The transcript interactome of skeletal muscle RNA binding protein motif 3 (RBM3).

Post-transcriptional regulation of gene expression represents a critical regulatory step in the production of a functional proteome. Elevated expression of post-transcriptional regulator RNA binding motif protein 3 (RBM3), an RNA binding protein in the cold-shock family, is positively correlated with skeletal muscle growth in adult mice. However, mechanisms through which RBM3 exerts its effects are largely unknown. The purpose of this study was to perform RNA immunoprecipitation followed by RNA sequencing (RIP-seq) and apply a network science approach to understand biological processes (BPs) most associated with RBM3-bound mRNAs. In addition, through nucleotide-sequence-scanning of enriched transcripts, we predicted the motif for skeletal muscle RBM3 binding. Gene set enrichment analysis followed by enrichment mapping of RBM3-bound transcripts (fold change >3; p.adj <0.01) revealed significant enrichment of BPs associated with "Contractile apparatus," "Translation initiation," and "Proteosome complex." Clusters were driven largely by enrichment of Myh1 (FC: 4.43), Eif4b (FC: 5.03), and Trim63 (FC: 5.84), respectively. Motif scanning of enriched sequences revealed a discrete 14 nucleotide-wide motif found most prominently at the junction between the protein coding region's termination sequence and the start of the 3' untranslated region (UTR; E-Value: 1.1 e-015 ). Proof of concept investigation of motif location along enriched transcripts Myh1 and Myl4 revealed 3' UTR binding, suggesting RBM3 involvement in transcript half-life regulation. Together, these results demonstrate the potential influence of RBM3 in reshaping the skeletal muscle proteome through post-transcriptional regulation of mRNAs crucial to muscle adaptations.

The genetic and biochemical determinants of mRNA degradation rates in mammals.

BACKGROUND: Degradation rate is a fundamental aspect of mRNA metabolism, and the factors governing it remain poorly characterized. Understanding the genetic and biochemical determinants of mRNA half-life would enable more precise identification of variants that perturb gene expression through post-transcriptional gene regulatory mechanisms. RESULTS: We establish a compendium of 39 human and 27 mouse transcriptome-wide mRNA decay rate datasets. A meta-analysis of these data identified a prevalence of technical noise and measurement bias, induced partially by the underlying experimental strategy. Correcting for these biases allowed us to derive more precise, consensus measurements of half-life which exhibit enhanced consistency between species. We trained substantially improved statistical models based upon genetic and biochemical features to better predict half-life and characterize the factors molding it. Our state-of-the-art model, Saluki, is a hybrid convolutional and recurrent deep neural network which relies only upon an mRNA sequence annotated with coding frame and splice sites to predict half-life (r=0.77). The key novel principle learned by Saluki is that the spatial positioning of splice sites, codons, and RNA-binding motifs within an mRNA is strongly associated with mRNA half-life. Saluki predicts the impact of RNA sequences and genetic mutations therein on mRNA stability, in agreement with functional measurements derived from massively parallel reporter assays. CONCLUSIONS: Our work produces a more robust ground truth for transcriptome-wide mRNA half-lives in mammalian cells. Using these revised measurements, we trained Saluki, a model that is over 50% more accurate in predicting half-life from sequence than existing models. Saluki succinctly captures many of the known determinants of mRNA half-life and can be rapidly deployed to predict the functional consequences of arbitrary mutations in the transcriptome.

Cytidine-containing tails robustly enhance and prolong protein production of synthetic mRNA in cell and in vivo.

Synthetic mRNAs are rising rapidly as alternative therapeutic agents for delivery of proteins. However, the practical use of synthetic mRNAs has been restricted by their low cellular stability as well as poor protein production efficiency. The key roles of poly(A) tail on mRNA biology inspire us to explore the optimization of tail sequence to overcome the aforementioned limitations. Here, the systematic substitution of non-A nucleotides in the tails revealed that cytidine-containing tails can substantially enhance the protein production rate and duration of synthetic mRNAs both in vitro and in vivo. Such C-containing tails shield synthetic mRNAs from deadenylase CCR4-NOT transcription complex, as the catalytic CNOT proteins, especially CNOT6L and CNOT7, have lower efficiency in trimming of cytidine. Consistently, these enhancement effects of C-containing tails were observed on all synthetic mRNAs tested and were independent of transfection reagents and cell types. As the C-containing tails can be used along with other mRNA enhancement technologies to synergically boost protein production, we believe that these tails can be broadly used on synthetic mRNAs to directly promote their clinical applications.

Monitoring mRNA Half-Life in Arabidopsis Using Droplet Digital PCR.

mRNA decay is an important process in post-transcriptional regulation; in addition, it plays a crucial role in plant development and response to stress. The development of new tools to quantify mRNA decay intermediates is thus important to better characterize the dynamic of mRNA decay in various conditions. Here, we applied droplet digital PCR (ddPCR), a recent and precise PCR technology, to determine mRNA half-life in Arabidopsis seedlings. We demonstrated that ddPCR can correctly assess mRNA half-life from a wide variety of transcripts in a reproducible manner. We also demonstrated that thanks to multiplexing mRNA, the half-life of multiple transcripts can be followed in the same reaction. As ddPCR allows precise quantification, we proposed that this approach is highly suitable when a low amount of RNA is available; for the detection of many targets or for the analysis of lowly expressed transcripts.

Combinations of slow-translating codon clusters can increase mRNA half-life in Saccharomyces cerevisiae.

The presence of a single cluster of nonoptimal codons was found to decrease a transcript's half-life through the interaction of the ribosome-associated quality control machinery with stalled ribosomes in Saccharomyces cerevisiae The impact of multiple nonoptimal codon clusters on a transcript's half-life, however, is unknown. Using a kinetic model, we predict that inserting a second nonoptimal cluster near the 5' end can lead to synergistic effects that increase a messenger RNA's (mRNA's) half-life in S. cerevisiae Specifically, the 5' end cluster suppresses the formation of ribosome queues, reducing the interaction of ribosome-associated quality control factors with stalled ribosomes. We experimentally validate this prediction by introducing two nonoptimal clusters into three different genes and find that their mRNA half-life increases up to fourfold. The model also predicts that in the presence of two clusters, the cluster closest to the 5' end is the primary determinant of mRNA half-life. These results suggest the "translational ramp," in which nonoptimal codons are located near the start codon and increase translational efficiency, may have the additional biological benefit of allowing downstream slow-codon clusters to be present without decreasing mRNA half-life. These results indicate that codon usage bias plays a more nuanced role in controlling cellular protein levels than previously thought.

Dynamics of the canonical RNA degradosome components during glucose stress.

The RNA Degradosome (RNAD) is a multi-enzyme complex, which performs important functions in post-transcriptional regulation in Escherichia coli with the assistance of regulatory sRNAs and the RNA chaperone Hfq. Although the interaction of the canonical RNAD components with RNase E has been extensively studied, the dynamic nature of the interactions in vivo remains largely unknown. In this work, we explored the rearrangements upon glucose stress using fluorescence energy transfer (hetero-FRET). Results revealed differences in the proximity of the canonical components with 1% (55.5 mM) glucose concentration, with the helicase RhlB and the glycolytic enzyme Enolase exhibiting the largest changes to the C-terminus of RNase E, followed by PNPase. We quantified ptsG mRNA decay and SgrS sRNA synthesis as they mediate bacterial adaptation to glucose stress conditions. We propose that once the mRNA degradation is completed, the RhlB, Enolase and PNPase decrease their proximity to the C-terminus of RNase E. Based on the results, we present a model where the canonical components of the RNAD coalesce when the bacteria is under glucose-6-phosphate stress and associate it with RNA decay. Our results demonstrate that FRET is a helpful tool to study conformational rearrangements in enzymatic complexes in bacteria in vivo.

An integrative approach uncovers transcriptome-wide determinants of mRNA stability regulation in Saccharomyces cerevisiae.

mRNA degradation rate is one of the key stages of gene expression regulation in eukaryotic cells. To date, intertwined processes of post-transcriptional control have been widely investigated, but focused rather on the examination of mechanisms controlling stability of particular protein-coding transcripts. Currently, a wealth of information from structural, biochemical, and high-throughput studies makes it tempting to define general rules governing mRNA stability that could be considered as versatile and valid on a genome-wide scale. Basu et al. analyzed multiple experimental and computational data on Saccharomyces cerevisiae mRNA half-lives as well as on secondary structures and protein-binding sites within transcripts, and collated it with available structures of ribonucleases, that is, enzymes responsible for mRNA degradation. This approach allowed to conclude how particular mRNA features such as lengths of unstructured terminal or internal regions or sequestration into ribonucleoprotein complexes impact half-lives of protein-coding transcripts and to define genome-scale principles of mRNA stability control in yeast.

Widespread Protection of RNA Cleavage Sites by a Riboswitch Aptamer that Folds as a Compact Obstacle to Scanning by RNase E.

Riboswitches are thought generally to function by modulating transcription elongation or translation initiation. In rare instances, ligand binding to a riboswitch has been found to alter the rate of RNA degradation by directly stimulating or inhibiting nearby cleavage. Here, we show that guanidine-induced pseudoknot formation by the aptamer domain of a guanidine III riboswitch from Legionella pneumophila has a different effect, stabilizing mRNA by protecting distal cleavage sites en masse from ribonuclease attack. It does so by creating a coaxially base-paired obstacle that impedes scanning from a monophosphorylated 5' end to those sites by the regulatory endonuclease RNase E. Ligand binding by other riboswitch aptamers peripheral to the path traveled by RNase E does not inhibit distal cleavage. These findings reveal that a riboswitch aptamer can function independently of any overlapping expression platform to regulate gene expression by acting directly to prolong mRNA longevity in response to ligand binding.

Gene Regulatory Strategies that Decode the Duration of NFκB Dynamics Contribute to LPS- versus TNF-Specific Gene Expression.

Pathogen-derived lipopolysaccharide (LPS) and cytokine tumor necrosis factor (TNF) activate NFκB with distinct duration dynamics, but how immune response genes decode NFκB duration to produce stimulus-specific expression remains unclear. Here, detailed transcriptomic profiling of combinatorial and temporal control mutants identified 81 genes that depend on stimulus-specific NFκB duration for their stimulus-specificity. Combining quantitative experimentation with mathematical modeling, we found that for some genes a long mRNA half-life allowed effective decoding, but for many genes this was insufficient to account for the data; instead, we found that chromatin mechanisms, such as a slow transition rate between inactive and RelA-bound enhancer states, could also decode NFκB dynamics. Chromatin-mediated decoding is favored by genes acting as immune effectors (e.g., tissue remodelers and T cell recruiters) rather than immune regulators (e.g., signaling proteins and monocyte recruiters). Overall, our results delineate two gene regulatory strategies that decode stimulus-specific NFκB dynamics and determine distinct biological functions.

Identification of Novel Compounds Enhancing SR-BI mRNA Stability through High-Throughput Screening.

Atherosclerosis is the pathological basis of most cardiovascular diseases. Reverse cholesterol transport (RCT) is a main mechanism of cholesterol homeostasis and involves the direct transport of high-density lipoprotein (HDL) cholesteryl ester by selective cholesterol uptake. Hepatic scavenger receptor class B member 1 (SR-BI) overexpression can effectively promote RCT and reduce atherosclerosis. SR-BI may be an important target for prevention or treatment of atherosclerotic disease. In our study, we inserted human SR-BI mRNA 3' untranslated region (3'UTR) downstream of the luciferase reporter gene, to establish a high-throughput screening model based on stably transfected HepG2 cells and to screen small-molecule compounds that can significantly enhance the mRNA stability of the SR-BI gene. Through multiple screenings of 25 755 compounds, the top five active compounds that have similar structures were obtained, with a positive rate of 0.19%. The five positive compounds could enhance the SR-BI expression and uptake of DiI-HDL in the hepatocyte HepG2. E238B-63 could also effectively extend the half-life of SR-BI mRNA and enhance the SR-BI mRNA and protein level and the uptake of DiI-HDL in hepatocytes in a time-dependent and dose-dependent manner. The structure-activity relationship analysis showed that the structure N-(3-hydroxy-2-pyridyl) carboxamide is possibly the key pharmacophore of the active compound, providing reference for acquiring candidate compounds with better activity. The positive small molecular compounds obtained in this study might become new drug candidates or lead compounds for the treatment of cardiovascular diseases and contribute to the further study of the posttranscriptional regulation mechanism of the SR-BI gene.

Design and Evaluation of Synthetic Terminators for Regulating Mammalian Cell Transgene Expression.

Tuning heterologous gene expression in mammalian production hosts has predominantly relied upon engineering the promoter elements driving the transcription of the transgene. Moreover, most regulatory elements have borrowed genetic sequences from viral elements. Here, we generate a set of 10 rational and 30 synthetic terminators derived from nonviral elements and evaluate them in the HT1080 and HEK293 cell lines to demonstrate that they are comparable in terms of tuning gene expression/protein output to the viral SV40 element and often require less sequence footprint. The mode of action of these terminators is determined to be an increase in mRNA half-life. Furthermore, we demonstrate that constructs comprising completely nonviral regulatory elements ( i.e., promoters and terminators) can outperform commonly used, strong viral based elements by nearly 2-fold. Ultimately, this novel set of terminators expanded our genetic toolkit for engineering mammalian host cells.

Fast Translation within the First 45 Codons Decreases mRNA Stability and Increases Premature Transcription Termination in E. coli.

We show here that the specific use of fast or slowly translated codons in the early coding region of a gene may influence both the mRNA stability and premature transcription termination. We first inserted a pair of nearly identical 42-base-pair (bp)-long sequences into codon 3 of the Escherichia coli lacZ gene. The only difference between the two inserts was that the first base in one was moved to become the last base in the other, providing a difference in the reading frame, one of which had the biased codons typical for ribosomal protein genes and which previously was shown to be faster translated than average. This insert reduced the mRNA stability and increased premature transcription termination and together resulted in a hundred-fold difference in lacZ expression. We next generated lacZ variants with 7, 14 or 21 fast translated, ribosomal-type codons inserted into codon 13 of lacZ. This gave progressively more unstable mRNAs and also progressively increased transcription termination up to 90%. By modeling, based on estimates of the translation rate of individual codons, we can explain these observations by an increased susceptibility of the mRNA to degradation, determined by the length and degree of the early mRNA being uncovered by ribosomes. Thus, we suggest that the translation rate differences among the synonymous codons early in a gene enable a "velocity code" within the amino acid coding ability, where the translation rate differences encode the mRNA stability and the premature termination of the RNA polymerase.

Extensive Structural Differences of Closely Related 3' mRNA Isoforms: Links to Pab1 Binding and mRNA Stability.

Alternative polyadenylation generates numerous 3' mRNA isoforms that can vary in biological properties, such as stability and localization. We developed methods to obtain transcriptome-scale structural information and protein binding on individual 3' mRNA isoforms in vivo. Strikingly, near-identical mRNA isoforms can possess dramatically different structures throughout the 3' UTR. Analyses of identical mRNAs in different species or refolded in vitro indicate that structural differences in vivo are often due to trans-acting factors. The level of Pab1 binding to poly(A)-containing isoforms is surprisingly variable, and differences in Pab1 binding correlate with the extent of structural variation for closely spaced isoforms. A pattern encompassing single-strandedness near the 3' terminus, double-strandedness of the poly(A) tail, and low Pab1 binding is associated with mRNA stability. Thus, individual 3' mRNA isoforms can be remarkably different physical entities in vivo. Sequences responsible for isoform-specific structures, differential Pab1 binding, and mRNA stability are evolutionarily conserved, indicating biological function.

Measuring mRNA Decay in Budding Yeast Using Single Molecule FISH.

Cellular mRNA levels are determined by the rates of mRNA synthesis and mRNA decay. Typically, mRNA degradation kinetics are measured on a population of cells that are either chemically treated or genetically engineered to inhibit transcription. However, these manipulations can affect the mRNA decay process itself by inhibiting regulatory mechanisms that govern mRNA degradation, especially if they occur on short time-scales. Recently, single molecule fluorescent in situ hybridization (smFISH) approaches have been implemented to quantify mRNA decay rates in single, unperturbed cells. Here, we provide a step-by-step protocol that allows quantification of mRNA decay in single Saccharomyces cerevisiae using smFISH. Our approach relies on fluorescent labeling of single cytoplasmic mRNAs and nascent mRNAs found at active sites of transcription, coupled with mathematical modeling to derive mRNA half-lives. Commercially available, single-stranded smFISH DNA oligonucleotides (smFISH probes) are used to fluorescently label mRNAs followed by the quantification of cellular and nascent mRNAs using freely available spot detection algorithms. Our method enables quantification of mRNA decay of any mRNA in single, unperturbed yeast cells and can be implemented to quantify mRNA turnover in a variety of cell types as well as tissues.

mRNA Stability Assay Using transcription inhibition by Actinomycin D in Mouse Pluripotent Stem Cells.

Gene expression is regulated through multiple steps at both transcriptional and post-transcriptional levels. The net abundance of mature mRNA species in cells is determined by the balance between transcription and degradation. Thus, the regulation of mRNA stability is a key post-transcriptional event that can greatly affect the net level of mRNAs in cells. The mRNA stability within cells can be measured indirectly by analyzing the mRNA half-life following transcription inhibition, where changes in mRNA levels are assumed to reflect mRNA degradation. Determination of mRNA half-life as a measure of mRNA stability is useful in understanding gene expression changes and underlying mechanisms regulating the level of transcripts at different physiological conditions or developmental stages. The protocol described here presents the analysis of mRNA decay as a measure for determining mRNA stability after transcriptional inhibition with Actinomycin D treatment in control and SRSF3 depleted mouse induced pluripotent stem cells (iPSC).

Determinants and Regulation of Protein Turnover in Yeast.

Protein turnover maintains the recycling needs of the proteome, and its malfunction has been linked to aging and age-related diseases. However, not all proteins turnover equally, and the factors that contribute to accelerate or slow down turnover are mostly unknown. We measured turnover rates for 3,160 proteins in exponentially growing yeast and analyzed their dependence on physical, functional, and genetic properties. We found that functional characteristics, including protein localization, complex membership, and connectivity, have greater effect on turnover than sequence elements. We also found that protein turnover and mRNA turnover are correlated. Analysis under nutrient perturbation and osmotic stress revealed that protein turnover highly depends on cellular state and is faster when proteins are being actively used. Finally, stress-induced changes in protein and transcript abundance correlated with changes in protein turnover. This study provides a resource of protein turnover rates and principles to understand the recycling needs of the proteome under basal conditions and perturbation.

Cis-regulatory elements explain most of the mRNA stability variation across genes in yeast.

The stability of mRNA is one of the major determinants of gene expression. Although a wealth of sequence elements regulating mRNA stability has been described, their quantitative contributions to half-life are unknown. Here, we built a quantitative model for Saccharomyces cerevisiae based on functional mRNA sequence features that explains 59% of the half-life variation between genes and predicts half-life at a median relative error of 30%. The model revealed a new destabilizing 3' UTR motif, ATATTC, which we functionally validated. Codon usage proves to be the major determinant of mRNA stability. Nonetheless, single-nucleotide variations have the largest effect when occurring on 3' UTR motifs or upstream AUGs. Analyzing mRNA half-life data of 34 knockout strains showed that the effect of codon usage not only requires functional decapping and deadenylation, but also the 5'-to-3' exonuclease Xrn1, the nonsense-mediated decay genes, but not no-go decay. Altogether, this study quantitatively delineates the contributions of mRNA sequence features on stability in yeast, reveals their functional dependencies on degradation pathways, and allows accurate prediction of half-life from mRNA sequence.

An immediate-late gene expression module decodes ERK signal duration.

The RAF-MEK-ERK signalling pathway controls fundamental, often opposing cellular processes such as proliferation and apoptosis. Signal duration has been identified to play a decisive role in these cell fate decisions. However, it remains unclear how the different early and late responding gene expression modules can discriminate short and long signals. We obtained both protein phosphorylation and gene expression time course data from HEK293 cells carrying an inducible construct of the proto-oncogene RAF By mathematical modelling, we identified a new gene expression module of immediate-late genes (ILGs) distinct in gene expression dynamics and function. We find that mRNA longevity enables these ILGs to respond late and thus translate ERK signal duration into response amplitude. Despite their late response, their GC-rich promoter structure suggested and metabolic labelling with 4SU confirmed that transcription of ILGs is induced immediately. A comparative analysis shows that the principle of duration decoding is conserved in PC12 cells and MCF7 cells, two paradigm cell systems for ERK signal duration. Altogether, our findings suggest that ILGs function as a gene expression module to decode ERK signal duration.

A novel method for quantitative measurements of gene expression in single living cells.

Gene expression is at the heart of virtually any biological process, and its deregulation is at the source of numerous pathological conditions. While impressive progress has been made in genome-wide measurements of mRNA and protein expression levels, it is still challenging to obtain highly quantitative measurements in single living cells. Here we describe a novel approach based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse microscopy. Using luminescence microscopy, fluctuations of protein expression levels can be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. The integrated protein decay reporter allows measuring protein degradation rates in the absence of protein synthesis inhibitors, and in combination with absolute protein levels allows determining absolute amounts of proteins synthesized over the cell cycle. Finally, the internal tag can be excised by inducible expression of Cre recombinase, which enables to estimate endogenous mRNA half-lives. Our method thus opens new avenues in quantitative analysis of gene expression in single living cells.

Co-ordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat-binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.