TRiC/CCT Chaperonin Regulates RNA Polymerase II in the Nucleus [4tU-Seq]
The molecular chaperonin TRiC/CCT is a large hetero-oligomeric structure that serves an essential role in eukaryotic cells by minimally supporting protein homeostasis including the folding of nascent polypeptides and the assembly/disassembly of protein complexes. TRiC/CCT is typically considered a strict cytosolic machine. Here, we investigated the influence of TRiC/CCT on nuclear features including epigenetic marks, chromatin accessibility, and transcription. Despite being linked to several chromatin modifiers, our work indicates TRiC/CCT does not have a sustained role with these factors. TRiC/CCT did actively contribute to transcription. Inactivation of TRiC/CCT resulted in a significant increase in the production of RNA leading to an accumulation of noncoding transcripts. Our data support a direct role for TRiC/CCT with RNA polymerase II as the chaperonin modulated nascent RNA production both in vivo and in vitro. Overall, our studies reveal a new avenue by which TRiC/CCT contributes to cell homeostasis by regulating the activity of nuclear RNA polymerase II. Overall design: To assess the influence of the CCT chaperonin on nuclear events, we examined active RNA transcripttion in the presence and absence of functional CCT. To inactivate CCT, we used the temperature sensitive strain cct1-2. WT and cct1-2 and an established temperature regime of 4 h at 37°C, which prompts cessation of cell growth that is reversible following a return to a permissive temperature (Ursic et al. 1994). In brief, exponentially growing parental and cct1-2 yeast were shifted to 37°C for 4 h. The cells were then spiked with 4tU (or DMSO for control) for 5 min. 4tU-containing RNA was isolated, RNA libraries were constructed and subjected to NGS. Active transcription analysis was performed by comparing steady-state (RNA-Seq) and 4tU-Seq samples in WT and cct1-2; regions with elevated or reduced active transcription were identified. The unitless constant of RNA half-life was estimated from the number of transcripts (RNA-Seq) divided by the production of new mRNA (4tU-Seq).