Nazanin Farahi

The androgen receptor (AR) is a transcription factor whose overactivation is a primary driver of prostate cancer. Although AR interactions with several long non-coding RNAs (lncRNAs) have been implicated in castration-resistant prostate cancer, their underlying molecular mechanisms and functional consequences remain poorly understood. Here, we identify the N-terminal 37 residues of the intrinsically disordered AR N-terminal domain as the primary RNA-binding region that mediates selective interactions with the lncRNAs HOTAIR and SLNCR1. Residue-level mapping and mutational analysis define Y11, R13, and Q24 as key determinants of RNA recognition. This RNA-binding region partially overlaps with the F23QNLF27 motif, previously shown to mediate N/C interdomain communication with the ligand-binding domain through folding upon binding. We observe a partner-dependent binding mode in which this motif remains dynamically disordered upon RNA binding. LncRNA binding promotes phase separation of the N-terminal domain, indicating that lncRNAs upregulated in late-stage prostate cancer may lower the threshold for AR condensate formation and contribute to ligand-independent AR signaling. LncRNAs modulate communication between the N-terminal and ligand-binding domains within condensates, suggesting that lncRNA binding may tune hormone-dependent full-length AR signaling. These findings provide a mechanistic framework for AR-lncRNA regulation, laying the groundwork for future therapeutic strategies against advanced prostate cancer.

Numerous cellular processes rely on biomolecular condensates formed through liquid-liquid phase separation (LLPS), thus, perturbations of LLPS underlie various diseases. We found that proteins initiating LLPS are frequently implicated in somatic cancers, even surpassing their involvement in neurodegeneration. Cancer-associated LLPS scaffolds are connected to all cancer hallmarks and tend to be oncogenes with dominant genetic effects lacking therapeutic options. Since most of them act as oncogenic fusion proteins (OFPs), we undertook a systematic analysis of cancer driver OFPs by assessing their module-level molecular functions. We identified both known and novel combinations of molecular functions that are specific to OFPs and thus have a high potential for driving tumorigenesis. Protein regions driving condensate formation show an increased association with DNA- or chromatin-binding domains of transcription regulators within OFPs, indicating a common molecular mechanism underlying several soft tissue sarcomas and hematologic malignancies where phase-separation-prone OFPs form abnormal, ectopic condensates along the DNA, and thereby dysregulate gene expression programs.

The Database of Intrinsically Disordered Proteins (DisProt, URL: https://disprot.org) is the major repository of manually curated annotations of intrinsically disordered proteins and regions from the literature. We report here recent updates of DisProt version 9, including a restyled web interface, refactored Intrinsically Disordered Proteins Ontology (IDPO), improvements in the curation process and significant content growth of around 30%. Higher quality and consistency of annotations is provided by a newly implemented reviewing process and training of curators. The increased curation capacity is fostered by the integration of DisProt with APICURON, a dedicated resource for the proper attribution and recognition of biocuration efforts. Better interoperability is provided through the adoption of the Minimum Information About Disorder (MIADE) standard, an active collaboration with the Gene Ontology (GO) and Evidence and Conclusion Ontology (ECO) consortia and the support of the ELIXIR infrastructure.

Almost twenty years after its initial release, the Eukaryotic Linear Motif (ELM) resource remains an invaluable source of information for the study of motif-mediated protein-protein interactions. ELM provides a comprehensive, regularly updated and well-organised repository of manually curated, experimentally validated short linear motifs (SLiMs). An increasing number of SLiM-mediated interactions are discovered each year and keeping the resource up-to-date continues to be a great challenge. In the current update, 30 novel motif classes have been added and five existing classes have undergone major revisions. The update includes 411 new motif instances mostly focused on cell-cycle regulation, control of the actin cytoskeleton, membrane remodelling and vesicle trafficking pathways, liquid-liquid phase separation and integrin signalling. Many of the newly annotated motif-mediated interactions are targets of pathogenic motif mimicry by viral, bacterial or eukaryotic pathogens, providing invaluable insights into the molecular mechanisms underlying infectious diseases. The current ELM release includes 317 motif classes incorporating 3934 individual motif instances manually curated from 3867 scientific publications. ELM is available at: http://elm.eu.org.

Liquid–liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles, representing functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integrating the data on LLPS-associated proteins from dedicated databases revealed only modest agreement between them and yielded a high-confidence dataset of 89 human LLPS drivers. Analysis of the supporting evidence for our dataset uncovered a systematic and potentially concerning difference between protein concentrations used in a good fraction of the in vitro LLPS experiments, a key parameter that governs the phase behavior, and the proteomics-derived cellular abundance levels of the corresponding proteins. Closer scrutiny of the underlying experimental data enabled us to offer a sound rationale for this systematic difference, which draws on our current understanding of the cellular organization of the proteome and the LLPS process. In support of this rationale, we find that genes coding for our human LLPS drivers tend to be dosage-sensitive, suggesting that their cellular availability is tightly regulated to preserve their functional role in direct or indirect relation to condensate formation. Our analysis offers guideposts for increasing agreement between in vitro and in vivo studies, probing the roles of proteins in LLPS.

Liquid-liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles (MLOs), i.e. functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integration of data on LLPS-associated proteins from dedicated databases revealed only modest overlap between them and resulted in a confident set of 89 human LLPS driver proteins. Since LLPS is highly concentration-sensitive, the underlying experiments are often criticized for applying higher-than-physiological protein concentrations. To clarify this issue, we performed a naive comparison of in vitro applied and quantitative proteomics-derived protein concentrations and discuss a number of considerations that rationalize the choice of apparently high in vitro concentrations in most LLPS studies. The validity of in vitro LLPS experiments is further supported by in vivo phase-separation experiments and by the observation that the corresponding genes show a strong propensity for dosage sensitivity. This observation implies that the availability of the respective proteins is tightly regulated in cells to avoid erroneous condensate formation. In all, we propose that although local protein concentrations are practically impossible to determine in cells, proteomics-derived cellular concentrations should rather be considered as lower limits of protein concentrations, than strict upper bounds, to be respected by in vitro experiments.