BioProject

In cells, transcription is tightly regulated on multiple layers. The condensation of the transcription machinery into distinct phases is hypothesized to spatio-temporally fine tune RNA polymerase II behaviour during two key stages, transcription initiation and the elongation of the nascent RNA transcripts. More...

In cells, transcription is tightly regulated on multiple layers. The condensation of the transcription machinery into distinct phases is hypothesized to spatio-temporally fine tune RNA polymerase II behaviour during two key stages, transcription initiation and the elongation of the nascent RNA transcripts. However, it has remained unclear whether these phases would mix when present at the same time or remain distinct chemical environments; either as multi-phase condensates or by forming entirely separate condensates. Here we combine particle-based multi-scale simulations and experiments in the model organism C. elegans to characterise the biophysical properties of RNA polymerase II condensates. Both simulations and the in vivo work describe a lower critical solution temperature (LCST) behaviour of RNA Polymerase II, with condensates dissolving at lower temperatures whereas higher temperatures promote condensate stability, which highlights that these condensates are physio-chemically distinct from heterochromatin condensates. The LCST behavior of CTD correlates with gradual shifts in the transcription program but is largely uncoupled from the classical stress response. Expanding the simulations we model how the degree of phosphorylation of the disordered C-terminal domain of RNA polymerase II (CTD), which is characteristic for each step of transcription, controls the existence and morphology of multi-phasic condensates. We show that the two phases putatively underpinning the initiation of transcription and transcription elongation constitute distinct chemical environments and are in agreement with RNA polymerase II condensates observed in C. elegans embryos by super resolution microscopy. Our analysis shows how depending on its post transcriptional modifications and its interaction partner a single protein can form multiple partially engulfed condensates, potentially promoting the selective recruitment of additional factors to these two phases. Overall design: RNA was extracted from early wild-type (N2 Bristol) C. elegans embryos from a synchronized culture grown at 20°C and exposed to indicated temperatures for 2 h, using Trizol. Embryos were freeze cracked five times, then RNA was extracted with choloroform followed by isopropanol precipitation. Further purification was performed with the RNA Clean and Concentrator kit (Zymo). Libraries were produced using the Stranded Total RNA Prep Ligation with Ribo-Zero Plus Kit (Illumina). rRNA was depleted using the Ribo-Zero Plus kit supplemented with custom made C. elegans specific rDNA oligos (IDT). Libraries were profiled in a DNA 1000 Chip on a 2100 Bioanalyser (Agilent technologies) and quantified using the Qubit 1x dsDNA HS Assay Kit, in a Qubit 4.0 Fluorometer (Life technologies). Equimolar amounts of indexed libraries were pooled and sequenced on a NextSeq 2000 (Illumina). Less...