The role of a novel DNA-binding protein WDHD1 in RNA processing and its link to diseases

Background: Alternative splicing of pre-mRNA is a widespread process by which functionally diverse protein isoforms can be generated in a spatiotemporal manner. It is a precise and powerful mechanism for expanding genomic plasticity as well as proteomic complexity. Accuracy of intron removal is thus essential, and disruption of normal splicing patterns has been linked to various genetic diseases and cancers. Recent results from our lab have uncovered a potentially novel and important regulator of post-transcriptional RNA processing. Through proteomic and functional genomic approaches, we have found that WDHD1, a HMG domain-containing protein whose function is largely uncharacterized, stably associates with the spliceosome complexes and may be involved in controlling alternative splicing. We propose a set of synergistic experiments to further explore this function and, more importantly, its (patho)physiological relevance. Objective: First, after completing a comprehensive profile of the pre-mRNA targets of WDHD1 via microarray analysis, we will perform validation on selected candidate genes. Next, the exact molecular mechanism underlying WDHD1’s splicing function will be thoroughly dissected. Finally, physiological significance of WDHD1 can be illustrated by assessing tumorigenic and/or disease association of the splicing targets. Methods & Results: Based on the results of the genome-wide splicing-sensitive microarray platform, we have preliminarily selected a few candidate target genes for further analysis and designed more than 3 primers for each gene. Two splicing targets, PSME3 and MER5, were repeatedly confirmed by end-point and real-time PCR assays (Figure 1, A & B; Figure 2, A & B). Previous studies have implicated PSME3 (proteasome activator complex subunit 3) in facilitating p53 ubiquitination and degradation by promoting MDM2 and p53 interaction. Results from this study have shown that WDHD1 knockdown led to an increase in the expression of a specific splicing form of PSME3 transcript and ultimately reduced PSME3 protein expression (Fig. 1, C & D). In line with previous findings, downregulation of PSME3 coincided with elevated p53 protein level (Fig. 1D), suggesting moderated p53 degradation. MER5, also called Peroxiredoxin-3 (Prdx3), is a mitochondrial member of the antioxidant family of thioredoxin peroxidases that scavenge hydrogen peroxide (H2O2). It exerts such antioxidant role through using mitochondrial thioredoxin-2 (Trx2) as a source of reducing equivalents. MER5 has been shown to confer a resistance to hypoxia-induced H2O2 formation and apoptosis and thus is considered an important part of the cellular antioxidant defense system. Our results demonstrated that WDHD1 knockdown caused an exon-skipped variant form of the MER5 RNA transcript (Figure 2C). Such alteration consequently resulted in a lower expression of the protein product (Figure 2D). In addition to pre-mRNA splicing, our recent findings have also uncovered a role of WDHD1 in the processing of centromere-encoding non-coding RNA and the regulation of centromere function. The centromere is a highly specialized chromosomal element that is essential for chromosome segregation during mitosis. Centromere integrity is properly preserved by and strictly dependent upon the establishment and maintenance of surrounding chromatin structure. We showed that WDHD1 associates with centromeres in a cell cycle-dependent manner, coinciding with mid-to-late S phase. Moreover, we demonstrated that a possible underlying mechanism of WDHD1’s involvement lies in the proper generation of the small non-coding RNAs encoded by the centromeric satellite repeats. This role is mediated at the post-transcriptional level and likely through stabilizing Dicer association with centromeric RNA. WDHD1 down-regulation compromises HP1a localization to pericentric heterochromatin and leads to altered expression of epigenetic markers associated with this chromatin region. As a consequence, such reduced epigenetic silencing is manifested in disrupted heterochromatic state of the centromere and a defective mitosis. Collectively, these findings suggest that WDHD1 may be a critical component of the RNA-dependent epigenetic control mechanism that sustains centromere integrity and genomic stability. Conclusions: Our current findings strongly suggest that WDHD1 may be closely linked to different cellular processes, such as p53 signaling and antioxidant response, through modulating splicing and expression of key players of the respective processes. Ongoing studies have been focused on elucidating the cell biological consequence of altered splicing and protein expression of these WDHD1 target genes. The ultimate aim is to clarify the mechanism underlying the regulation of WDHD1 as well as the functional roles of such splicing regulation in cells. By establishing the roles of WDHD1 in pre-mRNA splicing and RNA processing, these studies will expand our view on the post-transcriptional regulation of gene expression, and potentially open new avenues for therapeutic development.

Project ID:PG10012-0226
External Project ID:NHRI-EX101-9923SC