(E) DDX6 staining in wild-type ESCs. The wide range of mRNA stabilities are regulated by both intrinsic sequence features as well as the binding of regulatory factors such as microRNAs and RNA-binding proteins (Cheng et al., 2017; Hasan et al., 2014; Wu and Brewer, 2012). The emergence of small RNA-mediated gene silencing preceded the onset of multicellularity and was followed by a drastic expansion of the miRNA repertoire in conjunction with the evolution of complexity in the plant and animal kingdoms. Here, we studied these mechanisms in embryonic stem cells (ESCs). The stabilized transcripts were not specifically enriched within the lowly translated transcripts and instead they occurred across all levels of translation. Furthermore, these data uncover a central role for translational repression independent of transcript destabilization in defining the downstream consequences of microRNA loss. We have added a sentence discussing these reporters and citing Kuzuoglu-Ozturk et al. These data show that DDX6 is an essential effector for miRNA-driven translational repression, but not mRNA destabilization. TREX selectively binds mRNA maturation marks and licenses mRNA for nuclear export by loading the export factor NXF1–NXT1. The emerging roles of WBP2 oncogene in human cancers. The extent to which an mRNA is utilized is determined by its lifespan and rate of translation. Additional rounds of Ddx6 KO and matched wild-type 4sU samples were sequenced with paired-end reads and counts were merged with single end reads. MicroRNAs work to.... control patterns of alternative splicing increase rates of transcription decrease the binding of ribosomes to the 5' cap destroy mRNA or block its translation We next compared the polysome profiling data to the mRNA stability data. * indicates p<0.05 using a t-test, error bars are standard deviation. It is not fully understood how DDX6 directly represses translation of miRNA targets or if it recruits additional effector molecules. Alternatively, some RNA-binding proteins may sense slowly translating transcripts and accelerate their degradation as recently described for DHH1 (Radhakrishnan et al., 2016). miRNAs (microRNAs) are short non-coding RNAs that regulate gene expression post-transcriptionally. It was recently shown that DDX6 interacts with 4E-T, which competes with eIF4G for binding to the translation initiation factor eIF4E and leads to translational repression (Kamenska et al., 2016; Ozgur et al., 2015). To measure transcription, nascent transcripts were labeled with a 30-min 4sU pulse, biotinylated, pulled down with streptavidin, and sequenced (4sU-Seq). However, in our data, the loss of the mammalian homolog of DHH1, DDX6, did not appear to link low levels of translation with low mRNA stability. The translation rates measured are relative translation rates normalized for mRNA levels. Therefore, DDX6 does not appear to play a major role in transcript destabilization downstream of miRNAs. Before the mRNA leaves the nucleus, it is given two protective “caps” that prevent the end of the strand from degrading during its journey. This section now reads: “This situation has been observed for synthetic miRNA reporters that cannot undergo deadenylation and subsequent degradation but can still be translationally repressed (Kuzuoğlu-Öztürk et al., 2016). Sanger sequencing confirmed a single nucleotide insertion in one clone and a large deletion in a second clone, both of which produce a premature stop (Figure 3—figure supplement 1A). We generated Ddx6 KO ESCs to determine whether DDX6 links translation to mRNA stability. 2) There is a confusing aspect in that the authors report upregulation in translation of miRNA targets without an increase in mRNA stability; this is mechanistically counterintuitive. ... All of the choices are correct. However, P-bodies may also serve as repositories for the temporary and reversible storage of untranslated mRNA, and reducing the expression (knockdown) of several distinct P-body protein components can alleviate miRNA-mediated repression of gene expression. Although not directly measured, protein levels of miRNA targets are likely higher in Dgcr8 KO cells than in Ddx6 KO cells as the former leads to both mRNA stabilization and translational derepression of miRNA targets, while the later only influences translation (Figure 5D). We thank the following people for critical reading of the manuscript: Marco Conti, Stephen Floor, Raga Krishnakumar, Brian DeVeale, and Deniz Goekbuget. miRNAs can bind to target messenger RNA (mRNA) transcripts of protein-coding genes and negatively control their translation or cause mRNA degradation. See also Figure 2—figure supplement 1. We apologize for this oversight. Interestingly, analysis of the 4sU-Seq data showed that long non-coding RNAs (lncRNAs) were significantly less stable than protein coding genes (p<2.22*10−16, Mann-Whitney test) (Figure 2E). Whether translational repression or mRNA destabilization is the predominant effect of miRNAs is controversial as it is difficult to separate the two (Iwakawa and Tomari, 2015; Jonas and Izaurralde, 2015). Your article has been reviewed by James Manley as the Senior Editor, a Reviewing Editor, and three reviewers. The Ddx6 knockout cells were phenotypically and molecularly similar to cells lacking all microRNAs (Dgcr8 knockout ESCs). The stAI metric takes into account tRNA copy number and a tRNA’s ability to wobble base pair with different codons (Radhakrishnan et al., 2016; Sabi and Tuller, 2014). We have clarified these points in the text. The p value calculated with correlation significance test. Cells were blocked with 2% BSA and 1% goat serum in PBST. 8) Many references are listed multiple times in the references list. ESCs were grown in Knockout DMEM (Invitrogen) supplemented with 15% Fetal Bovine Serum, LIF and 2i (Peprotech PD0325901 and CHIR99021). Cells were fixed with 4% PFA 10 min at room temperature. RFP+/GFP+ cells were gated in FlowJo and median RFP/GFP ratios were calculated. Metazoan miRNAs were previously thought to down-regulate protein expression by inhibiting target mRNA translation at some stage after the translation initiation step, without much effect on mRNA abundance. miRNAs function posttranscriptionally by usually base-pairing to the mRNA 3′-untranslated regions to repress protein synthesis by mechanisms that are not fully understood. This project was funded by the National Institutes of Health (R01 GM101180, R01 GM122439) to RB, and a Genentech Predoctoral Research Fellowship to JWF. However, a large number of ESCC targets were still in the top 50% of the most stable genes (Figure 2—figure supplement 1A). This site needs JavaScript to work properly. USA.gov. Additionally, the Spearman correlation was calculated between each feature and mRNA stability. While transcriptional regulation is well studied, less is known about how post-transcriptional events contribute to overall mRNA levels. A number of mechanisms have been proposed. However, there were striking morphological changes in the cells (Figure 3D). Several aspects of the Ddx6 KO phenotype, including the cell morphology changes and growth defects, resemble the phenotype of Dgcr8 KO cells (Wang et al., 2007). (A) Western blot of DDX6 in two Ddx6 knockout (KO) lines. false. c. Therefore, we can independently quantify changes in translation level versus changes in mRNA stability. Genes were cloned into the pBUTR (piggyBac-based 3′ UnTranslated Region reporter) using gateway cloning as outlined in Chaudhury et al. (Bottom) Normalized median RFP/GFP ratios versus mRNA stability for endogenous genes as measured by 4sU-Seq. Overall, the conclusion that translational repression can be separated from mRNA destabilization needs to be better explained in reference to the existing literature. This is in contrast to endogenous miRNA targets that can be deadenylated and degraded. The p value was calculated with Mann-Whitney test. (B) The correlation between sequence features and mRNA stability in ESCs. 3) The authors should include in the Discussion section a paragraph better describing previous work demonstrating the involvement of DDX6 in translational repression by miRNAs. 2020 Oct;9(10):689-700. doi: 10.1302/2046-3758.910.BJR-2020-0140.R1. (2014). Epub 2005 Nov 15. Ddx6 KO lines were generated using the protocol from (Ran et al., 2013). RFP/GFP ratios were standardized between days to accounts for differences in laser power. DDX6 contact with the CCR4-NOT complex has been linked to 'pure' translational repression; however, this is always assessed in the context of a miRNA-targeted reporter mRNA that cannot be deadenylated and is therefore highly stable. RNA-Seq from the monosome, low polysome (2–4 ribosomes), and high polysome (4 + ribosomes were collected) fractions was mapped as above. RNA is transcribed, but must be processed into a mature form before translation can begin. The p value was calculated with correlation significance test. MiRNA-induced translational repression in the absence of mRNA destabilization has also been observed in the early zebrafish embryo, but the mechanism underlying the phenomenon remains unclear (Bazzini et al., 2012). In ESCs, the embryonic stem-cell-enriched cell cycle (ESCC) family of miRNAs represent a predominant fraction of all miRNAs in ESCs (Greve et al., 2013; Houbaviy et al., 2003; Marson et al., 2008). Detailed reviews describing work presented at the annual Cold Spring Harbor Symposia on Quantitative Biology In addition, miRNAs generally induce a smaller degree of repression (around two to three times) compared with site-specific RNAi-based cleavage. miRNA sites were defined as below. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. This list was filtered for genes that are targeted by the miR-291–3 p/294–3 p/295–3 p/302–3 p family yielding 765 target genes. The relative stabilities predicted by the two approaches were highly correlated. In support of this model, RNA-Seq of the 5’ end of decapped RNA degradation intermediates shows a three nucleotide periodicity consistent with exonucleases running into the ribosome on a final round of translation (Pelechano et al., 2015). Similarly, the treatment of H9C2 rat myoblasts with DOX at 0.1 and 1 µM for 24 h showed a decrease of Sipa1 mRNA and an increase of endogenous miR-34c . rRNA was depleted from Total RNA using the Ribo-Zero Gold kit (Illumina). Until recently, it was believed this mechanism operated almost exclusively at a step in translation. (E) MA plot of translational efficiency (TE) changes during the ESC to EpiLC transition. In DDX6-depleted cells, repression of a miRNA reporter cannot be rescued by DDX6 mutants that cannot bind to CNOT1 (Chen et al., 2014; Kuzuoğlu-Öztürk et al., 2016; Mathys et al., 2014; Rouya et al., 2014). The need to locate first one and then another tRNA for that amino acid slows down the rate of translation. Epub 2010 Apr 21. For each gene with multiple isoforms, the APPRIS principle isoform was used. It has been argued that mRNA changes are the dominant effect of miRNAs, since miRNA-induced changes in mRNA levels are often larger than changes in translational efficiency (Eichhorn et al., 2014; Guo et al., 2010). How TREX integrates these marks and achieves high selectivity for mature mRNA is poorly understood. We show that sensitivity to GSK3 inhibition is likely due to stabilization of β-catenin in cohesin-mutant cells, and that Wnt-responsive gene expression is highly sensitized in STAG2-mutant CMK leukemia cells. Additionally, the authors find that during ESC differentiation transcriptional changes drive gene expression changes, which contrasts with conclusions from previous studies performed in Lemischa's lab. In this study, we sought to uncover how mRNA stability and translation are regulated within the ESC state and during differentiation. However, the Ddx6 KO cells demonstrate that mRNA destabilization can occur independent of translational repression in a context where both forms of repression normally occur. Within those genes, we took the top 20% (top) and bottom 20% (bottom) of mRNA stability changes in Ddx6 KO ESCs and calculated codon usage frequency within each group. However, recent studies have questioned these suppositions. To measure translation of all genes, we performed ribosome profiling to collect Ribosome Protected Footprints (RPFs) and matched total mRNA (Ingolia et al., 2011). To get around this problem when analyzing the Ddx6 KO data, we focused on codons that were enriched in unstable genes in wild-type cells and asked if they were enriched in genes that were stabilized in Ddx6 KO cells. Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. Oncogene. MiRNA induced translational repression in the absence of mRNA destabilization has also been observed in the early zebrafish embryo, but the mechanism underlying the phenomenon remains unclear (Bazzini et al., 2012).”. Cells were collected in RIPA buffer with Protease Inhibitor Cocktail (Roche). miRNAs can also inhibit translation initiation, specifically the function of the cap-binding initiation factor, eIF4E. We repeated the 4sU-Seq and polysome profiling in Ddx6 KO and matched wild-type cells to measure changes in mRNA stability and translation levels.  |  The most important finding of the work is the demonstration that knock-out of Ddx6 in ESCs relieves translational repression induced by miRNAs and that the phenotype of Ddx6 KO cells is similar to that of cells in which miRNA population is eliminated through inactivation of DGCR8. Future studies will likely identify factors that can decouple translational repression and mRNA destabilization in the other direction so that miRNA targets are translationally repressed without inducing mRNA destabilization. Although there were minimal changes in mRNA stability during the ESC to EpiLC transition, there was a wide range of mRNA stabilities within ESCs. Since the Ddx6 KO and Dgcr8 KO cells have partially overlapping phenotypes, we compared global changes in mRNA stability, mRNA levels, and translation levels. 2007 Oct 30;8:396. doi: 10.1186/1471-2164-8-396. Tethering experiments in human cells demonstrate that DDX6 also represses translation (Kuzuoğlu-Öztürk et al., 2016). We have updated that sentence to include the new data and it now reads “Nascent transcriptional changes between Ddx6 KO and Dgcr8 KO measured by 4sU-Seq are also well correlated (Figure 5—figure supplement 1A) showing that the correlation in mRNA changes is due to transcriptional changes, likely secondary to the direct effects of Ddx6 and Dgcr8 loss on the translation of transcriptional regulators.”, We apologize for this oversight. For codon usage frequency for mRNA stability changes in Ddx6 KO cells, we first filtered for genes in the bottom 20% of wild-type stability as defined above. Thank you for submitting your article "Decoupling the impact of microRNAs on translational repression versus RNA degradation in embryonic stem cells" for consideration by eLife. For KO versus wild-type analysis, a linear model was used for each condition in limma and significant changes in stability are based on the interaction term. Function and localization of microRNAs in mammalian cells. Summary: RNA-Dependent Intergenerational Inheritance of Enhanced Synaptic Plasticity after Environmental Enrichment was not linked to sympatric speciation via The Bull Sperm MicroRNAome and the Effect of Fescue Toxicosis on Sperm MicroRNA Expression Something has gone horribly wrong. (E) RNA stability of long non-coding RNAs (lncRNAs) compared to protein-coding RNAs. Using RNA-Seq, metabolic labeling (4sU-Seq), and ribosome profiling, we found that most changes during ESC differentiation are driven at the level of transcription. The mammalian homolog of DHH1, DDX6, has been shown to associate with both the mRNA decapping and deadenylation complex, also consistent with a potential link between mRNA stability and translation (Chen et al., 2014; Mathys et al., 2014; Rouya et al., 2014). Since yeast DHH1 destabilizes lowly translated transcripts enriched in non-optimal codons, we expected that lowly translated genes might be stabilized in Ddx6 KO ESCs (Radhakrishnan et al., 2016). There has been extensive debate about whether miRNAs primarily inhibit translation or induce destabilization of their target transcripts (Iwakawa and Tomari, 2015; Jonas and Izaurralde, 2015). Cells were incubated with goat 488 secondary for 1 hr at room temperature. Thus, nascent transcription, not mRNA stability, underlies the mRNA changes associated with the ESC to EpiLC transition. 2020 Nov;48(11):300060520969550. doi: 10.1177/0300060520969550. We also acknowledge Indiana University for access to their Mason cluster of computers, supported by the National Science Foundation (DBI #1458641). This binding information directly links miRNA target recognition (AGO-mRNA binding) to translational repression through DDX6, via CNOT1-CNOT9-TNRC6 binding and is consistent with our findings. Although some of the studies are mentioned in two sentences in the Introduction and Results section, it would also be interesting to discuss (for example, in the Discussion section), that the role of DDX6 is documented by structural studies (Chen et al., 2014, Mathys et al., 2014), importance of the CNOT1 ~ DDX6 interaction for activation of the DDX6 ATPase activity, which in turn is needed for translational repression (Mathys et al., 2014; Kuzyoglu-Ozturk et al., 2016). Cells were treated with 5 ug/ml Actinomycin D (Fisher Scientific). We measure translation as the ratio of polysome reads to monosome reads, which normalizes for any changes in mRNA levels. Each of these features and mRNA stability were used in a multiple linear regression using the lm function in R version 3.4.2. Therapeutic Strategies in the Development of Anti-viral Drugs and Vaccines Against SARS-CoV-2 Infection. In that work, differentiation was induced by expressing a shRNA to Nanog in ESCs grown in LIF. Unexpectedly, one of the top ‘hits’ was a GSK3 inhibitor, an agonist of Wnt signaling. MicroRNAs (miRNAs) are endogenously encoded small noncoding RNAs, derived by processing of short RNA hairpins, that can inhibit the translation of mRNAs bearing partially complementary target sequences. The accession number for the sequencing data reported in this paper is GEO: GSE112767. miRNAs can also inhibit translation initiation, specifically the function of the cap-binding initiation factor, eIF4E. How to evaluate its activity? MicroRNAs (miRNA) are small non-coding RNA molecules, which bind to the 3’UTR of target mRNA and regulate gene expression by suppressing their translation (Kloosterman and Plasterk, 2006). With some targets, an increase in the rate of mRNA degradation by the normal decay pathway contributes to the decrease in protein expression. These data suggested that DDX6 does not link mRNA stability with translation levels across all genes. Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks in vertebrates. A comparison of the predictive performance of eighteen in … (D) Brightfield images of wild-type and Ddx6 KO ESCs. n = 3 for each ESC and EpiLC seq experiment. We isolated monosome fractions, low polysome fractions containing 2–4 ribosomes, and high polysome fractions containing 4+ ribosomes and performed RNA-Seq (Figure 2—figure supplement 1B). As expected, RPFs showed a strong three nucleotide phasing of reads that was not present in the mRNA samples, confirming the quality of the data (Figure 1—figure supplement 1E). The difference may be explained by the different approaches used and the fact that Lemischka and colleagues focused their analysis on nuclear protein changes. As expected, protein coding genes had a much higher translation level compared to lncRNAs (p<2.22*10−16, Mann-Whitney test) (Figure 2—figure supplement 1C). Antibodies: DDX6 1:1000 (A300-460A-T), GAPDH 1:1000 (SC 25778), ACTIN 1:1000 (A4700). To study the effects of miR-124 on translation of targeted mRNAs, we estimated the change in the translation rates between miR-124-transfected and mock-transfected cells (Tr) for each mRNA as: (1) where multiplying O, the fraction of the mRNA that is ribosome-bound (ribosome occupancy), by D, the average number of ribosomes per 100 nts for bound mRNAs (ribosome … We have added statistical significance for differences in codon frequency using the Mann–Whitney test followed by Bonferroni correction. Ma F, Liu X, Li D, Wang P, Li N, Lu L, Cao X. J Immunol. Last year, a three-way collaboration between three groups at Stanford, led by Pat Brown, set out to measure just how much effect miRNAs have on protein translation, and how much on mRNA levels. Could they show such relationship within their own datasets? These data suggest that, while miRNAs are strong destabilizers, they can only explain a small portion of the large range of mRNA stabilities seen in the cells. Our structural and biochemical results suggest a conserved model for TREX complex function that depends on multivalent interactions between proteins and mRNA. 4sU-Seq and total RNA-Seq showed that while there was a minimal reduction of nascent Ddx6 mRNA, there was a drastic loss of mature Ddx6 mRNA in the Ddx6 KO cells (Figure 3B). Unlike wild-type ESCs which form tight domed colonies, Ddx6 KO cells grew in a jagged, dispersed monolayer (Figure 3D). 50,000 cells were plated in multiple wells of a six well on day 0. Although the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Significant changes are shown as red dots (Adjusted p value < 0.05 and |log2 fold change| > 1) in B, C, E. Dashed lines indicated a twofold change. Differential effects of translational inhibition in Cis and in trans on the decay of the unstable yeast MFA2 mRNA, GRHL2-Dependent enhancer switching maintains a pluripotent stem cell transcriptional subnetwork after exit from naive pluripotency. RNA was isolated using RNeasy Micro kits (Qiagen). We are not aware of complications of the interpretation of our translational efficiency data; if the reviewers think we are missing something we would be happy to take it into account. A guide RNA (CATGTGGTGATCGCTACCCC) was cloned into PX458, transfected into ESCs using Fugene 6, and then GFP-positive cells were sorted at clonal density. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. The transfection of miR-34c mimics in H9c2 showed a statistically significant decrease of Sipa1 mRNA, as the transfection of miR-34c hairpin inhibitor (HI) showed a statistically significant increase of Sipa1 mRNA ( Figure 5C ). 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