Summary Mutations in the tumor suppressor SPOP (Speckle-type POZ protein) cause prostate, breast and other solid tumors. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localizes to nuclear speckles. Although cancer-associated mutations in SPOP interfere with substrate recruitment to the ligase, mechanisms underlying assembly of SPOP with its substrates in liquid nuclear bodies, and effects of SPOP mutations on assembly are poorly understood. Here we show that substrates trigger phase separation of SPOP in vitro and co-localization in membraneless organelles in cells. Enzymatic activity correlates with cellular co-localization and in vitro mesoscale assembly formation. Diseaseassociated SPOP mutations that lead to the accumulation of proto-oncogenic proteins interfere with phase separation and co-localization in membraneless organelles, suggesting that substrate-directed phase separation of this E3 ligase underlies the regulation of ubiquitin-dependent proteostasis.

Castration-resistant prostate cancer is the lethal condition suffered by prostate cancer patients that become refractory to androgen deprivation therapy. EPI-001 is a recently identified compound active against this condition that modulates the activity of the androgen receptor, a nuclear receptor that is essential for disease progression. The mechanism by which this compound exerts its inhibitory activity is however not yet fully understood. Here we show, by using high resolution solution nuclear magnetic resonance spectroscopy, that EPI-001 selectively interacts with a partially folded region of the transactivation domain of the androgen receptor, known as transactivation unit 5, that is key for the ability of prostate cells to proliferate in the absence of androgens, a distinctive feature of castration-resistant prostate cancer. Our results can contribute to the development of more potent and less toxic novel androgen receptor antagonists for treating this disease.

Highlights d hnRNPDL requires both N-and C-terminal IDRs to phase separate d The absence of N-terminal IDR facilitates aggregation d The unique combination of IDRs in each isoform determines its cellular behavior d D378N/H mutations accelerate hnRNPDL aggregation and compromise its solubility

The androgen receptor is a transcription factor that plays a key role in the development of prostate cancer, and its interactions with general transcription regulators are therefore of potential therapeutic interest. The mechanistic basis of these interactions is poorly understood due to the intrinsically disordered nature of the transactivation domain of the androgen receptor and the generally transient nature of the protein-protein interactions that trigger transcription. Here, we identify a motif of the transactivation domain that contributes to transcriptional activity by recruiting the C-terminal domain of subunit 1 of the general transcription regulator TFIIF. These findings provide molecular insights into the regulation of androgen receptor function and suggest strategies for treating castration-resistant prostate cancer.

2The androgen receptor is a transcription factor that plays a key role in the development of prostate cancer and its interactions with general transcription regulators are therefore of potential therapeutic interest. The mechanistic basis of these interactions is poorly understood due to the intrinsically disordered nature of the transactivation domain of the androgen receptor and the generally transient nature of the protein-protein interactions that trigger transcription. Here we identify a motif of the transactivation domain that contributes to transcriptional activity by recruiting the C-terminal domain of subunit 1 of the general transcription regulator TFIIF. These findings provide new molecular insights into the regulation of androgen receptor function and suggest new strategies for treating prostate cancer.3 Highlights -A short motif in transactivation unit 5 recruits the transcription machinery to the AR -The motif is intrinsically disordered but folds into a helix upon binding -Phosphorylation of Ser 424 enhances the interaction and is essential for transcription -The interaction is a potential therapeutic target for castration-resistant prostate cancer 4 Graphical abstract 5 IntroductionThe activation of transcription relies on interactions between specific transcription factors and general transcription regulators that can mediated by transcriptional co-activators (Fuda et al., 2009). It is important to characterize these interactions because their inhibition by small molecules or other biological tools offers opportunities for therapeutic intervention in many disease areas, including oncology (Darnell, 2002). Since they involve intrinsically disordered transactivation domains the associated complexes are however transient, marginally stable and challenging to study (Wright and Dyson, 2015).One case where inhibiting these interactions is appealing is castration resistant prostate cancer (CRPC). This condition is suffered by prostate cancer patients that are refractory to hormone therapy, which is based on preventing the activation of the androgen receptor (AR). The mechanisms that allow cell proliferation under these conditions are not yet fully characterized but it is becoming clear that they include expression of constitutively active AR isoforms lacking the ligand binding domain (Miyamoto et al., 2015;Robinson et al., 2015).The complexes formed by the transactivation domain of AR (Lavery and McEwan, 2008a) and general transcription regulators are targets to interfere with CRPC (Sadar, 2011) because inhibiting their formation can lead to a decrease in AR transcriptional activity and in the proliferation of prostate cancer cells. Here we report the structural basis for the interaction of the transactivation domain of AR and the C-terminal domain of subunit 1 of the general transcription regulator TFIIF (RAP74-CTD), which involves the partial folding upon binding of a ca 10-residue motif in this receptor and contributes to the initiation of transcription (Choudhry et al., 2006;McEwan and Gustafsson, 1...