MicroRNA (miRNA) biogenesis is a cellular process that produces mature miRNAs from their primary transcripts, pri-miRNAs, via two RNAse III enzyme complexes: the Drosha-DGCR8 microprocessor complex in the nucleus and the Dicer-TRBP complex in the cytoplasm. Emerging evidence suggests that miRNA biogenesis is tightly regulated by posttranscriptional and posttranslational modifications and aberrant miRNA biogenesis is associated with various human diseases including cancer. DGCR8 has been shown to be modified by SUMOylation. Yet, the SUMO ligase mediating DGCR8 SUMOylation is currently unknown. Here, we report that USP36, a nucleolar ubiquitin-specific protease essential for ribosome biogenesis, is a novel regulator of DGCR8. USP36 interacts with the microprocessor complex and promotes DGCR8 SUMOylation, specifically modified by SUMO2. USP36-mediated SUMOylation does not affect the levels of DGCR8 and the formation of the Drosha-DGCR8 complex, but promotes the binding of DGCR8 to pri-miRNAs. Consistently, abolishing DGCR8 SUMOylation significantly attenuates its binding to pri-miRNAs and knockdown of USP36 attenuates pri-miRNA processing, resulting in marked reduction of tested mature miRNAs. Induced expression of a SUMOylation-defective mutant of DGCR8 inhibits cell proliferation. Together, these results suggest that USP36 plays an important role in regulating miRNA biogenesis by SUMOylating DGCR8.

<p>Supplementary Fig. S3 shows that the knockdown of Drosha does not affect USP36 interaction with DGCR8 and the knockdown of DGCR8 does not affect USP36 interaction with Drosha.</p>

<p>Supplementary Fig. S4 shows the effects of USP36 on DGCR8 ubiquitination and that USP36 SUMOylation of DGCR8 does not depend on its DUB activity.</p>

<p>Supplementary Fig. S1 shows that knockdown of USP36 decreases the levels of mature miRNAs in HeLa and IMR-90 cells.</p>

<p>Supplementary Fig. S2 shows that DGCR8 and Drosha can localize in the nucleolus and USP36 can colocalize with DGCR8 in the nucleolus.</p>