The human genome codes for thousands of long noncoding RNAs (lncRNAs), many of which are implicated in diseases ranging from cancer to neurodegenerative disorders. Despite the importance of lncRNAs, we only understand the function of a handful of them. Even for these select few that have been studied, our understanding lies at the genetic or molecular level and we lack a detailed structural understanding of how they function. This structural information is critical for validating proposed mechanisms, understanding and predicting the function of unstudied lncRNAs, and potentially targeting lncRNAs for therapeutic purposes. One of the most common functions of lncRNAs is the recruitment of chromatin modifying complexes. This function is consistent with their tissue specific expression and their implication in diseases related to cellular differentiation, such as cancer metastasis. Perhaps the best-characterized lncRNAs, Xist and its partially overlapping 5' transcript repA, function in this manner by recruiting PRC2 to silence the X- chromosome during dosage compensation in female mammals. Additional lncRNAs, such as HOTAIR, also recruit PRC2 via interaction with a 5' domain, suggesting a global mechanism of action. Recent reports in the literature, however, disagree about the mechanism and specificity of PRC2 recruitment. Thus, we will additionally utilize an analogous model system in flies to gain insight into the structural basis of lncRNA recruitment of histone modification complexes.
The first aim will be to determine the structural basis for the specific interaction between the roX lncRNAs and the MSL histone modification complex in Drosophila. The roX lncRNAs recruit the MSL complex to the X-chromosome in a fashion akin to the Xist/PRC2 system. In flies, however, the components of this complex have been well characterized and two protein components have been proposed to specifically interact with a defined RNA element. These models will be validated using in vitro biochemical approaches such as electrophoretic mobility shift assays and isothermal titration calorimetry. Ultimately, these biochemical data will facilitate the solution of the x-ray crystal structure of the complex responsible for conferring specificity in the roX/MSL complex.
The second aim will be to structurally characterize the lncRNA repA. repA will be transcribed in vitro, but will be subsequently purified by size-exclusion chromatography without the traditional denaturation step. This production method has been shown to produce homogenous, well-folded RNA amenable to structural characterization by chemical probing. Protein footprinting assays will also be used to determine those RNA structural elements that specifically interact with PRC2. Together, these aims will elucidate the conserved mechanisms of interaction between lncRNAs and histone modification complexes.

Public Health Relevance

Long non-coding RNAs (lncRNAs) are an abundant, but newly characterized class of molecules. Despite implications in diseases ranging from cancer to Alzheimer's disease, there is little known about the structural details of how they execute their functions. Our proposed characterization of lncRNA structure will aid in the prediction of function and the design and implementation of therapeutics for multiple diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Melillo, Amanda A
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Yale University
Schools of Arts and Sciences
New Haven
United States
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