RNA is bound and regulated from its transcription to decay by RNA binding proteins (RBPs). However, how RBPs regulate transcription and post-transcriptional processes in time and space remains largely unknown. My research integrates use of existing with development of novel transcriptome-wide approaches to study the mechanistic basis of RBP activity. Specifically, I am pursuing two aims: 1) to understand the regulatory roles of interactions between RBPs and effectors of RNA processing, and 2) development of a novel method for time-resolved studies of gene regulatory functions of RBPs (timeCLIP). In Aim 1, through integrative analysis of multiple transcriptome datasets, we found that Unkempt (UNK) is a potent and specific translational repressor of its target mRNAs, which it binds in a sequence-specific manner but exerts no detectable effect on their stability. We defined an intrinsically disordered region of UNK, and within it, specific peptide motifs required for functional associations of UNK with the cytoplasmic poly(A)-binding protein (PABPC) and the CCR4-NOT deadenylase complex. Notably, the recruited CCR4-NOT does not utilize its deadenylase activity but rather enables formation of a compact ribonucleoprotein (RNP) particle, restricting access of the targeted mRNA to ribosomes and thus repressing translation. In Aim 2, we developed timeCLIP, a method for time-resolved studies of RBPs in regulating gene transcription and post-transcriptional RNA processing. This method is based on metabolic labeling of cells with 4-thiouridine (4SU) for different periods of time, followed by analyses of RBP-bound labeled RNA and total labeled RNA. By controlling the duration of the 4SU pulse, we can regulate the time of RNA labeling in vivo and determine the age of RNA involved in RBP-RNA binding. We have benchmarked timeCLIP against three previously studied RBPs, RBFOX2, FUS, and RBM25, in HepG2 cells. We identified the canonical RNA binding sites and motifs for RBFOX2 and FUS at all times of labeling, but not in unlabeled samples. Importantly, timeCLIP distinguished early versus later RNA binding, illuminating time-resolved regulation of RNA processing, including alternative splicing and 3' end processing of mRNAs, as well as early binding to anti-sense RNA and enhancer RNA, events that cannot be readily studied by traditional, steady-state CLIP approaches.