Genome-wide studies of the noncoding functions and mechanisms of human mRNAs

Our genome repeatedly surprises us with novel ways of encoding genetic information, including introns, small regulatory RNAs, and more recently, long noncoding RNAs (lncRNAs). Each discovery not only reveals novel layers of gene regulation but also sheds light on disease mechanisms and potential therapeutic opportunities, clearly demonstrating the importance of studying the fundamental mechanisms of how genetic information is coded in the genome. Thousands of long noncoding RNAs (lncRNAs) have been discovered in mammalian cells, with many employing remarkably diverse mechanisms to control gene expression and cellular activities without being translated. LncRNAs share many characteristics with mRNAs, and increasing evidence has blurred the boundary between lncRNAs and mRNAs. This suggests a continuous functional landscape for lncRNA/mRNA, that mRNAs may act like lncRNAs without being translated into proteins. Known examples of mRNA with noncoding functions are very rare and challenging to predict. Nonetheless, more and more examples are being reported and each of those discoveries revealed a novel mechanism of gene regulation. Existing examples also suggest noncanonical translation-independent functions of mRNAs may be activated by stress and facilitated by reduced global translation activity. To systematically discover translation-independent functions of human mRNA, I will develop a CRISPR/dCas13-based system for inhibiting the translation of specific mRNAs, and then design genome-wide libraries for all human mRNAs and perform parallel dCas13/Cas13 screens to identify mRNAs showing strong cellular phenotype when the mRNA is cleaved by wild type Cas13 but not when the translation of this mRNA is blocked by dCas13. To systematically dissect the molecular mechanisms of the underlying translation-independent functions of mRNAs identified from the parallel CRISPR screens, I will perform Perturb-seq/CROP-seq to simultaneously compare the transcriptome responses to Cas13/dCas13 perturbations of each individual mRNA hit. This experiment will reveal targets of the mRNA noncoding functions that lead to a particular cellular phenotype. I will also develop a dCas13-based tiling and scan assay to identify functional elements in mRNAs that are necessary for their noncoding function. These novel assays will be used to discover and dissect mRNA noncoding functions that allow cancer cells to survive translation inhibition. The proposed study challenges existing paradigms on how mRNA works in the cell, and pushes multiple frontiers of technology development, including CRISPR/Cas13 and single-cell RNA sequencing. If successful, the results will have very profound impact on understanding gene regulation and human disease.