The development of hybridization mapping for the identification of transcriptomes and their novel transcripts

The search for non-coding RNAs (ncRNAs) and their various functions has been the focus of many recent investigations. The subsequent characterization of ncRNAs, having roles in a variety of cellular processes from development to disease, has required amendment of the central dogma which states that the only role for mRNA is to store coding information for proteins. However, questions remain regarding the actual extent and function of the ncRNA transcriptome. We hypothesize that cells contain a multitude of ncRNAs that may or may not assemble into protein complexes and that help to regulate cellular function and activity at every level. However, the majority of ncRNAs remain undiscovered due to biases that favor protein-coding genes. We tested this by developing an unbiased technique to define the complete stranded transcriptome (both coding and non-coding) of an organism. This technique is called HybMap for Hybridization Mapping because it utilizes an antibody that specifically recognizes RNA-DNA hybrids. We first used HybMap to determine the dynamic transcriptome of Schizosaccharomyces pombe across multiple conditions. Comprehensive analysis of the S. pombe dynamic transcriptome revealed several novel biological features, as well as thousands of novel transcripts. Next, we selected a subset of the novel transcripts for further characterization regarding their coding status and function. As part of this ongoing analysis, knock-out strains for each of the selected transcripts were generated and screened for growth phenotypes. We found that 10-15% of the selected transcripts are required for normal growth, highlighting the effectiveness of using HybMap for the identification of novel functional transcripts. Finally, we extended HybMap to evaluate known ncRNAs and to identify novel transcripts involved in various cancers. It has already been established that ncRNAs play an integral role in cancer initiation and progression. Thus, the ultimate goal of these final ongoing studies was to assess the applicability of HybMap for the determination of ncRNA presence and/or levels in clinical samples. Together these studies illustrate the complexity of an organism's transcriptome and inspire us to more closely examine the non-coding genome.