Project Summary Though triple helices were deduced to form in vitro over sixty years ago, the function of triple helices in cells is only beginning to be appreciated. My work will focus on pyrimidine motif triple helices, whereby a pyrimidine- rich third strand binds in the parallel orientation along the purine-rich strand in the major groove of a double helix. This work will focus on two key questions about triple helices: (i) which base triples stabilize an RNA•DNA-DNA triple helix? and (ii) how does a protein recognize a triple helix? Noncoding RNAs have been proposed to regulate gene expression by binding to genomic DNA via an RNA•DNA-DNA triple helix. However, beyond the canonical U•A-T and C•G-C base triples, the stability of base triples that compose RNA•DNA-DNA triple helices is unknown. Therefore, in Aim 1, I will systematically determine the stability of an RNA•DNA-DNA triple helix when a single base triple, Z•X-Y (where Z = C, U, A, G and X-Y = A-T, G-C, T-A, C-G), is varied in a U•A-T-rich triple helix, using a native electrophoretic mobility shift assay to examine the binding between the RNA and double-stranded DNA. Furthermore, I will test the stability of nine common RNA modifications at the same position in the RNA•DNA-DNA triple helix and compare the stabilities of modified RNA to its unmodified RNA counterpart. This study will be the first to show the relative stability of each Z•X-Y base triple in an RNA•DNA-DNA triple helix and will lead to a better understanding of how nature uses RNA•DNA-DNA triple helices to regulate gene expression. In Aim 2, I will use X-ray crystallography and cryogenic electron microscopy to solve a three-dimensional structure of methyltransferase-like protein 16 (METTL16) in complex with the triple helix from the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) long noncoding RNA. METTL16 is an essential human RNA methyltransferase that has been shown to bind to the 3' end of the MALAT1 long noncoding RNA both in vitro and in cell-based assays. Interestingly, the 3' end of MALAT1 forms a triple helix that functions to protect MALAT1 from 3'-end degradation. METTL16 is the first and only putative triple-stranded RNA-binding protein. Thus, the structure of the METTL16-MALAT1 triple helix complex will potentially uncover a novel class of triple-stranded RNA-binding proteins. Overall, this work will increase our knowledge of base triples that stabilize triple helices and of proteins that interact with triple helices.