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

Messenger RNA (mRNA) localization to specific cellular compartments is vital for efficient protein translation and delivery. Researchers have discovered a secretion-enhancing cis regulatory targeting element (SECReTE) that enriches mRNAs encoding secreted proteins, facilitating both protein synthesis and secretion. This highly degenerate motif comprises at least 10 consecutive NNY triplets, where N represents any nucleotide and Y is a pyrimidine, and can be specified by approximately 10^15 distinct sequences.

However, certain combinations may be more effective than others in regulating mRNA localization and protein secretion.

To elucidate the syntax of SECReTE and identify optimal combinations, researchers created a library of ~5 x 10^4 variants and integrated them into the 3' untranslated region (3 UTR) of two reporter genes: green fluorescent protein (GFP) and immunoglobulin light chain. They expressed each variant in Chinese hamster ovary cells and evaluated their activity using parallel assays to gauge the impact on mRNA partitioning between the endoplasmic reticulum (ER) and cytoplasm, as well as protein secretion.

The findings suggest that ER-associated mRNAs are enriched with SECReTE elements exhibiting a defined syntax. Specifically, RRY repeats (with R being a purine) were found to be superior to NNY repeats.

Variants that demonstrated a high mRNA ER localization score consistently enhanced protein secretion, establishing a mechanistic link between mRNA targeting to the ER and secretion efficiency. The correlation between variant performances across both reporters indicates a shared regulatory mechanism. Furthermore, researchers identified a novel secretion-promoting cis element, termed "split NNS," featuring S as either G or C. This study refines the syntactic definition of mRNA localization motifs that govern protein secretion in both natural and heterologous expression systems.