et al.. flanking sequences provided the specificity for enhanced pri-miRNA processing by the Microprocessor Drosha/DGCR8. Interestingly, while repressing Drosha expression, as reported earlier, we found that EWS was able to enhance the recruitment of Drosha to chromatin. Together, these findings suggest that EWS may positively and negatively regulate miRNA biogenesis via distinct mechanisms, thus providing a new foundation to understand the function of EWS in development and disease. INTRODUCTION EWS belongs to the TET family of RNA binding proteins (RBPs), consisting of FUS/TLS, EWS, and TAF15 (1,2). These RBPs have been implicated in multiple layers of regulated gene expression via their roles in modulating transcription (3C6), coupling between transcription and RNA processing (7) and mediating splice site selection during pre-mRNA splicing (8C11). Consequently, knockout of these RBPs causes severe developmental abnormality in mice (12,13). Importantly, various chromosome translocation events that involve and mutations in both and have been linked to specific human diseases (14,15). Given the ability of individual TET family members to bind RNAs, multiple groups have performed crosslinking immunoprecipitation coupled with deep sequencing (CLIP-seq) to characterize their RNA binding profiles on both cellular Cd33 and animal models (16,17). The initial analysis by PAR-CLIP on HEK293 cells showed related, but distinct RNA binding profiles of FUS/TLS, EWS and TAF15 (18). This study also revealed a general association of these RBPs with 3 splice sites in pre-mRNAs and a preference for both G-rich and AU-rich sequences. However, the association of these RBPs with 3 splice sites was not seen by a separate CLIP study of EWS on HeLa cells, which instead showed enriched RNA binding near EWS-regulated 5 splice sites (10). Two independent genome-wide analyses of FUS/TLS in mouse and human brain also found its prevalent coating on long pre-mRNA transcripts; however, most binding events detected in these studies did not seem to occur near induced alternative splicing events in FUS/TLS deficient cells (8,11). While it has been unclear about the sources of such discrepancies, the seemly degenerative sequence preference for the TET family members might be explained by the observation that FUS/TLS appears to bind certain secondary structures in RNAs, rather than specific motifs in exposed single-stranded RNA regions (18). More importantly, the biological meaning of most detected RNA binding events has been poorly understood. We were initially motivated to investigate various inconsistencies among published genome-wide RNA interactomes by the TET family members. Instead of relying on mining the existing datasets, we generated our own high quality EWS CLIP-seq libraries on HeLa cells and noted prevalent interaction of EWS with a large number of expressed pri-miRNAs, reminiscent of FUS/TLS binding to hairpin-containing RNAs as reported earlier (18). We therefore decided to focus on this new lead in the current study because it has been reported that a large number of miRNAs were induced while others suppressed in EWS knockout mouse embryonic fibroblasts (MEFs) (19). Interestingly, EWS deficiency has also been linked to elevated Drosha expression at both the mRNA and protein levels, and because Drosha Mitoquinone mesylate is the catalytic subunit of the Microprocesssor, which is Mitoquinone mesylate recruited to chromatin to facilitate co-transcriptional pri-miRNA processing in the nucleus (20,21), increased Drosha may therefore account for the induction of a specific set of miRNAs (19). However, how EWS deficiency would also cause the repression of other miRNAs has remained unknown. We now provide evidence for a direct role of EWS in enhancing pri-miRNA processing by the Microprocessor, thus joining EWS to the growing list of RBPs involved in modulating miRNA biogenesis in mammals (22C24). Unlike other RBPs involved in modulating miRNA biogenesis described earlier, Mitoquinone mesylate EWS appears to bind and modulate processing of a large number of pri-miRNAs. Coupled with EWS-mediated Drosha repression, this RBP appears to be capable of both stimulating and inhibiting miRNA biogenesis, but via distinct mechanisms, which we have dissected in this study. The newly elucidated function of EWS adds a new dimension in understanding the mechanisms underlying EWS mutation-induced cancers (5,25,26) and neurodegenerative diseases (27). MATERIALS AND METHODS Cell.