RNA binding proteins (RBPs) mediate constitutive RNA metabolism and gene 16 specific regulatory interactions. To identify RNA cis-regulatory elements, we developed 17GCLiPP, a biochemical technique for detecting RBP occupancy transcriptome-wide. GCLiPP 18 sequence tags corresponded with known RBP binding sites, specifically correlating to abundant 19 cytosolic RBPs. To demonstrate the utility of our occupancy profiles, we performed functional 20 dissection of 3' UTRs with CRISPR/Cas9 genome editing. Two RBP occupied sites in the CD69 21 3' UTR destabilized the transcript of this key regulator of lymphocyte tissue egress. Comparing 22human Jurkat T cells and mouse primary T cells uncovered hundreds of biochemically shared 23 peaks of GCLiPP signal across homologous regions of human and mouse 3' UTRs, including a 24 cis-regulatory element that governs the stability of the mRNA that encodes the proto-oncogene 25 PIM3 in both species. Our GCLiPP datasets provide a rich resource for investigation of post-26 transcriptional regulation in the immune system. 27 28 Introduction 29The life cycle of protein coding RNA transcripts involves their transcription from DNA, 30 5¢ capping, splicing, 3′ polyadenylation, nuclear export, targeting to the correct cellular 31 compartment, translation and degradation (Beelman and Parker, 1995; Martin and Ephrussi, 32 2009;Reed, 2003). RNA binding proteins (RBPs) coordinately regulate these processes through 33 interaction with RNA cis-regulatory elements, often in the 5′ and 3′ untranslated regions (UTRs) 34 2 whose sequences are not constrained by a functional coding sequence (Keene, 2007). 35Mammalian genomes encode hundreds of RBPs (Castello et al., 2012), and mutations in 36 individual RBPs or even individual binding sites can induce strong developmental, autoimmune 37 and neurological defects in human patients and mouse models (Bassell and Kelic, 2004; Kafasla 38 et al., 2014;Schwerk and Savan, 2015). As much as half of the extensive gene expression 39 changes that occur during T cell activation occur post-transcriptionally (Raghavan et al., 2002), 40 and several RBPs are known to be critical determinants of immune function and homeostasis 41 (Kafasla et al., 2014). 42 Methods like DNase I hypersensitivity and ATAC-seq that query regulatory element 43 accessibility and occupancy without prior knowledge of their protein binding partners have 44 proven themselves as powerful techniques for the systematic mapping of cis-regulatory 45 sequences in DNA (Buenrostro et al., 2013; Thurman et al., 2012). Their development has 46 allowed for comparisons in the regulatory structure of diverse cell types (Corces et al., 2016) and 47 Current methods for regulatory element identification in RNA have focused on specific 52 trans factors (Lee and Ule, 2018), although more recent technologies have also analyzed 53 secondary structure (Spitale et al., 2015) and interaction with chromatin (Li et al., 2017) 54 transcriptome wide. Protein precipitation (Baltz et al., 2012) and chemical biotinylat...