Abstract IA004: Biocondensates in action: The formation and reformation of EWSR1, FUS and TAF15 ribonucleoprotein networks

Abstract Several cancers arise because of chromosomal rearrangements that result in a fusion oncoprotein which includes the N-terminus of FUS, EWSR1, or TAF15, members of the FET family of RNA-binding proteins. Examples of fusion oncoproteins involving the FET proteins, include FUS::ATF1, FUS::CREB3L1, FUS::DDIT3, EWSR1::CREB1, EWSR1::ERG, EWSR1::FLI1, EWSR1::PATZ1, EWSR1::VEZF1, EWSR1::WT1, TAF15::ZNF384, and TAF15::NR4A3. The FET proteins have similar protein domains, specifically, a N-terminal low complexity domain (LCD), an RNA-recognition motif (RRM), a Zn-finger domain, and a C-terminal nuclear localization signal. Study of the LCDs of the FET proteins has proven essential to determining the biophysical properties of the interactions that define biocondensates, and functional studies have implicated FET proteins as regulators of transcription and RNA maturation. However, the exact functions of the FET proteins remain poorly defined. An enhanced understanding of FET protein function is critical to understanding how the biocondensate properties of these proteins and their functions may contribute to the regulation of gene expression and to tumorigenesis. To investigate the function of the FET proteins, we are using high-resolution microscopy to examine the cellular localization of these proteins at endogenous levels of expression, with an initial focus on EWSR1. To generate endogenous EWSR1 reporter cell lines, we used CRISPR-Cas9 gene editing to introduce a DNA cassette at the 5’ end of EWSR1 exon 1. The DNA cassette expressed a FLAG-FKBP12F36V (FF) peptide that enables the nanoscale imaging of EWSR1 using immunofluorescence (IF) and its depletion via compound-regulated (dTAG-13) proteasomal degradation. Using these modified EWSR1 reporter cell lines, we show that EWSR1 interacts with nascent RNA to form an essential, DNA-independent, non-random ribonucleoprotein network (RNP). Depletion of EWSR1 results in a rapid, but transient reduction in nascent RNA and metabolic activity. Loss of EWSR1 mediates no changes in a marker of transcriptional elongation. However, as EWSR1 is depleted, the expression of FUS and TAF15 increase and concurrently exhibit nuclear reorganization. The observed change in the localizations of FUS and TAF15 reestablishes the formation of non-random RNP networks and restores nascent RNA accumulation. We conclude that the EWSR1 RNP forms an essential scaffold for nascent RNA and in the absence of EWSR1, FUS and TAF15 safeguard this critical function. Citation Format: Soumya Sundara Rajan, Imran Khan, Tamara L. Jones, Langston Lim, Andy Tran, Michael J. Kruhlak, Natasha J. Caplen. Biocondensates in action: The formation and reformation of EWSR1, FUS and TAF15 ribonucleoprotein networks [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr IA004.