Abstract
Nuclear Factor 90 (NF90) is a double-stranded RNA-binding protein (RBP) found in both the nucleus and the cytoplasm that is involved in a plethora of different cellular processes and pathways, such as transcription, splicing, translation and mRNA stability or degradation. For this reason, NF90 compartmentalization and shuttling from the nucleus to the cytoplasm is a very important and strictly controlled process, guided by several stimuli such as viral infection or hypoxia. With this PhD we analyzed the role of NF90 in the microRNA (miRNA) biogenesis, in the nucleus, and its role in translational control and messenger RNA (mRNA) stability, in the cytoplasm.In the nucleus, NF90 was recently shown to be involved in miRNA biogenesis regulation by negatively affecting the Microprocessor activity. However, this mechanism, which our lab previously linked to ovarian carcinoma progression and metastasis, is not fully understood. Here, we show the extent of NF90-mediated pri-miRNA regulation in hepatocellular carcinoma (HCC), for which NF90 is associated with poor prognosis. Genome-wide approaches revealed that NF90 increases the abundance of 286 miRNAs in HepG2 cell line. Of these, 22 pri-miRNAs are directly associated with NF90 through their stem region, in a manner that is largely exclusive of Microprocessor. NF90-targeted pri-miRNAs are mainly intronic, highly stable and have lower free energy and fewer mismatches compared to all human pri-miRNAs. A group of mRNAs hosting NF90-bound and modulated pri-miRNAs were significantly downregulated after loss of NF90 or showed splicing defects in the introns containing the pri-miRNAs. These findings suggest that NF90 is involved in the biogenesis of a subset of highly stable, intronic miRNAs.In the cytoplasm, NF90 was shown to bind mRNAs, which increases their stability or influences their translation. However, the exact mechanism is still largely unknown. Here, we show that NF90 interacts with RBPs involved in RNA-induced silencing complex (RISC)-mediated silencing, such as Moloney leukemia virus 10 (MOV10) and Argonaute 2 (Ago2), in an RNA-dependent manner. Upon glycerol gradient sedimentation, we found that NF90, MOV10 and Ago2 can be found in the same complex in HEK293T cell line. Using published data of NF90 and MOV10 enhancedcrosslinkingimmunoprecipitation(eCLIP)and individual-nucleotide-resolution UV crosslinking (iCLIP), respectively, we identified a subset of mRNAs that can be bound by both NF90 and MOV10 in the 3’UTR. RNA immunoprecipitation (RIP) analyses suggest that the binding of MOV10 to target mRNAs might prevent or reduce the binding of NF90 on the same targets, and vice versa. Moreover, loss of NF90 increased association of Ago2 to the target mRNAs while reducing their abundance. These findings suggest that NF90 might have a role in RISC-mediated silencing by modulating Ago2 association with mRNAs and thereby enhancing their stability.