Coilin, the Cajal body marker protein, is regulated by SUMOylation and mediates expression of innate immune response genes in human (miRNA)
Cajal bodies (CBs) are membraneless organelles whose mechanism of formation is still not fully understood. Many proteins contribute to the formation of CBs, including Nopp140, WRAP53, and coilin. SUMOylation, a post-translational modification, of coilin involves multiple different lysine residues. Additionally, coilin is also found in the nucleoplasm where its role is still being understood. Here, we demonstrate a novel mechanism examining the interaction changes of coilin when its SUMOylation is disrupted. We look at both global SUMOylation inhibition and its effect on CB formation, as well as targeted SUMOylation inhibition of coilin itself in cells by developing a coilin SUMO mutant. We found upon two types of global SUMOylation inhibition, as well as upon transfection of the coilin SUMO mutant, that CBs increased in number and decreased in size. Additionally, we saw via coimmunoprecipitation the coilin SUMO mutant has altered interaction with Nopp140. This demonstrates increased mechanistic ties to CB formation and SUMOylation. Furthermore, RNA sequencing and small RNA sequencing on a primary cell line with few CBs showed that coilin impacts the response to lipopolysaccharide-induced stress. Collectively, our results identify SUMOylation as a novel mechanism driving CB formation and highlights a positive role for human coilin in innate immunity. Overall design: The Revvity Nextflex Small RNA NGS Kit v4 was employed to construct small RNA libraries from total RNA samples following the manufacturer's protocols. The procedure included adapter ligation, reverse transcription, and PCR amplification, culminating in size selection to enrich for small RNA species. Libraries were quantified, pooled (n=24), and sequenced on an Illumina platform to generate high-throughput sequencing data for downstream analysis. Library was sequenced with NextSeq 2000 P3 (50 cycle) flow cell (Illumina; San Diego, CA, USA)