FUS regulates the alternative splicing of cell proliferation genes related to atherosclerosis

Abstract

FUS plays a significant role as an RNA-binding protein in several cellular processes, including RNA splicing, DNA repair, and transcriptional regulation. However, the RNA-binding capacity of FUS in atherosclerosis is unclear. We aimed to study the functions of FUS in inflammatory regulation through the role of the splicing factor. We knocked down FUS with siRNA to further study the overall transcriptional level and select alternative splicing (AS) of FUS regulation in human umbilical vein endothelial cells (HUVECs) by RNA sequencing. The results suggested that the knockdown of FUS significantly affected gene expression in HUVECs. In addition, the knockdown of FUS resulted in 200 differentially expressed genes (DEGs) that were highly related to apoptotic process, signal transduction, multicellular organism development, cell adhesion and regulation of transcription, and DNA-templated pathways. Importantly, FUS extensively regulated 2870 AS events with a significant difference. Functional analysis of its modulated AS genes revealed they were highly enriched in cell cycle and cell population proliferation pathways. The qRT-PCR and RNA-seq data showed consistent results. Our findings suggested new knowledge of the mechanisms of FUS associated with atherosclerosis.

Keywords: FUS; RNA-binding protein; RNA-seq; alternative splicing; atherosclerosis.

Figure 1.

Differential gene expression responding to FUS knockdown in HUVECs. (A) Bar plot showing the knockdown efficiency for three siRNAs of FUS from RT-qPCR result (left panel) and RNA-seq data (right panel). (B) Western blot result showing the decreased level of FUS in HUVECs. (C) PCA result showing the global expression pattern of RNA-seq data. (D) Volcano plot showing the upregulated (red) and down-regulated (blue) genes. (E) Heatmap showing the high expression (red) and low expression (blue) of DEGs.

Figure 2.

FUS regulates the expression of apoptosis-related genes. (A) The top 10 GO terms of up or down-regulated genes. (B) Genes expression of FUS regulated from RNA-seq data and qRT-PCR validation.

Figure 3.

FUS regulates gene alternative splicing in HUVECs. (A) Frequency distribution of different alternative splicing events types regulated by FUS. (B) The top 10 GO biological processes of RASGs. (C) Venn diagram illustrating the overlap genes of RASGs and DEGs. (D and E) FUS regulates the alternative splicing of CD44 and FLT1. IGV-sashimi diagrams illustrate AS changes in FUS knockdown cells and control cells (left panel). The schematic plots describe the structures of ASEs (right panel, top). The constitutive exon sequences are represented by white boxes, and the intron sequences are represented by the horizontal line, while the alternative exons are represented by blue boxes. The bottom of the right panel shows the RNA-seq quantification and qRT-PCR validation of ASEs.