PROJECT SUMMARY/ ABSTRACT RNA localization is critical for a diverse set of biological processes. The localization of an RNA depends on cis-elements, features inherent to the transcript, and trans-factors, effectors independent of the target transcript, which are often RNA binding proteins. Cis-elements that regulate RNA localization, often called "zip-codes," are often found in the 3′ untranslated regions (UTRs) of transcripts. However, for the more than 99% of the known localized RNAs, the cis-elements that regulate their localization are unknown. Recent work from the Taliaferro lab identified several 260 nucleotide RNA sequences within the 3’ UTRs of some neurite-localized RNAs that were necessary and sufficient for neurite RNA transport. These were identified using a massively parallel reporter assay that screened approximately 10,000 RNA sequences drawn from endogenous 3’ UTRs for their ability to traffick a reporter transcript to neurites. Interestingly, 100 nt subsequences of these 260 nt active elements were not capable of directing RNA transport. This indicates that (1) the minimal regulatory elements are quite large (likely longer than 100 nt) and (2) the true character of the localization regulatory elements remains unknown. In this work, it is proposed to comprehensively characterize the previously identified RNA localization regulatory elements and zero in on their important features. This will be done by generating a pool of 10,000 RNA sequences based on the previously identified 260 nt zipcodes. Each sequence in this pool will contain defined deletions of varying sizes that span the length of the zipcode. By integrating these mutants into the 3’ UTR of a reporter transcript assaying which of them retain the ability to direct localization of the reporter to neurites, a quantitative readout of the functional importance of each nucleotide within the 260 nt zipcode will be obtained. From this, a clear picture of the important features that make up active localization elements will arise, facilitating their identification in other localized RNAs. The large size of these zipcodes suggests that their secondary may be important for their activity. To test this, their secondary structure will be determined using chemical probing techniques. To test the functionality of the structure, thousands of mutants that disrupt RNA structure as well as compensatory mutants that restore it will be generated. As above, the ability of each of these mutants to drive a reporter transcript to neurites will be tested. In this way, RNA structure and function will be directly related. Answering these questions will help in understanding the underlying mechanisms of RNA localization as very few examples of RNA localization have known mechanistic underpinnings. Identifying the mechanism of RNA localization under physiological conditions is important to being able to understand potential dysfunction in disease states.