You, S.-T. | Search bioRxiv

bioRxiv2026

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 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.

cell biology↗

bioRxiv2019

Experimental evolution reveals a general role for the methyltransferase Hmt1 in noise buffering

Cell-to-cell heterogeneity within an isogenic population has been observed in prokaryotic and eukaryotic cells. Such heterogeneity often manifests at the level of individual protein abundance and may have evolutionary benefits, especially for organisms in fluctuating environments. Although general features and the origins of cellular noise have been revealed, details of the molecular pathways underlying noise regulation remain elusive. Here, we used experimental evolution of Saccharomyces cerevisiae to select for mutations that increase reporter protein noise. By combining bulk segregant analysis and CRISPR/Cas9-based reconstitution, we identified the methyltransferase Hmt1 as a general regulator of noise buffering. Hmt1 methylation activity is critical for the evolved phenotype, and noise buffering is primarily achieved via two Hmt1 methylation targets. Hmt1 functions as an environmental sensor to adjust noise levels in response to environmental cues. Moreover, Hmt1-mediated noise buffering is conserved in an evolutionarily distant yeast species, suggesting broad significance of noise regulation.\n\nAuthor SummaryCell-to-cell heterogeneity within an isogenic population has been observed in prokaryotic and eukaryotic cells. Such heterogeneity often manifests at the level of individual protein abundance and may have evolutionary benefits, especially for organisms in fluctuating environments. Here, we used experimental evolution of Saccharomyces cerevisiae to select for mutations that increase reporter protein noise and identified the methyltransferase Hmt1 as a general regulator of noise buffering. Hmt1 is a central hub protein that is involved in multiple basic cellular pathways, including chromatin remodeling/transcription, translation, ribosome biogenesis, and post-transcriptional regulation. Our results show that Hmt1 constrains the noise level of multiple cellular pathways under normal conditions, so the physiology of individual cells in a population will not deviate too much from optimal peak fitness. However, when cells encounter environmental stresses, HMT1 is quickly down-regulated and expression noise is enhanced to increase the likelihood of population survival. Moreover, the noise buffering function of Hmt1 is conserved in Schizosaccharomyces pombe that diverged from the common ancestor of Saccharomyces cerevisiae more than 400 million years ago. Since the Hmt1 network is conserved from yeast cells to human, it is quite possible that Hmt1-mediated noise buffering also operates in multicellular organisms.

molecular biology↗