Biology subjects

Gerst, J. E.

Publications and source records attributed to Gerst, J. E..

10 recordsLinked to original sources

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↗

bioRxiv2024

Complementation of a human disease phenotype in vitro by intercellular mRNA transfer

There is growing evidence that full-length mRNAs undergo intercellular transfer through long, thin cytoplasmic connections called tunneling nanotubes (TNTs), but whether transferred mRNAs are translated and effect cellular changes post-transfer is unknown. Using multiple lines of evidence, we show that transferred mRNAs undergo translation and can fully complement the phenotype of genetic mutations in vitro. For example, the human peroxisome biogenesis disorder, Zellweger Syndrome, results from mutations in genes like PEX5 and PEX6. We demonstrate that the co-culture of patient-derived PEX6 mutant fibroblasts or PEX5 knock-out cells with wild-type cells leads to de novo peroxisome biogenesis. Complementation occurs by TNT-mediated mRNA transfer and translation in acceptor cells and not by peroxisome transfer. We provide additional examples of genetic complementation via transfer of mRNAs encoding the HSF1 transcription factor or CRE recombinase. Our study provides evidence for the physiological significance of mRNA transfer and suggests a new approach for mRNA therapeutics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/622258v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@fe4563org.highwire.dtl.DTLVardef@17d6d3forg.highwire.dtl.DTLVardef@18f47f7org.highwire.dtl.DTLVardef@1bdae08_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Conservation of mRNA operon formation in the control of the heat shock response in mammalian cells

Prokaryotic organisms rely on polycistronic transcription (i.e. operons) to express multiple mRNAs from a single promoter, enabling rapid and robust responses to stimuli. In yeast, a similar mechanism exists whereby monocistronic mRNAs are selectively assembled into ribonucleoprotein particles, termed RNA operons or transperons, to regulate the expression of genes involved in the same biological process. One example is the heat shock protein (HSP) transperon that confers the eukaryotic heat shock response (HSR), a conserved cellular mechanism that enables organisms to cope with proteotoxic stress. As it was unknown whether transperons exist in higher eukaryotes, we examined whether a similar mechanism operates in mammalian cells. Using both single-molecule fluorescent in situ hybridization and RNA pulldown techniques, we show that mammalian HSP mRNAs colocalize and multiplex to form mRNA assemblages during heat stress. These RNP assemblies are dependent on heat shock factor 1 transcriptional regulator and involve both inter- and intrachromosomal interactions among the HSP genes. Bioinformatic analysis identifies a conserved sequence motif within the coding regions of HSP mRNAs and mutational studies in yeast suggest that it is critical for mRNA multiplexing and the HSR. These findings emphasize the evolutionarily conserved nature of heat shock gene regulation across species and suggest that mammalian cells employ RNA operons/transperons in organizing their heat shock response.

cell biology↗

bioRxiv2024

EASI-ORC: A Pipeline for the Efficient Analysis and Segmentation of smFISH Images for Organelle-RNA Colocalization Measurements in Yeast

Analysis of single-molecule fluorescent in situ hybridization (smFISH) images is an arduous and time-consuming task that is important to perform accurately in order to translate image data into a quantifiable format. This task becomes increasingly more difficult greater the experimental scope and number of images captured. Although smFISH is the gold standard for RNA localization measurements, there are no freely available, user-friendly applications for assaying messenger RNA localization to sub-cellular structures, like the endoplasmic reticulum (ER) or mitochondria). We have developed a pipeline that allows for the automated analysis of multiple smFISH images in yeast cells: EASI-ORC (Efficient Analysis and Segmentation of smFISH Images for Organelle-RNA Colocalization). The EASI-ORC pipeline automates the segmentation of cells and sub-cellular structures (e.g. organelles), identifies bona fide smFISH signals, and measures the level of colocalization between an organelle and mRNA signals. Importantly, EASI-ORC works in a fast, accurate, and unbiased manner that is difficult to replicate manually. It also allows for the visualization of data filtering and outputs graphical representations of the colocalization data along with statistical analysis. EASI-ORC is based on existing ImageJ plugins and original scripts, thus, allowing free access and a relative ease of use. To circumvent any technical literacy issues, a step-by-step user guide is provided. EASI-ORC offers a robust solution for smFISH image analysis for both new and experienced researchers - one that saves time and effort, as well as providing more consistent overall measurements of RNA-organelle colocalization in yeast.

cell biology↗

bioRxiv2024

The 3'-untranslated regions of yeast ribosomal protein mRNAs determine paralog incorporation into ribosomes and recruit factors necessary for specialized functions

Ribosome heterogeneity arises via the differential incorporation of ribosomal protein (RP) paralogs, post-transcriptionally modified rRNA, post-translationally modified RPs, and ribosome-associated proteins (RAPs) into ribosomes. This has led to the hypothesis that heterogeneous or "specialized" ribosomes, which translate specific mRNA subsets, confer key roles in cell growth and development. While proven examples of functional ribosome heterogeneity in eukaryotes exist, there is no comprehensive analysis of specialized ribosome formation. We employed yeast RP paralog deletion libraries and high-throughput screening to investigate the functional specificity and redundancy between paralogs under various growth conditions. Composition and translatome analyses verified paralog specificity in the assembly and function of ribosomes specialized for growth on different carbon sources, and identified a novel RAP required for the efficient translation of peroxisomal proteins. Importantly, we also show that the mechanism by which specific RP paralogs incorporate into ribosomes requires their unique 3-untranslated regions to yield ribosomes that differ in composition and function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/585503v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@15ba935org.highwire.dtl.DTLVardef@c210b7org.highwire.dtl.DTLVardef@d77b7dorg.highwire.dtl.DTLVardef@ec72f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Global analysis of human-to-mouse intercellular RNA transfer in cell culture

Full-length mRNAs can transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the transferome) is unknown. Here, we analyzed whole transcriptome mRNA and lncRNA transfer between heterogeneous human-mouse cell populations in in vitro co-culture using RNA-sequencing. Our data indicate that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells (MCF7). These results were validated by both quantitative RT-PCR and in situ hybridization, and analysis shows that typically <1% of endogenous mRNAs and lncRNAs undergo transfer. Non-selective expression-dependent RNA transfer was further validated using synthetic RNA reporters. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/470233v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@13a7176org.highwire.dtl.DTLVardef@1ef1c75org.highwire.dtl.DTLVardef@1355eeforg.highwire.dtl.DTLVardef@ebc275_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Translational control as a novel regulator of gradient sensing and chemotropism in yeast

The yeast mating pathway regulates haploid cell fusion in response to pheromone signaling via a mitogen-activated protein kinase (MAPK) cascade that controls directional growth (chemotropism). However, the regulators of chemotropic morphogenesis are ill-defined. By using a non-biased genome-wide screen, we identified hundreds of genes that affect mating. An additional screens identified and validated >20 novel positive and negative regulators of pheromone gradient sensing, chemotropism, shmoo development, and mating. Aside from known regulators of exocytosis and endocytosis, genes involved in translational control downstream of the G-protein-regulated pheromone and filamentous growth MAPK pathways were identified. These include the Scp160 RNA-binding protein and the Asc1, Rpl12b, and Rpl19b ribosomal proteins (RPs). Importantly, we demonstrate that pheromone treatment and G (Gpa1) activation stimulate Scp160 binding to (and inhibition of) Asc1, which acts downstream of glucose-activated G (Gpa2) on the filamentous growth pathway. Moreover, we identify both Rpl12b and Rpl19b as RP paralog-specific positive regulators of translation of mating components, including Scp160. Thus, opposing MAPK pathways may converge at the level of translational control to regulate signaling output. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/422562v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@3944e1org.highwire.dtl.DTLVardef@1d9204eorg.highwire.dtl.DTLVardef@1d7e902org.highwire.dtl.DTLVardef@2bfc6_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Multiplexed mRNA assembly into ribonucleoprotein particles plays an operon-like role in the control of yeast cell physiology

Prokaryotes utilize polycistronic messages (operons) to co-translate proteins involved in the same biological process. Whether eukaryotes achieve similar regulation by selectively assembling monocistronic messages derived from different chromosomes is unclear. We employed transcript-specific RNA pulldowns and RNA-seq/RT-PCR to identify mRNAs that co-precipitate into ribonucleoprotein (RNP) particles in yeast. Consistent with the hypothesis of eukaryotic RNA operons, mRNAs encoding components of the mating pathway, heat shock proteins, and mitochondrial outer membrane proteins multiplex in trans to form discrete mRNP particles, termed transperons. Chromatin-capture experiments reveal that genes encoding multiplexed mRNAs physically interact, thus, RNA assembly may result from co-regulated gene expression. Transperon assembly and function depends upon H4 histones and their depletion leads to defects in RNA multiplexing, resulting in decreased pheromone responsiveness and mating, and increased heat shock sensitivity. We propose that intergenic associations and non-canonical H4 histone functions contribute to transperon formation in eukaryotic cells to regulate cell physiology. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cell biology↗

bioRxiv2020

Identification and enrichment of SECReTE cis-acting RNA elements in the Coronaviridae and other (+) single-strand RNA viruses

cis-acting RNA motifs play a major role in regulating many aspects of RNA biology including posttranscriptional processing, nuclear export, RNA localization, translation and degradation. Here we analyzed the genomes of SARS-CoV-2 and other single-strand RNA (ssRNA) viruses for the presence of a unique cis RNA element called SECReTE. This motif consists of 10 or more consecutive triplet nucleotide repeats where a pyrimidine nucleotide (C or U) in present every third base, and which we identified in mRNAs encoding secreted proteins in bacteria, yeast, and humans. This motif facilitates mRNA localization to the endoplasmic reticulum (ER), along with the enhanced translation and secretion of translated protein. We now examined for SECReTE presence in Group IV and V RNA viruses, the former including the Coronaviridae, like SARS-CoV-2 and other positive (+)ssRNA viruses, and the latter consisting of negative (-) ssRNA viruses. Interestingly, the SARS-CoV-2 genome contains 40 SECReTE motifs at an abundance of ~1.3 SECReTEs/kilobase (kb). Moreover, all ssRNA viruses we examined contain multiple copies of this motif and appears in (+)ssRNA viruses as non-random in occurrence and independent of genome length. Importantly, (+)ssRNA viruses (e.g. Coronaviruses and Hepaciviruses), which utilize ER membranes to create double membrane vesicles to serve as viral replication centers (VRCs), contain more SECReTE motifs per kb as compared to (-)ssRNA viruses (e.g. Rabies, Mumps, and Influenza), that replicate in the nucleus or the cytoplasm, or other (+)ssRNA viruses (e.g. Enteroviruses and Flaviviruses) which employ different organellar membranes. As predicted by our earlier work, SECReTE sequences are mostly found in membranal or ER-associated/secreted proteins. Thus, we propose that SECReTE motifs could be important for the efficient translation and secretion of secreted viral proteins, as well as for VRC formation. Future studies of SECReTE function and identification of SECReTE-binding proteins could provide new drug targets to treat COVID-19 and other (+)ssRNA related diseases.

bioinformatics↗

bioRxiv2017

Intercellular mRNA Trafficking Via Membrane Nanotubes In Mammalian Cells

RNAs have been shown to undergo transfer between mammalian cells, though the mechanism behind this phenomenon and its overall importance to cell physiology is not well understood. Numerous publications have suggested that RNAs (microRNAs and incomplete mRNAs) undergo transfer via extracellular vesicles (e.g. exosomes). However, in contrast to a diffusion-based transfer mechanism, we find that full-length mRNAs undergo direct cell-cell transfer via cytoplasmic extensions, called membrane nanotubes (mNTs), which connect donor and acceptor cells. By employing a simple co-culture experimental model and using single-molecule imaging, we provide quantitative data showing that mRNAs are transferred between cells in contact. Examples of mRNAs that undergo transfer include those encoding GFP, mouse {beta}-actin, and human Cyclin D1, BRCA1, MT2A, and HER2. We show that intercellular mRNA transfer occurs in all co-culture models tested (e.g. between primary cells, immortalized cells, and in co-cultures of immortalized human and murine cells). Rapid mRNA transfer is dependent upon actin, but independent of de novo protein synthesis, and is modulated by stress conditions and gene expression levels. Hence, this work supports the hypothesis that full-length mRNAs undergo transfer between cells through a refined structural connection. Importantly, unlike the transfer of miRNA or RNA fragments, this process of communication transfers genetic information that could potentially alter the acceptor cell proteome. This phenomenon may prove important for the proper development and functioning of tissues, as well as host-parasite or symbiotic interactions.\n\nSignificanceMessenger RNA (mRNA) molecules convey genetic information within cells, beginning from genes in the nucleus to ribosomes in the cell body, where they are translated into proteins. Here, we show a novel mode of transferring genetic information from one cell to another. Contrary to previous publications suggesting that mRNAs transfer via extracellular vesicles, we provide visual and quantitative data showing that mRNAs transfer via membrane nanotubes and direct cell-to-cell contact. We predict that this process has a major role in regulating local cellular environments with respect to tissue development and maintenance, cellular responses to stress, interactions with parasites, tissue transplants, and the tumor microenvironment.\n\nAuthor contributionsG.H., A.R. and R.H.S. conceived the research and designed the experiments; C.M.E. performed and analyzed the experiments with WM983b+/-GFP, including transwell and exosomes; M.C.D. and E.E. performed and analyzed the WM983b/NIH393 co-culture experiments; G.H. performed and analyzed all other experiments; and G.H., J.E.G, A.R. and R.H.S. wrote the paper.

cell biology↗