On the rise of TRIM8 as a multifunctional workhorse during the course of mitosis

The human TRI-partite Motif containing protein 8 (TRIM8), originally discovered as the Glioblastoma Expressed RING-finger Protein (GERP), is encoded by the TRIM8 gene and belongs to the TRIM family (earlier called as RBCC proteins) of E3 ubiquitin ligases. In my PhD research project, I attempted to understand the involvement of TRIM8 in mitosis in a cell cycle stage-specific manner with the aid of differential transcriptomic (single-cell RNA sequencing), and proteomic (LC-MS/MS) approaches along with the functional experimental and molecular studies. I standardised and optimised a pipeline based on already available machine-learning algorithms on SeqGeqTM platform to divide a population of cells from a single cell line into three phases, G1, S, and G2/M. The importance of the pipeline is that without eliminating cell-cycle heterogeneity (which is typically removed in standard scRNA-seq analysis from tissues) we can compare the impact of a gene silencing in a mitotic stage-specific manner and can also evaluate the impact on cell cycle phase specific marker genes without being biased by the cell-cycle heterogeneity of a single-cell RNA sequencing data from a single cell-line. For instance, high TOP2A expression, a marker of G2/M, has been compared in a stage specific manner in both control and TRIM8-silenced condition in single-cell BD Rhapsody RNA sequencing data from RPE cells. Upon implementation of this new pipeline on the single-cell transcriptomic (scRNA-seq) data from hTERT-immortalized retinal pigment epithelial cells (hTERT RPE-1 or RPE cells), I showed the impact of TRIM8-silencing in three different stages of mitosis (G1, S, and G2/M), and illustrated a very significant impact of TRIM8-silencing on the “Cell Cycle Control of Chromosomal Pathway”. Furthermore, based on the computational analysis of the scRNA-seq data, important candidate genes of the pathway, such as CDK4, CDK6, MCM2, RPA2, have been shown to be upregulated by experimental validation with RT-qPCR. With the indication from transcriptomic level data, proteomic level data has also been generated upon silencing of TRIM8 in RPE cells, and the “Cell Cycle Control of Chromosomal Replication” pathway is shown as the most significant hit also at the protein level. From both differential transcriptomic and proteomic study, I showed that DNA topoisomerase 2-alpha (TOP2A), essential for DNA replication and a key member of the aforementioned pathway, is upregulated at both RNA and protein level upon silencing of TRIM8. Furthermore, I showed that E3 ubiquitin ligase TRIM8 co-localises with centrosomal protein CEP170 during all phases of mitosis at the centrosomal region in HeLa cells, and silencing of TRIM8 upregulates CEP170 only at the protein level in RPE cells. And, it is also shown that TRIM8 localises at the site of primary cilium formation during G0 phase in RPE cells, and downregulation of TRIM8 shows clear impact on two ciliary proteins, NEDD9 and NEK7, in the single-cell RNA sequencing analysis. Overall, my PhD thesis work, along with the earlier published studies, brings out an impression of TRIM8 as a multifunctional workhorse during the course mitosis.