Cell-cycle dependent organization and dynamics of RNA Polymerase I in live human cells

RNA Polymerase I (Pol I) is responsible for over 60% of transcriptional output in human cells, yet basic questions concerning the spatial and temporal organization of the polymerase remain unanswered. Here we investigate how mammalian cells rely on Pol I organization throughout the cell cycle to balance different needs, from complete transcription shut down to massive increase in protein synthesis (and thus ribosomal RNA synthesis) before cell division. In contrast to our previous reports on RNA Polymerase II, Pol I clusters are stable with active transcription, and the presence of transient Pol I clusters correlates with inactive ribosomal transcription. Our results suggest that both stable and transient populations Pol I clusters co-exist in individual living cells, and their relative fraction may directly reflect the global gene expression need of the cell

Super-resolution imaging of fluorescently labeled, endogenous RNA Polymerase II in living cells with CRISPR/Cas9-mediated gene editing

Live cell imaging of mammalian RNA polymerase II (Pol II) has previously relied on random insertions of exogenous, mutant Pol II coupled with the degradation of endogenous Pol II using a toxin, α-amanitin. Therefore, it has been unclear whether over-expression of labeled Pol II under an exogenous promoter may have played a role in reported Pol II dynamics in vivo. Here we label the endogenous Pol II in mouse embryonic fibroblast (MEF) cells using the CRISPR/Cas9 gene editing system. Using single-molecule based super-resolution imaging in the living cells, we captured endogenous Pol II clusters. Consistent with previous studies, we observed that Pol II clusters were short-lived (cluster lifetime ~8 s) in living cells. Moreover, dynamic responses to serum-stimulation, and drug-mediated transcription inhibition were all in agreement with previous observations in the exogenous Pol II MEF cell line. Our findings suggest that previous exogenously tagged Pol II faithfully recapitulated the endogenous polymerase clustering dynamics in living cells, and our approach may in principle be used to directly label transcription factors for live cell imaging.National Cancer Institute (U.S.) (Award DP2CA195769)Massachusetts Institute of Technology. Department of Physic

Mechanism of Discrimination of 8‑Oxoguanosine versus Guanosine by <i>Escherichia coli</i> Fpg

The mutagenic 8-oxoguanosine monophosphate, the predominant product of DNA oxidation, is excised by formamidopyrimidine glycosylase (Fpg) in bacteria. The mechanism of recognition of 8-oxodG, which differs subtly from its normal counterpart, guanosine monophosphate (dG), by <i>Escherichia coli</i> Fpg remains elusive due to the lack of structural data of <i>E. coli</i> Fpg bound to 8-oxodG. Here, we present solution-state structure of 8-oxodG oligomer bound to <i>E. coli</i> E3Q Fpg using UV resonance Raman (UVRR) spectroscopy. The vibrational spectra report on the π-stacking and hydrogen bonding interactions established by 8-oxodG with <i>E. coli</i> E3Q Fpg. Furthermore, we report on the interactions of <i>E. coli</i> E3Q Fpg with the normal, undamaged nucleotide, dG. We show that <i>E. coli</i> Fpg recognizes 8-oxodG and dG through their C2-amino group but only 8-oxodG forms extensive contacts with <i>E. coli</i> Fpg. Our findings provide a basis for mechanism of lesion recognition by <i>E. coli</i> Fpg

qSR: a quantitative super-resolution analysis tool reveals the cell-cycle dependent organization of RNA Polymerase I in live human cells

We present qSR, an analytical tool for the quantitative analysis of single molecule based super-resolution data. The software is created as an open-source platform integrating multiple algorithms for rigorous spatial and temporal characterizations of protein clusters in super-resolution data of living cells. First, we illustrate qSR using a sample live cell data of RNA Polymerase II (Pol II) as an example of highly dynamic sub-diffractive clusters. Then we utilize qSR to investigate the organization and dynamics of endogenous RNA Polymerase I (Pol I) in live human cells, throughout the cell cycle. Our analysis reveals a previously uncharacterized transient clustering of Pol I. Both stable and transient populations of Pol I clusters co-exist in individual living cells, and their relative fraction vary during cell cycle, in a manner correlating with global gene expression. Thus, qSR serves to facilitate the study of protein organization and dynamics with very high spatial and temporal resolutions directly in live cell.National Institutes of Health (U.S.)National Cancer Institute (U.S.) (NIH Director’s New Innovator Award DP2-CA195769)Massachusetts Institute of Technology. Department of Physic