# Understanding the Structural Properties of Triple Helices and a Triple-Stranded RNA-Binding Protein

> **NIH NIH F31** · UNIVERSITY OF NOTRE DAME · 2021 · $42,156

## Abstract

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.

## Key facts

- **NIH application ID:** 10115519
- **Project number:** 5F31GM136163-02
- **Recipient organization:** UNIVERSITY OF NOTRE DAME
- **Principal Investigator:** Charlotte Nicole Kunkler
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $42,156
- **Award type:** 5
- **Project period:** 2020-03-01 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10115519

## Citation

> US National Institutes of Health, RePORTER application 10115519, Understanding the Structural Properties of Triple Helices and a Triple-Stranded RNA-Binding Protein (5F31GM136163-02). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10115519. Licensed CC0.

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