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

Alternative splicing of pre-mRNA is a widespread process by which functionally diverse protein isoforms can be expressed according to different cellular programs. It is a powerful mechanism for expanding genomic versatility as well as proteomic complexity. Precision 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. First, we will establish a comprehensive profile of the pre-mRNA targets of WDHD1 via microarray analysis. 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. By establishing the roles of WDHD1 in pre-mRNA splicing, these studies will expand our view on the post-transcriptional regulation of gene expression, and potentially open new avenues for therapeutic development. Based on the results of the genome-wide splicing-sensitive microarray platform, we have preliminarily selected 33 candidate target genes for further analysis and designed more than 3 primers for each gene. Four splicing targets, including KIAA0344, SUZ12, DCTN6 and BUB1B, were repeatedly confirmed by real-time PCR assays (Fig. 1A). The other selected genes, such as PRR11, NEK9 and RFWD2, showed no significant changes as predicted by the array data (Fig. 1B) and a large part of selected targets either couldn’t be detected by RT-PCR or real-time PCR, or are still under experimental validation (data not shown). All pertinent information of our selected genes is summarized in Table 1. On the other hand, we used structure model of splicearray to search for other possible target genes. Based on this approach, we identified and confirmed one potential target gene, F12 (Fig. 2). Cyclin D1 is a checkpoint protein at G1/S phase of cell cycle, and it has been reported that their two isoforms, including cyclin D1a and b, play the difference role at the process of cell cycle. To link WDHD1 to the regulation of this gene’s alternative splicing, we monitored the changes of mRNA isoform expression patterns in control vs. WDHD1 knockdown cells, by real-time RT PCR analysis (Fig. 3A). As the expression of WDHD1 was abrogated, level of cyclin D1a was up-regulated and that of cyclin D1b was down-regulated (Fig. 3B), strongly suggesting that the mRNA pattern of cyclin D1 is regulated by WDHD1. According to the function of three confirmed splicing targets, namely DCTN6, BUB1A and cyclin D1, WDHD1 may presumably regulate the cell cycle process through modulating the expression and splicing of these cell cycle-related mRNAs. To assess this hypothesis, we first monitored the protein levels of DCTN6 and BUB1B in the knockdown cells. While protein expression of WDHD1 was decreased by 60~70% in the HeLa knockdown cells, protein expression of DCTN6 and BUB1B were also down-regulated by 30~40% (Fig. 4). Therefore, defective splicing of these target gene mRNAs as a result of WDHD1 knockdown led to altered levels of final protein product. The structural probes of splicearray were designed to examine the splicing efficiency of intron which contains consensus splice site. The probes are categorized as X, Y, and Z (Fig. 5A). The value of probe X could reflect the efficiency of intron excision and the values of probe Y, and Z may reflect the efficiency of the intron retention. Based on this information, we calculated the ratio of X value to the geometric mean of Z and Y as a means to represent the splicing efficiency of a given junction. The summary of such analysis on the spliced introns for the F12 and Bub1b are now shown in Figure 5. Importantly, these measurements display a wide variation in the level of spliced introns in control vs. siWDHD1 knockdown cells (Fig. 5B & C). The degree of splicing efficiency of introns 6 & 10 of F12 mRNA, as well as intron 3 of Bub1b, was also confirmed (Fig. 1A, 2 & 5B). These results implied that WDHD1 may regulate the alternative splicing of different targets, like F12 and BUB1B, and may in turn affect processes such as cell cycle.

Project ID:PG10001-0176
External Project ID:NHRI-EX100-9923SC