Project Summary/Abstract Recently, several canonical tRNA modifications, including pseudouridine (Ψ ), N6-methyladenosine (m6A), N1- methyladenosine (m1A), and 5-methylcytosine (m5C) have been found in messenger RNA. The development of new high-throughput sequencing methods has revealed the location of individual mRNA modifications and paved the way for the interrogation of their function across the transcriptome. RNA modifications have multiple effects on mRNA during its lifespan: splicing, trafficking, translation and degradation can all be altered. Dihydrouridine (D) is a ubiquitous modified nucleotide found in tRNAs in every branch of the tree of life. Conserved dihydrouridine synthases (DUS) enzymes DUS1L and DUS3L, associate with mRNA in mammalian cells, suggesting that they modify mRNA. Dihydrouridine affects RNA secondary structure by distorting the pyrimidine ring, which is likely to profoundly influence mRNA metabolism. My preliminary data demonstrate that D is installed in mRNAs in yeast, and I hypothesize that this is also true in mammalian cells. I have developed a method (D-seq) to profile D at single-nucleotide resolution. The method works by reducing D with sodium borohydride, causing reverse transcriptase (RT) to fall off the template while traversing reduced D. To expand this technique to transcriptome scale, I will develop a two-step protocol to directly couple biotin to reduced D (eD-seq). This will permit enrichment of RNAs containing D, and comprehensive inspection of all RNA in the human transcriptome for D. To quantify the stoichiometry of modification at D positions, I will also develop a modified form of D-Seq that uses a different RT which misincorporates while traversing D (D-MaP- seq). To demonstrate the utility of these methods, and identify disease relevant D sites, I will comprehensively map and quantify D in the transcriptome of two types of cancer that have characteristic overexpression of DUS. The new D profiling tools that I will develop will identify the locations of DUS-dependent Ds across the transcriptome. They are likely to reveal that D is a previously unknown component of the human `epitranscriptome'.