4) Subsection “There is a wide range of RNA stabilities which are positively correlated with translation in ESCs”. In addition, miRNAs generally induce a smaller degree of repression (around two to three times) compared with site-specific RNAi-based cleavage. Mutations in genes encoding subunits of the cohesin complex are common in several cancers, but may also expose druggable vulnerabilities. RNA (mRNA) and inhibit translation or induce degradation. 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. DDX6 has been implicated as an effector of miRNA activity (Chen et al., 2014; Chu and Rana, 2006; Mathys et al., 2014; Rouya et al., 2014). miRNA sites were defined as below. To directly compare the two, we performed 4sU-Seq and polysome profiling in Dgcr8 KO ESCs and analyzed the data in parallel with that of the Ddx6 KO ESCs. The general lack of changes in mRNA stability suggested that transcription is the dominant regulator of the changing mRNA levels during the ESC to EpiLC transition. Further, this interaction is important for the translational repression of both CNOT1 tethered reporters and miRNA reporters. For each gene, the CDS region from the Gencode M14 annotation was used. 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 current annotation count on this page is, Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing—original draft, Writing—review and editing, "This ORCID iD identifies the author of this article:", Conceptualization, Resources, Supervision, Funding acquisition, Project administration, Writing—review and editing. This may aid in keeping ribosomes from bumping into each other on the polysome. These data show that DDX6 is an essential effector for miRNA-driven translational repression, but not mRNA destabilization. Additionally, through interactions with the CCR4-NOT complex and the decapping complex, DDX6 is thought to be involved in miRNA-mediated translational repression, but its exact role is not fully understood (Chen et al., 2014; Chu and Rana, 2006; Mathys et al., 2014; Rouya et al., 2014). The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. Epub 2010 Apr 21. Day 2 and day 3 counts were normalized to the day 1 count. Zhang A, Ma S, Yuan L, Wu S, Liu S, Wei X, Chen L, Ma C, Zhao H. Bone Joint Res. With some targets, an increase in the rate of mRNA degradation by the normal decay pathway contributes to the decrease in protein expression. There was a highly significant correlation between mRNA stability and translation levels across all genes (Spearman’s rho 0.2; p<2.22*10−16) (Figure 2F). While both ribosome profiling and polysome profiling measure global levels of translation, polysome profiling can be a more sensitive measure of translational regulation (Heyer and Moore, 2016). MicroRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay. Conversely, CNOT1 requires binding to DDX6 in order to repress translation of a reporter that is resistant to deadenylation and degradation, suggesting that DDX6 can repress translation without affecting mRNA levels (Kuzuoğlu-Öztürk et al., 2016; Mathys et al., 2014). However, depletion of 4E-T only partially alleviates DDX6 mediated translational repression (Kamenska et al., 2014; Kuzuoğlu-Öztürk et al., 2016). 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. This lack of changes was not because of noise among the replicates, as biological replicates were well correlated (Figure 1—figure supplement 1D). Therefore, like mRNA stability, there are few changes in translational efficiency in early ESC differentiation. Control of RNA Stability. Reads were mapped with STAR version 2.5.3a (Dobin et al., 2013) to the mm10/Gencode M14 genome with the following settings: --outFilterMultimapNmax 1 --outFilterMismatchNoverReadLmax 0.05 --seedSearchStartLmax 25 --winAnchorMultimapNmax 100. This is deduced from the simple fact that relative mRNA levels do not reflect the corresponding cellular ... the decay rates of mRNA for Further comparing changes in median codon frequency in stable versus unstable transcripts in wild-type cells with changes in median codon frequency in stabilized versus unstabilized transcripts in Ddx6 KO cells showed no correlation (Figure 4—figure supplement 1C). To complement Figure 2F showing the positive correlation between mRNA stability and translation level as measured by polysome profiling, we have made a similar scatter plot with translation efficiency as measured by the ribosome profiling data. Therefore, DDX6 likely interacts with additional unknown factors to inhibit translation initiation. We have clarified these points in the text. 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 ). (A–D) mRNA stability or translation level changes of ESCC miRNA targets versus all mRNAs. 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). AU-rich elements were defined as the number of UAUUUAU sequences in the 3’ UTR. n = 3 for wild-type, n = 4 for Ddx6 KO (2 replicates of each Ddx6 KO line), n = 3 for Dgcr8 KO. Conserved microRNA targets were downloaded from Targetscan mouse release 7.1. RNA-Seq showed 1890 genes significantly upregulated and 1532 genes significantly downregulated during the ESC to EpiLC transition (Figure 1B and F). To measure transcription, nascent transcripts were labeled with a 30-min 4sU pulse, biotinylated, pulled down with streptavidin, and sequenced (4sU-Seq). To further test this hypothesis, we performed polysome profiling (Arava et al., 2003). We have incorporated their feedback and added new graphs as well as added new text, both of which have improved the manuscript. 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/. This study measured mRNA abundance (using DNA microarrays) and translation rate for more than 8,000 genes. We measured translational efficiency, but not post-translational events; therefore, it remains plausible that protein degradation rates or protein localization are dynamic during ESC differentiation. Evidence of an increase in pre-mRNA or IVS-containing reads? These data show that translational repression alone can explain much of a miRNA’s function in ESCs. 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). MicroRNA-466l upregulates IL-10 expression in TLR-triggered macrophages by antagonizing RNA-binding protein tristetraprolin-mediated IL-10 mRNA degradation. 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). Your article has been reviewed by James Manley as the Senior Editor, a Reviewing Editor, and three reviewers. Each of these features and mRNA stability were used in a multiple linear regression using the lm function in R version 3.4.2. MicroRNAs have emerged as important post-transcriptional regulators of lipid metabolism, ... By altering mRNA stability and/or repressing mRNA translation, microRNAs represent an additional layer above transcriptional control for both fine-tuning and dramatically altering cell ... and an increase in the rate of fatty acid β-oxidation . 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). Adapter sequence used for trimming: AGATCGGAAGAGCACACGTCT. Arthritis Res Ther. While transcriptional regulation is well studied, less is known about how post-transcriptional events contribute to overall mRNA levels. These data show that the loss of DDX6 can separate the two canonical functions of microRNAs: translational repression and transcript destabilization. We also acknowledge Indiana University for access to their Mason cluster of computers, supported by the National Science Foundation (DBI #1458641). (G) Growth curves of wild-type and Ddx6 KO ESCs in ESC maintenance conditions (LIF/2i). (F) The number of significant increases or decreases in transcription, mRNA levels, mRNA stability, and translational efficiency during the ESC to EpiLC transition. In order to generate EpiLCs, 400,000 ESCs were plated in a 15 cm plate; 24 hr later LIF/2i media was removed, cells were washed with PBS, and EpiLCs were collected ~56 hr later (Krishnakumar et al., 2016). Cells were treated with 5 ug/ml Actinomycin D (Fisher Scientific). We calculated stAI values for mouse and asked if they could predict changes in transcript stability associated with DDX6 loss. The reviewers have opted to remain anonymous. They This result suggested a strong role for translation in regulating RNA stability in ESCs. 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). The accession number for the sequencing data reported in this paper is GEO: GSE112767. ... All of the choices are correct. MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. 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). c. 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. 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). Samples were labeled with 500 uM 4-thiouridine (4sU) (Sigma) for 30 min then extracted with TRIzol (Invitrogen) and split into two groups. In yeast, inhibition of translation initiation through either 5’ cap binding mutants or drug treatment leads to accelerated mRNA decay (Chan and Mugler, 2017; Huch and Nissan, 2014; Schwartz and Parker, 1999). 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. n = 3 for wild-type, n = 4 for Ddx6 KO (2 replicates of each Ddx6 KO line) (C) Expression of pluripotency genes in Ddx6 KO ESCs based on RNA-Seq.  |  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). Reverse transcription was performed with the Maxima first strand synthesis kit (Thermo Scientific). DDX6 localized to discrete punctate in the wild-type cells consistent with P-body localization, as previously reported (Figure 3E) (Ernoult-Lange et al., 2012; Hubstenberger et al., 2017; Minshall et al., 2009; Presnyak and Coller, 2013). Furthermore, experiments using miRNA reporters to examine the kinetics of miRNA repression suggest that translational repression precedes mRNA destabilization (Béthune et al., 2012; Djuranovic et al., 2012). It is not fully understood how DDX6 directly represses translation of miRNA targets or if it recruits additional effector molecules. However, in subsection “Transcriptional changes drive expression changes during the ESC to EpiLC transition” and elsewhere they use the term "levels" for ribosome profiling data. The export of mRNA from nucleus to cytoplasm requires the conserved and essential transcription and export (TREX) complex (THO–UAP56/DDX39B–ALYREF). Here, we studied these mechanisms in embryonic stem cells (ESCs). Images taken at 20X. 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). The loss of DDX6 had little impact on the expression of pluripotency markers (Figure 3C). One is that ribosomes sterically hinder the degradation machinery from accessing the transcript. 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. In yeast, the protein DHH1 has been shown to link translation to mRNA stability through codon optimality (Radhakrishnan et al., 2016). They were not (Figure 4—figure supplement 1B). Endogenous 3’ UTRs from the following genes were amplified from ESC cDNA: ENSMUSG00000021583, ENSMUSG00000029580, ENSMUSG00000043716, ENSMUSG00000010342, ENSMUSG00000021665, ENSMUSG00000024406, ENSMUSG00000052911, ENSMUSG00000058056, ENSMUSG00000020105, ENSMUSG00000026003, ENSMUSG00000020038, ENSMUSG00000025521, ENSMUSG00000031503. In contrast, small interfering RNAs (siRNAs), which are derived by processing of long double-stranded RNAs and are often of exogenous origin, degrade mRNAs bearing fully complementary … an activator exerting positive control. Two plates of 6 million V6.5 ESCs were seeded in a 15 cm plate 48 hr prior to collection (Eggan et al., 2001). 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). For each sample, the monosome, low polysome (2–4 ribosomes), and high polysome (4 + ribosomes were collected). 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. Mammalian mRNAs display a wide range of half-lives ranging from minutes to over a day (Schwanhäusser et al., 2011). NIH 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). Codon optimality is driven in part through tRNA abundance, which is cell type specific in mammals and can alter translation and mRNA stability in a cell type specific manner (Goodarzi et al., 2016; Gingold et al., 2014). To resolve this question, it is important to genetically separate the two functions. 2005 Nov 22;102(47):16961-6. doi: 10.1073/pnas.0506482102. Dashed lines divide bottom 1%, middle 50%, and top 1% of the data. Can some statistical significance be provided for this data? I suspect the overwhelming ignorance of biologically uninformed theorists is the problem because their … 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). How to evaluate its activity? For samples with multiple comparisons, a linear model was used for each condition in limma taking into account assay type (e.g. n = 3. We have gone through the manuscript and changed the text in several places to only use translational efficiency when referring to ribosome profiling data and to only use translation levels when referring to polysome profiling data. Known naive markers were downregulated, while known primed markers were upregulated confirming robust differentiation (Figure 1—figure supplement 1A) (Boroviak et al., 2015). We generated isogenic MCF10A cell lines with deletion mutations of genes encoding cohesin subunits SMC3, RAD21, and STAG2 and screened for synthetic lethality with 3009 FDA-approved compounds. Unfortunately, tRNA abundance data does not exist for ESCs. QuantSeq 3’ end counting was used for polysome profiling samples as well as matched wild-type, Ddx6 KO, and Dgcr8 KO mRNA samples (Figure 5C). Overall, the conclusion that translational repression can be separated from mRNA destabilization needs to be better explained in reference to the existing literature. 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. false. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Solving for this equation, degradation rates can be calculated using a production rate (in this case nascent RNA transcription as measured by 4sU incorporation) and the concentration of total mRNA in the cell (as measured by total RNA-Seq). Instead we suspect, though do not prove, that there was an increase in the rates of microRNA degradation in the synaptoneurosome fraction following SE. Any data supporting this? The target sites are almost invariably in the 3'-untranslated region of the messenger RNA (mRNA), often in multiple copies. However, the identity and how such features and regulatory factors impact mRNA stability are not well understood. Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central. We calculated translation using the ratio of RPF/mRNA, also known as translational efficiency (Ingolia et al., 2011). Effects of miR-146a-5p on chondrocyte interleukin-1. We have revisited these papers and have expanded our discussion to better discuss the previous literature regarding the role of DDX6 in the translational repression of miRNA reporters and the interaction of DDX6 with the CCR4-NOT complex. For the comparison between codon usage frequency in wild-type versus Ddx6 KO, we took the median codon usage frequency in stable - the median codon usage frequency in unstable for each codon and compared it to the Ddx6 KO median codon usage frequency in the bottom group - median codon usage frequency in the top group, using groups as defined above. 1,2 MicroRNAs bound to Argonaute proteins recruit other protein components of RNA-induced silencing complexes (RISCs) to specific sites on target mRNAs. How TREX integrates these marks and achieves high selectivity for mature mRNA is poorly understood. We have added statistical significance for differences in codon frequency using the Mann–Whitney test followed by Bonferroni correction. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. The p value was calculated with correlation significance test. Simultaneous RNA-Seq and ribosome profiling experiments across a number of contexts show that miRNAs produce larger changes in mRNA levels than in translational efficiency, leading to the suggestion that mRNA destabilization is the dominant effect of miRNA repression (Eichhorn et al., 2014; Guo et al., 2010). Therefore, we next asked whether Ddx6 KO cells have similar downstream molecular consequences as Dgcr8 KO cells. 80 ug of RNA was biotinylated according to the following protocol Rädle et al. 2006;71:523-30. doi: 10.1101/sqb.2006.71.013. 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 KO versus wild-type); significant changes in stability or translation are based on the interaction term. (B) MA plot of mRNA changes during the ESC to EpiLC transition. 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. We have now clarified this issue in the text. The screen identified several compounds that interfere with transcription, DNA damage repair and the cell cycle. Therefore, the loss of DDX6 is able to separate the two central functions of miRNAs: translational repression and mRNA destabilization. It is assumed that across the population of cells there is no change in mRNA levels over time for a given state. The exact mechanism of … However, CNOT1 does not require DDX6 to degrade a reporter with a poly(A) tail, suggesting that CCR4-NOT can recruit degradation factors even when the interaction with DDX6 is disrupted (Kuzuoğlu-Öztürk et al., 2016; Mathys et al., 2014). MiRNAs are one regulatory factor that bind to the 3’ UTR of target mRNAs and recruit a complex of proteins that then destabilize the transcripts (Fabian and Sonenberg, 2012; Jonas and Izaurralde, 2015). This finding contrasts Lemischka and colleagues’ conclusion that post-transcriptional changes underlie many changes in protein levels during ESC differentiation (Lu et al., 2009). These data show that unlike yeast DHH1, the primary function of mammalian DDX6 is not to link codon optimality with transcript stability. Mol Neurobiol. Furthermore, these data uncover a central role for translational repression independent of transcript destabilization in defining the downstream consequences of microRNA loss. Clusters of dots indicate an endogenous 3’ UTR, individual dots within a cluster represent biological replicates (n = 3). 2020 Nov;57(11):4856-4877. doi: 10.1007/s12035-020-02074-2. To separate the contribution of transcription versus mRNA stability to changing mRNA levels during the ESC to EpiLC transition, we used metabolic labeling with 4-thiouridine (4sU) (Dölken et al., 2008; Rabani et al., 2011; Windhager et al., 2012). 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. (E) DDX6 staining in wild-type ESCs. Discordant changes between mRNA expression and nuclear protein levels could reflect changes in translational efficiency, protein stability, or protein localization (Liu et al., 2016). These studies found that the DDX6 RecA domain directly interacts with the CNOT1 MIF4G domain (Chen et al., 2014; Mathys et al., 2014; Rouya et al., 2014). Unexpectedly, one of the top ‘hits’ was a GSK3 inhibitor, an agonist of Wnt signaling. A number of mechanisms have been proposed. (E) Translation level changes of individual ESCC miRNA targets in Dgcr8 KO and Ddx6 KO ESCs. From ( Ran et al., 2010 ) usage frequency stability data suggested that DDX6 also represses translation ( et. Into account assay type ( e.g were cloned into the pBUTR ( 3′... Versus all mRNAs target gene expression is determined by its lifespan and rate of deadenylation does not for. Bumping into each other on the consequence of Dgcr8 and DDX6 loss led... Also represses translation ( Kuzuoğlu-Öztürk et al., 2003 ) we asked whether KO. Components of RNA-induced silencing complexes ( RISCs ) to specific sites on target.... 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Following sources: Crossref, Scopus, PubMed central additional effector molecules 5’ UTR consequences of microRNA loss 6 8... Jacobson, 2013 ) targets, without concurrent changes in mRNA stability is (! Metric of codon optimality miR-140 in cartilage matrix degradation by the miR-291–3 p/294–3 p/295–3 p/302–3 family! A step in translation level ( high polysome/monosome as translation levels across genes... It to take advantage of the P-body marker DCP1a ( Figure 3C ) translation these.