A Cross-Tissue Investigation of Molecular Targets and Physiological Functions of Nsun6 Using Knockout Mice.

The 5-methylcytosine (m5C) modification on an mRNA molecule is deposited by Nsun2 and its paralog Nsun6. While the physiological functions of Nsun2 have been carefully studied using gene knockout (KO) mice, the physiological functions of Nsun6 remain elusive. In this study, we generated an Nsun6-KO mouse strain, which exhibited no apparent phenotype in both the development and adult stages as compared to wild-type mice. Taking advantage of this mouse strain, we identified 80 high-confident Nsun6-dependent m5C sites by mRNA bisulfite sequencing in five different tissues and systematically analyzed the transcriptomic phenotypes of Nsun6-KO tissues by mRNA sequencing. Our data indicated that Nsun6 is not required for the homeostasis of these organs under laboratory housing conditions, but its loss may affect immune response in the spleen and oxidoreductive reaction in the liver under certain conditions. Additionally, we further investigated T-cell-dependent B cell activation in KO mice and found that Nsun6 is not essential for the germinal center B cell formation but is associated with the formation of antibody-secreting plasma cells. Finally, we found that Nsun6-mediated m5C modification does not have any evident influence on the stability of Nsun6 target mRNAs, suggesting that Nsun6-KO-induced phenotypes may be associated with other functions of the m5C modification or Nsun6 protein.

CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators.

Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m6 A and m5 C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that modulate the modification rate. However, a high-throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR-seq). Using CIGAR-seq, we discovered NSUN6 as a novel mRNA m5 C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on non-overlapping subsets of mRNA m5 C sites and together contributed to almost all the m5 C modification in mRNA. Finally, using m1 A as an example, we demonstrated that CIGAR-seq can be easily adapted for identifying regulators of other mRNA modification.

The SWI/SNF chromatin remodeling factor DPF3 regulates metastasis of ccRCC by modulating TGF-ß signaling.

DPF3, a component of the SWI/SNF chromatin remodeling complex, has been associated with clear cell renal cell carcinoma (ccRCC) in a genome-wide association study. However, the functional role of DPF3 in ccRCC development and progression remains unknown. In this study, we demonstrate that DPF3a, the short isoform of DPF3, promotes kidney cancer cell migration both in vitro and in vivo, consistent with the clinical observation that DPF3a is significantly upregulated in ccRCC patients with metastases. Mechanistically, DPF3a specifically interacts with SNIP1, via which it forms a complex with SMAD4 and p300 histone acetyltransferase (HAT), the major transcriptional regulators of TGF-ß signaling pathway. Moreover, the binding of DPF3a releases the repressive effect of SNIP1 on p300 HAT activity, leading to the increase in local histone acetylation and the activation of cell movement related genes. Overall, our findings reveal a metastasis-promoting function of DPF3, and further establish the link between SWI/SNF components and ccRCC.