Transcription-Dependent Formation of Nuclear Granules Containing FUS and RNA Pol II

Abstract

Purified recombinant FUsed in Sarcoma (FUS) assembles into an oligomeric state in an RNA-dependent manner to form large condensates. FUS condensates bind and concentrate the C-terminal domain of RNA polymerase II (RNA Pol II). We asked whether a granule in cells contained FUS and RNA Pol II as suggested by the binding of FUS condensates to the polymerase. We developed cross-linking protocols to recover protein particles containing FUS from cells and separated them by size exclusion chromatography. We found a significant fraction of RNA Pol II in large granules containing FUS with diameters of >50 nm or twice that of the RNA Pol II holoenzyme. Inhibition of transcription prevented the polymerase from associating with the granules. Altogether, we found physical evidence of granules containing FUS and RNA Pol II in cells that possess properties comparable to those of in vitro FUS condensates.

The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid-liquid phase separation

“…Indeed, it was previously reported that the N-terminus of FUS is required for binding of FUS to chromatin (Yang et al, 2014), where FUS localises to granules and regulates gene expression in a transcription-dependent manner. Moreover, inhibition of transcription leads to cytoplasmic relocalisation of FUS, suggesting that active transcription tethers FUS to newly synthesized RNA bound to chromatin (Patel et al, 2015;Thompson et al, 2018;Zinszner et al, 1994). This is consistent with the recent finding that FUS leaves the nucleus through passive diffusion .…”

“…Importantly, several proteins enriched with phase separated FUS, such as DDX3X, DHX9, FMR1, TIAR-1 and SMN1 (compare Supplementary Table 1), have already been observed by others to co-localize with FUS into cytoplasmic granules (Blokhuis et al, 2016;Bosco et al, 2010;Groen et al, 2013;Kamelgarn et al, 2016). Moreover, proteins found in nuclear granules, specifically paraspeckles (Shelkovnikova et al, 2014) and transcription-dependent granules containing FUS and RNA Pol II (Thompson et al, 2018) are highly enriched under LLPS conditions compared to non-LLPS conditions (compare data in Supplementary Table 1).…”

“…Consistently, LLPS deficient FUS mostly dissociates from chromatin and partially re-localizes to the cytoplasm. Most likely, LLPS FUS binds to chromatin in a transcription-dependent manner, since previous studies reported re-localization of FUS to the cytoplasm upon transcription inhibition (Patel et al, 2015;Zinszner et al, 1994) and FUS was found in transcription-dependent granules together with RNA Pol II (Thompson et al, 2018). Our data provides further evidence that LLPS is the driving force for FUS binding to chromatin and the cytoplasmic relocalisation of LLPS deficient FUS, as a consequence of losing its nuclear tether, is consistent with recent data , which suggests that FUS leaves the nucleus predominantly through passive diffusion In addition, we show that LLPS of FUS is required for FUS autoregulation, indicating that during transcription FUS is recruited through LLPS to the FUS gene to regulate its own expression.…”

“…Interestingly, numerous recent studies reported that transcription factors recruit the mediator coactivator complex through phase separation using their activation domains leading to recruitment of RNA Pol II binding RNA Pol II C-terminal domain (CTD) (Boehning et al, 2018;Boija et al, 2018;Cho et al, 2018;Chong et al, 2018;Sabari et al, 2018). Noteworthy, FUS was previously reported to interact with the CTD of RNA Pol II (Schwartz et al, 2012) and was identified in transcription-dependent granules together with RNA Pol II (Thompson et al, 2018). Moreover, the N-terminal domain of FUS, which was identified as the transcriptional activator in FUS-CHOP and FUS-ERG fusion proteins in liposarcoma and myeloid sarcoma respectively (Prasad et al, 1994;Zinszner et al, 1994), was recently shown to be sufficient to contact the SWI/SNF chromatin remodelling complex (Linden et al, 2019).…”

“…Furthermore, proteins involved in chromatin remodelling and DNA damage response as well as mitochondrial proteins were almost exclusively detectable together with phase-separated FUS. Interestingly, nuclear FUS granules have already been reported to associate with RNA polymerase II (RNA Pol II) and the N-terminus of FUS, which can phase-separate and is also the transcriptional activator domain of FUS-CHOP and FUS-ERG fusion proteins observed in cancer, is sufficient to target the SWI/SNF chromatin remodelling complex (Burke et al, 2015;Kato et al, 2012;Linden et al, 2019;Prasad et al, 1994;Thompson et al, 2018;Zinszner et al, 1994). Moreover, chromatin remodelling is an important aspect of DNA damage response and FUS granules have already been reported at sites of DNA damage (Patel et al, 2015).…”