Protein Translation Efficiency Research Articles

Influence of 5′-UTR nucleotide composition on translation efficiency in Escherichiacoli

Translation initiation for 5′-UTR contributes primarily to the efficient protein expression in Escherichia coli. Many studies have focused on constructing random 5′-UTR libraries to investigate the impact of mRNA features on protein translation efficiency. However, the study on the effect of the absence of specific types of nucleotides in the entire 5′-UTR region on translation efficiency has not yet been reported. Here, we constructed four reporter plasmid libraries encoding the sfGFP fluorescent protein, each preceded by 5′-UTRs that lack one specific nucleotide (25B, 25D, 25H, 25V). Each library was transformed into E. coli cells, and the fluorescence distribution among the different libraries was analyzed by flow cytometer. Additionally, we quantified the activity of 256 unique 5′-UTR sequences and analyzed the impact of the corresponding mRNA sequence features on translation efficiency. We found that the 25D library, which lacks the C nucleotide, exhibited the highest overall translation efficiency compared to the other three libraries. Moreover, the minimum free energy and 16S rRNA hybridization energy of the 5′-UTR sequence could work coordinately to influence translation efficiency. The 5′-UTR sequences lacking the C nucleotide also achieve efficient protein translation. These findings may provide several guiding principles for precisely tuning protein expression.

Integrated stress response mediates HSP70 to inhibit testosterone synthesis in aging testicular Leydig cells

The integrated stress response (ISR) is implicated in age-related diseases, while the molecular chaperone heat shock protein 70 (HSP70) can facilitate proper protein folding. However, the regulatory mechanism of ISR in insufficient testosterone synthesis of aging Leydig cells (LCs) remains unclear. This study aims to elucidate the regulatory role of ISR in inadequate testosterone synthesis of aging LCs. We observed a positive correlation between testosterone and HSP70 levels, which were found to be decreased in elderly men. ISR was detected in testicular tissue from old mice. The expression of testosterone synthesis related protein and the content of testosterone decreased in testicular tissue of old mice. Conversely, inhibition of the integrated stress response in testicular tissue led to an increase in steroid synthase expression among old mice. Furthermore, inhibiting ISR specifically within aging LCs resulted in enhanced protein translation efficiency and increased expression levels of new HSP70 and steroidogenic acute regulatory protein (StAR). These findings suggest that ISR occurrence within aging LCs affects StAR protein expression through regulation of HSP70-mediated translation, consequently impairing testosterone synthesis.

TRNA lysidinylation is essential for the minimal translation system found in the apicoplast of Plasmodium falciparum.

For decades, researchers have sought to define minimal genomes to elucidate the fundamental principles of life and advance biotechnology. tRNAs, essential components of this machinery, decode mRNA codons into amino acids. The apicoplast of malaria parasites encodes 25 tRNA isotypes in its organellar genome - the lowest number found in known translation systems. Efficient translation in such minimal systems depends heavily on post-transcriptional tRNA modifications, especially at the wobble anticodon position. Lysidine modification at the wobble position (C34) of tRNACAU distinguishes between methionine (AUG) and isoleucine (AUA) codons, altering the amino acid delivered by this tRNA and ensuring accurate protein synthesis. Lysidine is formed by the enzyme tRNA isoleucine lysidine synthetase (TilS) and is nearly ubiquitous in bacteria and essential for cellular viability. We identified a TilS ortholog (PfTilS) located in the apicoplast of Plasmodium falciparum parasites. By complementing PfTilS with a bacterial ortholog, we demonstrated that the lysidinylation activity of PfTilS is critical for parasite survival and apicoplast maintenance, likely due to its impact on apicoplast protein translation. Our findings represent the first characterization of TilS in an endosymbiotic organelle, advancing eukaryotic organelle research and our understanding of minimal translational machinery. Due to the absence of lysidine modifications in humans, this research also exposes a potential vulnerability in malaria parasites that could be targeted by antimalarial strategies.

RNase-mediated reprogramming of Yersinia virulence.

RNA degradation is an essential process that allows bacteria to regulate gene expression and has emerged as an important mechanism for controlling virulence. However, the individual contributions of RNases in this process are mostly unknown. Here, we tested the influence of 11 potential RNases in the intestinal pathogen Yersinia pseudotuberculosis on the expression of its type III secretion system (T3SS) and associated effectors (Yops) that are encoded on the Yersinia virulence plasmid. We found that exoribonuclease PNPase and endoribonuclease RNase III inhibit T3SS and yop gene transcription by repressing the synthesis of LcrF, the master activator of Yop-T3SS. Loss of both RNases led to an increase in lcrF mRNA levels. Our work indicates that PNPase exerts its influence via YopD, which accelerates lcrF mRNA degradation. Loss of RNase III, on the other hand, results in the downregulation of the CsrB and CsrC RNAs, thereby increasing the availability of active CsrA, which has been shown previously to enhance lcrF mRNA translation and stability. This CsrA-promoted increase of lcrF mRNA translation could be supported by other factors promoting the protein translation efficiency (e.g. IF-3, RimM, RsmG) that were also found to be repressed by RNase III. Transcriptomic profiling further revealed that Ysc-T3SS-mediated Yop secretion leads to global reprogramming of the Yersinia transcriptome with a massive shift of the expression from chromosomal to virulence plasmid-encoded genes. A similar reprogramming was also observed in the RNase III-deficient mutant under non-secretion conditions. Overall, our work revealed a complex control system where RNases orchestrate the expression of the T3SS/Yop machinery on multiple levels to antagonize phagocytic uptake and elimination by innate immune cells.

Time-Resolved Inspection of Ionizable Lipid-Facilitated Lipid Nanoparticle Disintegration and Cargo Release at an Early Endosomal Membrane Mimic.

Advances in lipid nanoparticle (LNP) design have contributed notably to the emergence of the current clinically approved mRNA-based vaccines and are of high relevance for delivering mRNA to combat diseases where therapeutic alternatives are sparse. LNP-assisted mRNA delivery utilizes ionizable lipid-mediated cargo translocation across the endosomal membrane driven by the acidification of the endosomal environment. However, this process occurs at a low efficiency, a few percent at the best. Utilizing surface-sensitive fluorescence microscopy with a single LNP and mRNA resolution, we have investigated pH-controlled interactions between individual LNPs and a planar anionic supported lipid bilayer (SLB) formed on nanoporous silica, mimicking the electrostatic conditions of the early endosomal membrane. For LNPs with an average diameter of 140 nm, fusion with the anionic SLB preferentially occurred when the pH was reduced from 6.6 to 6.0. Furthermore, there was a delay in the onset of LNP fusion after the pH drop, and upon fusion, a significant fraction (>70%) of mRNA was released into the acidic solution representing the endosomal lumen, while a fraction of mRNA remained bound to the SLB even after reversing the pH to neutral cytosolic conditions. Finally, a comparison of the fusion efficiency of two LNP formulations with different surface concentrations of gel-forming lipids correlated with differences in the protein translation efficiency previously observed in human primary cell transfection studies. Together, these findings emphasize the relevance of biophysical investigations of ionizable lipid-containing LNP-assisted mRNA delivery mechanisms while potentially also offering means to optimize the design of LNPs with enhanced endosomal escape capabilities.

N6-Methyladenosine enhances the translation of ENO1 to promote the progression of bladder cancer by inhibiting PCNA ubiquitination

The mechanism underlying N6-methyladenosine (m6A) modification in bladder cancer (BC) remains elusive. We identified that the RBM15/METTL3 complex enhances m6A modification and promotes the ENO1 protein translation efficiency through its 359A site by depending on YTHDF1 in BC cells. In the tumor microenvironment, TGF-β effectively stimulates RBM15/METTL3 expression to improve ENO1 mRNA m6A modification through the Smad2/3 pathway. Reduced ENO1 m6A levels hamper tumor proliferation both in vitro and in vivo. Mechanistically, ENO1 augments PCNA protein stability by reducing its K48-linked ubiquitination and thus prevents protein degradation through the endoplasmic reticulum-associated degradation pathway. According to the subsequent experiments, the ENO1 inhibitor significantly reduced tumor proliferation both in vitro and in vivo. Our study highlights the significance of RBM15/METTL3 complex-mediated ENO1 mRNA m6A modification in ENO1 expression. It also reveals a novel mechanism by which ENO1 promotes BC progression, thereby suggesting that ENO1 can be a therapeutic target for BC.

A role for the C. elegans Argonaute protein CSR-1 in small nuclear RNA 3' processing.

The Integrator is a multi-subunit protein complex that catalyzes the maturation of snRNA transcripts via 3' cleavage, a step required for snRNA incorporation with snRNP for spliceosome biogenesis. Here we developed a GFP based in vivo snRNA misprocessing reporter as a readout of Integrator function and performed a genome-wide RNAi screen for Integrator regulators. We found that loss of the Argonaute encoding csr-1 gene resulted in widespread 3' misprocessing of snRNA transcripts that is accompanied by a significant increase in alternative splicing. Loss of the csr-1 gene down-regulates the germline expression of Integrator subunits 4 and 6 and is accompanied by a reduced protein translation efficiency of multiple Integrator catalytic and non-catalytic subunits. Through isoform and motif mutant analysis, we determined that CSR-1's effect on snRNA processing is dependent on its catalytic slicer activity but does not involve the CSR-1a isoform. Moreover, mRNA-sequencing revealed high similarity in the transcriptome profile between csr-1 and Integrator subunit knockdown via RNAi. Together, our findings reveal CSR-1 as a new regulator of the Integrator complex and implicate a novel role of this Argonaute protein in snRNA 3' processing.

Identification of an N-terminal tag (580N) that improves the biosynthesis of fluorescent proteins in Francisella tularensis and other Gram-negative bacteria

Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFPFt that had been codon-optimized for F. tularensis, yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.

Synaptopathy: presynaptic convergence in frontotemporal dementia and amyotrophic lateral sclerosis.

Frontotemporal dementia and amyotrophic lateral sclerosis are common forms of neurodegenerative disease that share overlapping genetics and pathologies. Crucially, no significantly disease-modifying treatments are available for either disease. Identifying the earliest changes that initiate neuronal dysfunction is important for designing effective intervention therapeutics. The genes mutated in genetic forms of frontotemporal dementia and amyotrophic lateral sclerosis have diverse cellular functions, and multiple disease mechanisms have been proposed for both. Identification of a convergent disease mechanism in frontotemporal dementia and amyotrophic lateral sclerosis would focus research for a targetable pathway, which could potentially effectively treat all forms of frontotemporal dementia and amyotrophic lateral sclerosis (both familial and sporadic). Synaptopathies are diseases resulting from physiological dysfunction of synapses, and define the earliest stages in multiple neuronal diseases, with synapse loss a key feature in dementia. At the presynapse, the process of synaptic vesicle recruitment, fusion and recycling is necessary for activity-dependent neurotransmitter release. The unique distal location of the presynaptic terminal means the tight spatio-temporal control of presynaptic homeostasis is dependent on efficient local protein translation and degradation. Recently, numerous publications have shown that mutations associated with frontotemporal dementia and amyotrophic lateral sclerosis present with synaptopathy characterized by presynaptic dysfunction. This review will describe the complex local signalling and membrane trafficking events that occur at the presynapse to facilitate neurotransmission and will summarize recent publications linking frontotemporal dementia/amyotrophic lateral sclerosis genetic mutations to presynaptic function. This evidence indicates that presynaptic synaptopathy is an early and convergent event in frontotemporal dementia and amyotrophic lateral sclerosis and illustrates the need for further research in this area, to identify potential therapeutic targets with the ability to impact this convergent pathomechanism.

M6A modification regulates tumor suppressor DIRAS1 expression in cervical cancer cells

ABSTRACT DIRAS family GTPase 1 (DIRAS1) has been reported as a potential tumor suppressor in other human cancer. However, its expression pattern and role in cervical cancer remain unknown. Knockdown of DIRAS1 significantly promoted the proliferation, growth, migration, and invasion of C33A and SiHa cells cultured in vitro. Overexpression of DIRAS1 significantly inhibited the viability and motility of C33A and SiHa cells. Compared with normal cervical tissues, DIRAS1 mRNA levels were significantly lower in cervical cancer tissues. DIRAS1 protein expression was also significantly reduced in cervical cancer tissues compared with para-cancerous tissues. In addition, DIRAS1 expression level in tumor tissues was significantly negatively correlated with the pathological grades of cervical cancer patients. DNA methylation inhibitor (5-Azacytidine) and histone deacetylation inhibitor (SAHA) resulted in a significant increase in DIRAS1 mRNA levels in C33A and SiHa cells, but did not affect DIRAS1 protein levels. FTO inhibitor (FB23–2) significantly down-regulated intracellular DIRAS1 mRNA levels, but significantly up-regulated DIRAS1 protein levels. Moreover, the down-regulation of METTL3 and METTL14 expression significantly inhibited DIRAS1 protein expression, whereas the down-regulation of FTO and ALKBH5 expression significantly increased DIRAS1 protein expression. In conclusion, DIRAS1 exerts a significant anti-oncogenic function and its expression is significantly downregulated in cervical cancer cells. The m6A modification may be a key mechanism to regulate DIRAS1 mRNA stability and protein translation efficiency in cervical cancer.

Profile analysis of N4-acetylcytidine (ac4C) on mRNA of human lung adenocarcinoma and paired adjacent non-tumor tissues

BackgroundRNA modification, a major component of post-transcriptional modification, plays an essential role in tumor initiation and progression. N4-acetylcytidine (ac4C) present in different species as a highly conserved RNA modification. ac4C on mRNA increases the stability of mRNA and the efficiency of protein translation. However, the mRNA profiling of ac4C in lung adenocarcinoma (LUAD) is unknown. MethodsNAT10 expression was tested using immunohistochemistry in tissue microarray (TMA). The ac4C peaks on mRNA were identified through acetylated RNA immunoprecipitation sequencing in both human LUAD tissues and adjacent non-tumor tissues, and differences of acetylation and mRNA between the two groups were analyzed. Furthermore, the function of AC4C-specific acetylated transcripts was analyzed bioinformatically. And a ac-RIP-PCR was used to verify the ac4C acetylation sites of TFAP2A. ResultsThe expression of acetylated key enzyme NAT10 was obviously increased in LUAD group. Then we found noticeable differences in ac4C mRNA modification between LUAD and adjacent non-tumor tissues. In addition, bioinformatics analysis showed that the distinctive distribution pattern of mRNA ac4C in LUAD affects a variety of cellular functions, such as protein sumoylation and transmembrane transporter activity. Importantly, we verified the ac4C level of TFAP2A was up-regulated in LUAD. ConclusionsOur study revealed that the degree of ac4C in mRNA in LUAD was significantly higher than in adjacent tissues and was concentrated mainly in the coding sequences with a implications in a wide range of cellular functions. The ac4C may become a new molecular marker and treatment target for lung cancer.

Investigations of Single-Subunit tRNA Methyltransferases from Yeast.

tRNA methylations, including base modification and 2'-O-methylation of ribose moiety, play critical roles in the structural stabilization of tRNAs and the fidelity and efficiency of protein translation. These modifications are catalyzed by tRNA methyltransferases (TRMs). Some of the TRMs from yeast can fully function only by a single subunit. In this study, after performing the primary bioinformatic analyses, the progress of the studies of yeast single-subunit TRMs, as well as the studies of their homologues from yeast and other types of eukaryotes and the corresponding TRMs from other types of organisms was systematically reviewed, which will facilitate the understanding of the evolutionary origin of functional diversity of eukaryotic single-subunit TRM.

Length-Dependent Translation Efficiency of ER-Destined Proteins.

Gene expression is a fundamental process that enables cells to produce specific proteins in a timely and spatially dependent manner. In eukaryotic cells, the complex organization of the cell body requires precise control of protein synthesis and localization. Certain mRNAs encode proteins with an N-terminal signal sequences that direct the translation apparatus toward a specific organelle. Here, we focus on the mechanisms governing the translation of mRNAs, which encode proteins with an endoplasmic reticulum (ER) signal in human cells. The binding of a signal-recognition particle (SRP) to the translation machinery halts protein synthesis until the mRNA-ribosome complex reaches the ER membrane. The commonly accepted model suggests that mRNA that encodes a protein that contains an ER signal peptide continuously repeats the cycle of SRP binding followed by association and dissociation with the ER. In contrast to the current view, we show that the long mRNAs remain on the ER while being translated. On the other hand, due to low ribosome occupancy, the short mRNAs continue the cycle, always facing a translation pause. Ultimately, this leads to a significant drop in the translation efficiency of small, ER-targeted proteins. The proposed mechanism advances our understanding of selective protein synthesis in eukaryotic cells and provides new avenues to enhance protein production in biotechnological settings.

ALKBH4 is a novel enzyme that promotes translation through modified uridine regulation

Epitranscriptomics studies the mechanisms of acquired RNA modifications. The epitranscriptome is dynamically regulated by specific enzymatic reactions, and the proper execution of these enzymatic RNA modifications regulates a variety of physiological RNA functions. However, the lack of experimental tools, such as antibodies for RNA modification, limits the development of epitranscriptomic research. Furthermore, the regulatory enzymes of many RNA modifications have not yet been identified. Herein, we aimed to identify new molecular mechanisms involved in RNA modification by focusing on the ALKBH family molecules, a family of RNA demethylases. We demonstrated that ALKBH4 interacts with small RNA, regulating the formation and metabolism of the (R)-5-carboxyhydroxymethyl uridine methyl ester. We also found that the reaction of ALKBH4 with small RNA enhances protein translation efficiency in an in vitro assay system. These findings indicate that ALKBH4 is involved in the regulation of uridine modification and expand on the role of tRNA-mediated translation control through ALKBH4.

Mettl13 protects against cardiac contractile dysfunction by negatively regulating C-Cbl-mediated ubiquitination of SERCA2a in ischemic heart failure.

Ischemic heart failure (HF) remains a leading cause of morbidity and mortality. Maintaining homeostasis of cardiac function and preventing cardiac remodeling deterioration are critical to halting HF progression. Methyltransferase-like protein 13 (Mettl13) has been shown to regulate protein translation efficiency by acting as a protein lysine methyltransferase, but its role in cardiac pathology remains unexplored. This study aims to characterize the roles and mechanisms of Mettl13 in cardiac contractile function and HF. We found that Mettl13 was downregulated in the failing hearts of mice post-myocardial infarction (MI) and in a cellular model of oxidative stress. Cardiomyocyte-specific overexpression of Mettl13 mediated by AAV9-Mettl13 attenuated cardiac contractile dysfunction and fibrosis in response to MI, while silencing of Mettl13 impaired cardiac function in normal mice. Moreover, Mettl13 overexpression abrogated the reduction in cell shortening, Ca2+ transient amplitude and SERCA2a protein levels in the cardiomyocytes of adult mice with MI. Conversely, knockdown of Mettl13 impaired the contractility of cardiomyocytes, and decreased Ca2+ transient amplitude and SERCA2a protein expression in vivo and in vitro. Mechanistically, Mettl13 impaired the stability of c-Cbl by inducing lysine methylation of c-Cbl, which in turn inhibited ubiquitination-dependent degradation of SERCA2a. Furthermore, the inhibitory effects of knocking down Mettl13 on SERCA2a protein expression and Ca2+ transients were partially rescued by silencing c-Cbl in H2O2-treated cardiomyocytes. In conclusion, our study uncovers a novel mechanism that involves the Mettl13/c-Cbl/SERCA2a axis in regulating cardiac contractile function and remodeling, and identifies Mettl13 as a novel therapeutic target for ischemic HF.

Ac4C acetylation regulates mRNA stability and translation efficiency in osteosarcoma

ObjectiveN4-acetylcytidine (ac4C) acetylation can promote target gene expression through improved mRNA stability. To explore the role of ac4C acetylation in osteosarcoma, U2OS and MG63 cell lines were treated with the N-acetyltransferase 10 (NAT10) inhibitor Remodelin. Reverse transcription–polymerase chain reaction (RT-PCR) and Western blot were used to test the gene and protein expression efficiency. MethodsThe proliferation rate of osteosarcoma cells was measured by a cell counting kit-8 (CCK8) assay. The cell cycle and apoptosis were analyzed by flow cytometry. The invasiveness of osteosarcoma cells was detected by a transwell invasion assay. The ac4C acetylation of target genes was screened by acetylated RNA immunoprecipitation and sequencing (acRIP-seq). ResultsWe found that when osteosarcoma cells were treated with Remodelin at the optimal concentration, their NAT10 expression and the cell proliferation was inhibited, the cells in the G1 phase increased (P < 0.05) but those in the S phase decreased, the apoptotic cells in the early and late stages increased, and the cells invasiveness decreased (P < 0.05). ConclusionsThe farnesyltransferase subunit beta gene (FNTB) was identified by acRIP-seq as one of the target genes of ac4C acetylation and was further verified by RT-PCR and Western blot analyses. Remodelin was demonstrated to reduce the stability and protein translation efficiency of target gene mRNA in osteosarcoma cells. In conclusion, inhibition of ac4C acetylation in osteosarcoma can block proliferation and metastasis as well as promote apoptosis and cell cycle arrest. Ac4C acetylation contributes to the stability and protein translation efficiency of the downstream target gene mRNA.

Translation initiation consistency between in vivo and in vitro bacterial protein expression systems.

Strict on-demand control of protein synthesis is a crucial aspect of synthetic biology. The 5'-terminal untranslated region (5'-UTR) is an essential bacterial genetic element that can be designed for the regulation of translation initiation. However, there is insufficient systematical data on the consistency of 5'-UTR function among various bacterial cells and in vitro protein synthesis systems, which is crucial for the standardization and modularization of genetic elements in synthetic biology. Here, more than 400 expression cassettes comprising the GFP gene under the regulation of various 5'-UTRs were systematically characterized to evaluate the protein translation consistency in the two popular Escherichia coli strains of JM109 and BL21, as well as an in vitro protein expression system based on cell lysate. In contrast to the very strong correlation between the two cellular systems, the consistency between in vivo and in vitro protein translation was lost, whereby both in vivo and in vitro translation evidently deviated from the estimation of the standard statistical thermodynamic model. Finally, we found that the absence of nucleotide C and complex secondary structure in the 5'-UTR significantly improve the efficiency of protein translation, both in vitro and in vivo.

Circular RNA vaccine induces potent T cell responses

Circular RNAs (circRNAs) are a class of RNAs commonly found across eukaryotes and viruses, characterized by their resistance to exonuclease-mediated degradation. Their superior stability compared to linear RNAs, combined with previous work showing that engineered circRNAs serve as efficient protein translation templates, make circRNA a promising candidate for RNA medicine. Here, we systematically examine the adjuvant activity, route of administration, and antigen-specific immunity of circRNA vaccination in mice. Potent circRNA adjuvant activity is associated with RNA uptake and activation of myeloid cells in the draining lymph nodes and transient cytokine release. Immunization of mice with engineered circRNA encoding a protein antigen delivered by a charge-altering releasable transporter induced innate activation of dendritic cells, robust antigen-specific CD8 T cell responses in lymph nodes and tissues, and strong antitumor efficacy as a therapeutic cancer vaccine. These results highlight the potential utility of circRNA vaccines for stimulating potent innate and T cell responses in tissues.

METTL3 is essential for normal progesterone signaling during embryo implantation via m6A-mediated translation control of progesterone receptor

Embryo implantation, a crucial step in human reproduction, is tightly controlled by estrogen and progesterone (P4) via estrogen receptor alpha and progesterone receptor (PGR), respectively. Here, we report that N6-methyladenosine (m6A), the most abundant mRNA modification in eukaryotes, plays an essential role in embryo implantation through the maintenance of P4signaling. Conditional deletion of methyltransferase-like 3 (Mettl3), encoding the m6A writer METTL3, in the female reproductive tract using a Cre mouse line with Pgr promoter (Pgr-Cre) resulted in complete implantation failure due to pre-implantation embryo loss and defective uterine receptivity. Moreover, the uterus of Mettl3 null mice failed to respond to artificial decidualization. We further found that Mettl3 deletion was accompanied by a marked decrease in PGR protein expression. Mechanistically, we found that Pgr mRNA is a direct target for METTL3-mediated m6A modification. A luciferase assay revealed that the m6A modification in the 5' untranslated region (5'-UTR) of Pgr mRNA enhances PGR protein translation efficiency in a YTHDF1-dependent manner. Finally, we demonstrated that METTL3 is required for human endometrial stromal cell decidualization invitro and that the METTL3-PGR axis is conserved between mice and humans. In summary, this study provides evidence that METTL3 is essential for normal P4 signaling during embryo implantation via m6A-mediated translation control of Pgr mRNA.

Modelling ribosome kinetics and translational control on dynamic mRNA

The control of protein synthesis and the overall levels of various proteins in the cell is critical for achieving homoeostasis. Regulation of protein levels can occur at the transcriptional level, where the total number of messenger RNAs in the overall transcriptome are controlled, or at the translational level, where interactions of proteins and ribosomes with the messenger RNA determine protein translational efficiency. Although transcriptional control of mRNA levels is the most commonly used regulatory control mechanism in cells, positive-sense single-stranded RNA viruses often utilise translational control mechanisms to regulate their proteins in the host cell. Here I detail a computational method for stochastically simulating protein synthesis on a dynamic messenger RNA using the Gillespie algorithm, where the mRNA is allowed to co-translationally fold in response to ribosome movement. Applying the model to the test case of the bacteriophage MS2 virus, I show that the models ability to accurately reproduce experimental measurements of coat protein production and translational repression of the viral RNA dependant RNA polymerase at high coat protein concentrations. The computational techniques reported here open up the potential to examine the infection dynamics of a ssRNA virus in a host cell at the level of the genomic RNA, as well as examine general translation control mechanisms present in polycistronic mRNAs.