PROJECT SUMMARY AND ABSTRACT RNA surveillance pathways regulate the quality of cellular RNAs and their abundance. In the nucleus, aberrant, misfolded, or defective RNAs are degraded primarily by the 3¢-5¢ decay machinery that includes the RNA exosome, a multi-subunit protein complex that catalyzes 3¢ to 5¢ RNA degradation. The nuclear RNA exosome targeting substrate is mediated by several protein complexes containing the MTR4 helicase. These include the nuclear exosome targeting (NEXT) complex and the poly(A) exosome targeting (PAXT) connection which lie upstream of the RNA exosome. The RNA exosome mutants or overexpressed cofactors contribute to numerous human diseases, including cancer. Although several studies illuminated the human exosome structure and functions, there are limited studies on how human NEXT and PAXT engage and prepare their substrates for their delivery to the RNA exosome. This research will engage biochemical, structural, and functional approaches to characterize the core PAXT complex (Aim 1) and determine how substrates are recognized as ribonucleoprotein (RNP) complexes through nuclear cap-binding complex and poly(A) binding protein (Aim 2). I will leverage the use of reagents and skills developed in Aim 1 and Aim 2 during the K99 phase to investigate microRNA (miRNA) processing and turnover by PAXT connection (Aim 3). The outcome of this study will provide novel insights into the molecular mechanism of target RNP recognition by PAXT and their interaction with the RNA exosome in RNA decay pathways. Moreover, the study will be expanding to miRNA biogenesis. As RNA decay pathways and miRNA biogenesis play a fundamental role in gene regulation and cancer, this study can impact human health and the NCI mission to develop scientific knowledge and support all people to live longer and healthier. This proposal describes a comprehensive training plan to develop my academic career by investigating how PAXT recognizes its RNP substrates to stimulate downstream RNA decay and primary miRNA processing. The purpose of this award is to encourage and prepare my independent research program, and I will use this period to advance my scientific knowledge and professional skills. During the K99 phase, I will employ biochemical approaches to reconstitute the PAXT connection with its target and combine them with cryo-electron microscopy under Dr. Christopher D. Lima’s mentoring. That will extend my technical repertoire and biological knowledge in RNA biology, cancer biology, and structural biology for future research. Also, Memorial Sloan Kettering Cancer Center will provide institutional support, including laboratory research resources, an open scientific environment, and career development to achieve my goals.

PROJECT NARRATIVE RNA surveillance pathways monitor the quality of cellular RNAs and regulate them by degradation. The nuclear RNA exosome and its cofactors contribute to catalyzing 3¢ to 5¢ processing and degradation of RNAs in the surveillance pathways. This proposal seeks to study the upstream factors of the RNA exosome in RNA decay pathways and miRNA processing through structural, biochemical, and functional approaches, which provide valuable insights into the nuclear RNA surveillance mechanisms underlying defects associated with human diseases, such as cancer.