative Proteomics

Efficient determination of protein interactions and cellular
localization
remains a challenge in higher order eukaryotes and creates a need
for robust technologies for functional proteomics studies. To address
this, the HaloTag technology was developed for highly efficient and
rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength
of this technology by simultaneous capture of human eukaryotic RNA
polymerases (RNAP) I, II, and III using a shared subunit, POLR2H,
fused to the HaloTag. Affinity purifications showed successful isolation,
as determined using quantitative proteomics, of all RNAP core subunits,
even at expression levels near endogenous. Transient known RNAP II
interacting partners were identified as well as three previously uncharacterized
interactors. These interactions were validated and further functionally
characterized using cellular imaging. The multiple capabilities of
the HaloTag technology demonstrate the ability to efficiently isolate
highly challenging multiprotein complexes, discover new interactions,
and characterize cellular localization.