Novel ciliary protein TRIM8 is a multifunctional workhorse during mitosis

Abstract

TRIM8 is an E3 ubiquitin ligase that functions as both a tumour suppressor and an oncoprotein. Earlier, we reported that TRIM8 interacts with key regulators of mitotic spindle assembly, and that TRIM8 knockdown results in mitotic delay and aneuploidy. In this study, we implemented a multi-omics strategy with differential transcriptomic (single-cell RNA sequencing or scRNA-seq), translatomic (polysome profiling with RNA-seq), and proteomic (LC-MS/MS) approaches to elucidate the involvement of TRIM8 in different levels (transcription, translation, post-translation) and stages (G0/G1, S, G2/M) of mitotic cell cycle regulation and progression. With the aid of differential transcriptomic (scRNA-seq) and proteomic (LC-MS/MS) approaches, we show that depletion of TRIM8 perturbs the canonical "Cell Cycle Control of Chromosomal Replication" pathway and demonstrate that TRIM8 negatively regulates the expression of TOP2A, known to be essential for genomic integrity. We also show that TRIM8 downregulation induces substantial alterations in the translation activity of cells and results in the upregulation of polysome-bound MALAT1 lncRNA by means of significant changes in polysome profiling coupled with RNA-sequencing. Moreover, we unveil endogenous TRIM8 as a novel ciliary protein that co-localizes with CEP170, required for ciliary function, in the centrosomal region throughout all mitotic phases. Our work shows the dynamic role played by a TRIM family protein across various stages of mitosis for the first time, laying the foundation for exploring the therapeutic potential of TRIM8 in addressing cell cycle-related diseases, including cancer. HIGHLIGHTSO_LITRIM8 is involved in transcriptional and post-translational regulation of "Cell Cycle Control of Chromosomal Replication" pathway and oversees the expression of TOP2A, essential for mitotic chromosome structure maintenance. C_LIO_LIThe silencing of TRIM8 induces changes in cellular translation activity and alters the expression pattern of key translational proteins. Additionally, TRIM8-silencing leads to an elevation of long non-coding RNA (lncRNA) MALAT1 in the polysome-bound fraction. C_LIO_LITRIM8 is identified as a novel ciliary protein. C_LIO_LISilencing of TRIM8 results in the upregulation of the centrosomal protein CEP170, and both proteins co-localize in the centrosomal region throughout all stages of mitosis. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/646005v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1b03f1borg.highwire.dtl.DTLVardef@1ff16e4org.highwire.dtl.DTLVardef@37f07borg.highwire.dtl.DTLVardef@8d28af_HPS_FORMAT_FIGEXP M_FIG C_FIG