A massively parallel reporter screen defines the syntax of SECReTE, a secretion-enhancing cis regulatory targeting element, in mammalian cells

Abstract

Messenger RNA (mRNA) localization to specific subcellular regions is crucial for proper protein translation and localization. Potential means to localize mRNAs include cis-acting or structural motifs embedded within the sequence. We previously identified a secretion-enhancing cis regulatory targeting element (SECReTE) enriched in mRNAs encoding secreted proteins from prokaryotes, eukaryotes, and viruses, found to enhance both protein synthesis and secretion by localizing mRNAs to the endoplasmic reticulum (ER) membrane. Defined as at least 10 consecutive NNY triplets (where N is any nucleotide and Y is a pyrimidine), this highly degenerate motif can be specified by approximately ~10^15 different sequence combinations. Yet, some combinations may prove more efficient than others in regulating mRNA localization and protein secretion. To better understand SECReTE syntax and to identify robust combinations, we designed and synthesized a library of ~5 x 10^4 variants and inserted them into the 3' untranslated region (3'UTR) of two reporter genes, green fluorescent protein (GFP) and immunoglobulin light chain. We expressed each variant in Chinese hamster ovary cells and assayed their activity using parallel assays to measure the effect of each variant upon mRNA partitioning between the ER and cytoplasm, and protein secretion. Our results reveal that ER-associated mRNAs are enriched with SECReTE elements of a defined syntax. Notably, RRY repeats (where R is purine) were superior to NNY repeats. Variants conferring a high mRNA ER localization score consistently enhanced protein secretion, confirming the mechanistic relationship between mRNA targeting to the ER and secretion efficiency. The correlation between variant performances across both reporters suggests a shared regulatory mechanism. Additional analysis identified a novel secretion-promoting cis element, called "split NNS" (where S is G or C). This work refines the syntactic definition of mRNA localization motifs that regulate protein secretion in both natural and heterologous expression systems.