Completion of the DNA sequences of the human genome and that of the nematode Caenorhabditis elegans allows the large-scale identification and analysis of orthologs of human genes in an organism amenable to detailed genetic and molecular analyses. We are determining gene expression profiles in specific cells, tissues, and developmental stages in C. elegans. Our ultimate goal is not only to describe detailed gene expression profiles, but also to gain a greater understanding of the organization of gene regulatory networks and to determine how they control cell function during development and differentiation. The use of C. elegans as a platform to investigate the details of gene regulatory networks has several major advantages. Two key advantages are that it is the simplest multicellular organism for which there is a complete sequence (C. elegans Sequencing Consortium 1998), and it is the only multicellular organism for which there is a completely documented cell lineage (Sulston and Horvitz 1977; Sulston et al. 1983). C. elegans is amenable to both forward and reverse genetics (for review, see Riddle et al. 1997). A 2-week life span and generation time of just 3 days for C. elegans allows experimental procedures to be much shorter, more flexible, and more cost-effective compared to the use of mouse or zebrafish models for genomic analyses. Finally, the small size, transparency, and limited cell number of the worm make it possible to observe many complex cellular and developmental processes that cannot easily be observed in more complex organisms. Morphogenesis of organs and tissues can be observed at the level of a single cell (White et al. 1986). As events have shown, investigating the details of C. elegans biology can lead to fundamental observations about human health and biology (Sulston 1976; Hedgecock et al. 1983; Ellis and Horvitz 1986). We are using complementary approaches to examine gene expression in C. elegans. We are constructing transgenic animals containing promoter green fluorescent protein (GFP) fusions of nematode orthologs of human genes. These transgenic animals are examined to determine the time and tissue expression pattern of the promoter::GFP constructs. Concurrently, we are undertaking serial analysis of gene expression (SAGE) on all developmental stages of intact animals and on selected purified cells. Tissues and selected cells are isolated using a fluorescence activated cell sorter (FACS) to sort promoter::GFP marked cell populations. To date we have purified to near homogeneity cell populations for embryonic muscle, gut, and a subset of neurons. The SAGE and promoter::GFP expression data are publicly available at http://elegans.bcgsc.bc.ca.

The current Caenorhabditis elegans genomic annotation has many genes organized in operons. Using directionally stitched promoterϻGFP methodology, we have conducted the largest survey to date on the regulatory regions of annotated C. elegans operons and identified 65, over 25% of those studied, with internal promoters. We have termed these operons "hybrid operons." GFP expression patterns driven from internal promoters differ in tissue specificity from expression of operon promoters, and serial analysis of gene expression data reveals that there is a lack of expression correlation between genes in many hybrid operons. The average length of intergenic regions with putative promoter activity in hybrid operons is larger than previous estimates for operons as a whole. Genes with internal promoters are more commonly involved in gene duplications and have a significantly lower incidence of alternative splicing than genes without internal promoters, although we have observed almost all trans-splicing patterns in these two distinct groups. Finally, internal promoter constructs are able to rescue lethal knockout phenotypes, demonstrating their necessity in gene regulation and survival. Our work suggests that hybrid operons are common in the C. elegans genome and that internal promoters influence not only gene organization and expression but also operon evolution.

BackgroundPolycystic ovary syndrome (PCOS) is a complex, multifactor disorder in women of reproductive age worldwide. Although RNA editing may contribute to a variety of diseases, its role in PCOS remains unclear.MethodsA discovery RNA-Seq dataset was obtained from the NCBI Gene Expression Omnibus database of granulosa cells from women with PCOS and women without PCOS (controls). A validation RNA-Seq dataset downloaded from the European Nucleotide Archive Databank was used to validate differential editing. Transcriptome-wide investigation was conducted to analyze adenosine-to-inosine (A-to-I) RNA editing in PCOS and control samples.ResultsA total of 17,395 high-confidence A-to-I RNA editing sites were identified in 3,644 genes in all GC samples. As for differential RNA editing, there were 545 differential RNA editing (DRE) sites in 259 genes with Nucleoporin 43 (NUP43), Retinoblastoma Binding Protein 4 (RBBP4), and leckstrin homology-like domain family A member 1 (PHLDA) showing the most significant three 3′-untranslated region (3′UTR) editing. Furthermore, we identified 20 DRE sites that demonstrated a significant correlation between editing levels and gene expression levels. Notably, MIR193b-365a Host Gene (MIR193BHG) and Hook Microtubule Tethering Protein 3 (HOOK3) exhibited significant differential expression between PCOS and controls. Functional enrichment analysis showed that these 259 differentially edited genes were mainly related to apoptosis and necroptosis pathways. RNA binding protein (RBP) analysis revealed that RNA Binding Motif Protein 45 (RBM45) was predicted as the most frequent RBP binding with RNA editing sites. Additionally, we observed a correlation between editing levels of differential editing sites and the expression level of the RNA editing enzyme Adenosine Deaminase RNA Specific B1 (ADARB1). Moreover, the existence of 55 common differentially edited genes and nine differential editing sites were confirmed in the validation dataset.ConclusionOur current study highlighted the potential role of RNA editing in the pathophysiology of PCOS as an epigenetic process. These findings could provide valuable insights into the development of more targeted and effective treatment options for PCOS.