The ability of an animal to cope with environmental and chemical stress is paramount to its survival, and failure to properly respond to stress eventually leads to genetic mutations and cell death. The heat shock response is a conserved biological process present in almost every living organism and is activated not only by heat shock but also by a variety of cellular stresses. Interestingly, the heat shock response is also activated during tumor progression, neurodegeneration and viral infection, indicating a fundamental link between the heat shock response and human disease. Using genome-wide biochemical approaches and molecular genetic techniques in the worm C. elegans, I will focus on the role of long non-coding RNAs (ncRNAs) in the heat shock response. The recent modENCODE and ENCODE projects have shown that ncRNAs represent a significant portion of transcribed genomes, with many of these ncRNAs being upregulated in cancer cells. Specifically, I will focus on a large class of long ncRNAs whose expression is dependent on the function of Argonaute, the core effector of the conserved RNA-induced silencing complex. These Argonaute-dependent (AD) ncRNAs will be annotated using high-throughput RNA sequencing, ribosome profiling, rapid amplification of cDNA ends (RACE), and biochemical validation assays. A technique called iCLIP for extracting transcriptome-wide Argonaute-binding sites will be used to determine the role of Argonaute in the processing, regulation and activity of AD ncRNAs. Furthermore, ncRNA secondary structures in vivo will be annotated using a genome- wide method called SHAPE-seq for isolating and sequencing transcripts containing chemically modified ribonucleotides. To probe the specific function of AD ncRNAs, I will use the recently developed CRISPR/Cas9 genome-editing technology to generate appropriate transgenic strains and assay loss-of-function and gain-of- function phenotypes. Gene expression profiling and tissue-expression studies in select ncRNA-mutant strains will be used to determine the specific genetic pathways that are modulated by these AD ncRNAs. Finally, conservation studies will be carried out to determine the specific sequence features and structural motifs that are conserved among human non-coding RNAs. This will provide a foundation for future studies on the role of specific non-coding RNAs in the human heat shock response, and their functional relevance to human cancer and disease.
This project will use high-throughput RNA sequencing-based methods and molecular genetic assays to annotate and probe the function of long non-coding RNAs in the heat shock response of Caenorhabditis elegans. Since the heat shock response is conserved in humans and active in many cancers, viral infecitons, and neurodegenerative diseases, the study of heat shock genetics is critical to human health. Furthermore, the high-throughput, genome-wide techniques and workflow developed in this project should be useful to biomedical researchers and clinicians seeking to identify key genetic factors and biomarkers in cancer and disease.
