Human NOP2/NSUN1 regulates ribosome biogenesis through non-catalytic complex formation with box C/D snoRNPs.

5-Methylcytosine (m5C) is a base modification broadly found on various RNAs in the human transcriptome. In eukaryotes, m5C is catalyzed by enzymes of the NSUN family composed of seven human members (NSUN1-7). NOP2/NSUN1 has been primarily characterized in budding yeast as an essential ribosome biogenesis factor required for the deposition of m5C on the 25S ribosomal RNA (rRNA). Although human NOP2/NSUN1 has been known to be an oncogene overexpressed in several types of cancer, its functions and substrates remain poorly characterized. Here, we used a miCLIP-seq approach to identify human NOP2/NSUN1 RNA substrates. Our analysis revealed that NOP2/NSUN1 catalyzes the deposition of m5C at position 4447 on the 28S rRNA. We also find that NOP2/NSUN1 binds to the 5'ETS region of the pre-rRNA transcript and regulates pre-rRNA processing through non-catalytic complex formation with box C/D snoRNAs. We provide evidence that NOP2/NSUN1 facilitates the recruitment of U3 and U8 snoRNAs to pre-90S ribosomal particles and their stable assembly into snoRNP complexes. Remarkably, expression of both WT and catalytically inactive NOP2/NSUN1 in knockdown background rescues the rRNA processing defects and the stable assembly of box C/D snoRNP complexes, suggesting that NOP2/NSUN1-mediated deposition of m5C on rRNA is not required for ribosome synthesis.

p53 induces a survival transcriptional response after nucleolar stress.

Accumulating evidence indicates that increased ribosome biogenesis is a hallmark of cancer. It is well established that inhibition of any steps of ribosome biogenesis induces nucleolar stress characterized by p53 activation and subsequent cell cycle arrest and/or cell death. However, cells derived from solid tumors have demonstrated different degrees of sensitivity to ribosome biogenesis inhibition, where cytostatic effects rather than apoptosis are observed. The reason for this is not clear, and the p53-specific transcriptional program induced after nucleolar stress has not been previously investigated. Here we demonstrate that blocking rRNA synthesis by depletion of essential rRNA processing factors such as LAS1L, PELP1, and NOP2 or by inhibition of RNA Pol I with the specific small molecule inhibitor CX-5461, mainly induce cell cycle arrest accompanied by autophagy in solid tumor-derived cell lines. Using gene expression analysis, we find that p53 orchestrates a transcriptional program involved in promoting metabolic remodeling and autophagy to help cells survive under nucleolar stress. Importantly, our study demonstrates that blocking autophagy significantly sensitizes cancer cells to RNA Pol I inhibition by CX-5461, suggesting that interfering with autophagy should be considered a strategy to heighten the responsiveness of ribosome biogenesis-targeted therapies in p53-positive tumors.

CD10 expression identifies a subset of human perivascular progenitor cells with high proliferation and calcification potentials.

The tunica adventitia ensheathes arteries and veins and contains presumptive mesenchymal stem cells (MSCs) involved in vascular remodeling. We show here that a subset of human adventitial cells express the CD10/CALLA cell surface metalloprotease. Both CD10+ and CD10- adventitial cells displayed phenotypic features of MSCs when expanded in culture. However, CD10+ adventitial cells exhibited higher proliferation, clonogenic and osteogenic potentials in comparison to their CD10- counterparts. CD10+ adventitial cells increased expression of the cell cycle protein CCND2 via ERK1/2 signaling and osteoblastogenic gene expression via NF-κB signaling. CD10 expression was upregulated in adventitial cells through sonic hedgehog-mediated GLI1 signaling. These results suggest that CD10, which marks rapidly dividing cells in other normal and malignant cell lineages, plays a role in perivascular MSC function and cell fate specification. These findings also point to a role for CD10+ perivascular cells in vascular remodeling and calcification.