The sequence-binding-function map of an RNA nuclear enrichment element

The functions of long RNAs, including mRNAs and long noncoding RNAs (lncRNAs) critically depend on their subcellular localization. The identity of the sequences that dictate subcellular localization and their high-resolution anatomy remain largely unknown. We used a suite of massively parallel RNA assays and libraries containing thousands of sequence variants for pinpointing the functional features within the SIRLOIN element, which dictates nuclear enrichment through hnRNPK recruitment. We profiled the endogenous SIRLOIN RNA nucleoprotein complex, and identified the nuclear RNA binding proteins SLTM and SNRNP70 as novel SIRLOIN binders. Using massively parallel assays we identified the features that dictate binding of hnRNPK, SLTM, and SNRNP70 to SIRLOIN, and found that these factors are jointly required for SIRLOIN activity. Our study provides a roadmap for high-throughput dissection of functional sequence elements in long RNAs.