Project Summary/Abstract Long noncoding RNAs (lncRNAs) are vital components of gene expression programs controlling cellular differentiation and function. lncRNAs act as scaffolds to recruit or sequester effector proteins. They may act in cis and trans at multiple genomic sites. lncRNAs modulate transcription, chromatin organization, RNA processing, and translation, and many questions remain unanswered regarding how they influence gene expression. Recent reviews have detailed several lncRNAs that play key roles in prostate cancer including regulation of processes in cancer cells such as proliferative signaling, replicative immortality, invasion and metastasis, evasion of growth suppressors, induction of angiogenesis and resistance to apoptosis. While much remains to be learned regarding the mechanisms of action involved in lncRNA function, it is abundantly clear that lncRNAs act in concert with associated proteins to carry out their roles. Distinguishing between aggressive and indolent prostate cancer is a major conundrum in cancer. In roughly one-third of the 140,000 intermediate grade cancers (Gleason 7) diagnosed annually, the disease follows a more aggressive course developing rapid progression of prostate specific antigen (PSA) after treatment and earlier metastasis and death. During the prior three-year grant period, we discovered several dozen lncRNAs that distinguish aggressive and indolent prostate cancers, of which four were validated using RT-qPCR. We also developed the HyPR-MS strategy to permit elucidation of specific RNA-protein interactomes. In this renewal proposal we seek to further develop and apply a suite of powerful new proteomics tools to study these and other prostate cancer-relevant lncRNAs and the proteins that associate with them, and use this capability to reveal important proteomic signatures to distinguish aggressive versus indolent prostate cancer. These studies will provide a novel and unprecedented view into the nature of the proteoform–lncRNA interactions that underlie lncRNA function. The delineation of differentially expressed lncRNAs and their associated proteins/proteoforms will provide insight into the biological mechanisms underlying disease progression and provide novel therapeutic targets for further development.