This project will develop novel ways of finding which RNA molecules are found near a given kind of protein in eukaryotic cells. As the first test case, we will ask what species of RNA are enriched around one kind of formation in the nucleus, called nuclear speckles. The approach that we will develop will be applied later to determine the RNA composition in other cellular domains. This project will serve as a platform for teaching undergraduate, community college, and high school students to appreciate biology, and also for mentoring graduate students.
The localization and targeting of RNAs to specific cellular regions and compartments has been identified as an important mechanism in controlling several cellular processes such as cell migration, neuronal signaling and development in a wide array of model organisms. A major impediment to the understanding of RNA localization studies, however, is the current absence of a good genome-wide method to identify RNAs localized in specific cellular compartments. The objective of the present project is to develop a novel and robust genomic method, TSA-RNA-seq to identify RNAs that are enriched in a specific cellular compartment. TSA-RNA-seq will be developed in the context of a specific sub nuclear compartment called nuclear speckle. Nuclear speckles are dynamic structures enriched with proteins and RNAs involved in mRNA metabolism. Characterization of the mechanism/s that governs specific interactions between speckle-associated genes and RNAs would unravel the principles that define the contribution of nuclear speckles in gene expression and RNA maturation. The primary objectives of the project include: 1) Development of TSA-RNA-seq to identify nuclear speckle-resident RNAs. 2) Development of biochemical purification of speckles and to identify speckle-localized RNA. 3) Validation of speckle-resident RNA. The accomplishment of the objectives in this project would result in development of excellent tools that could be used to identify and to understand the transcriptome associated with any subnuclear domain.
