Therefore, we next asked whether Ddx6 KO cells have similar downstream molecular consequences as Dgcr8 KO cells. The binding of CNOT1 changes the conformation of DDX6 and stimulates DDX6 ATPase activity (Mathys et al., 2014). To validate the impact of 3’ UTRs on mRNA stability, we used a dual reporter system that contains a control GFP for normalization and a RFP with a cloned endogenous 3’ UTR from 12 representative genes (Figure 2D) (Chaudhury et al., 2014). Detailed reviews describing work presented at the annual Cold Spring Harbor Symposia on Quantitative Biology Indeed, fold changes in total mRNA levels correlated extremely well with fold changes in 4sU-labeled nascent transcripts (Spearman’s rho 0.88; p<2.22*10−16) (Figure 1D). MicroRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay. This ATPase activity is essential for translational repression as DDX6 that contains mutations in the ATPase domain can no longer repress miRNA reporters (Mathys et al., 2014). Given that the 4sU-Seq approach avoids the secondary effects associated with blocking all transcription, we used those data for genome-wide analysis (Bensaude, 2011; Lugowski et al., 2018). 2006;71:29-38. doi: 10.1101/sqb.2006.71.049. The Ddx6 knockout cells were phenotypically and molecularly similar to cells lacking all microRNAs (Dgcr8 knockout ESCs). 80 ug of RNA was biotinylated according to the following protocol Rädle et al. See also Figure 2—figure supplement 1. 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. (Bottom) Normalized median RFP/GFP ratios versus mRNA stability for endogenous genes as measured by 4sU-Seq. Several recent studies implicated DDX6 in the translational repression of miRNA reporters downstream of the CCR4-NOT complex (Chen et al., 2014; Kuzuoğlu-Öztürk et al., 2016; Mathys et al., 2014; Rouya et al., 2014). 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. MicroRNA. 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. Images taken at 20X. Therefore, codon optimality may in part explain the link between translation levels and mRNA stability. Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks in vertebrates. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.  |  Direct tethering of DDX6 represses translation of a reporter and this repression is only mildly disrupted by mutations that prevent DDX6 from interacting with CNOT1 suggesting that DDX6 acts downstream of the CCR4-NOT complex (Kuzuoğlu-Öztürk et al., 2016). Inhibiting eukaryotic transcription: Which compound to choose? In this study, we sought to uncover how mRNA stability and translation are regulated within the ESC state and during differentiation. Epub 2020 Aug 18. Reporters were transfected into ESCs using Fugene 6 (Promega). The translation rates measured are relative translation rates normalized for mRNA levels. RFP+/GFP+ cells were gated in FlowJo and median RFP/GFP ratios were calculated. 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 Each sample was normalized to 18S rRNA and its 0 hr time point. The translation rates measured are relative translation rates normalized for mRNA levels. Conversely, treatment with cycloheximide, which blocks ribosome elongation, stabilizes mRNAs (Beelman and Parker, 1994; Chan and Mugler, 2017; Huch and Nissan, 2014). Global analysis showed very few changes in translational efficiencies between the ESC and EpiLC states (Figure 1E and F). 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). These studies raise the question of whether translational repression is the direct mode of miRNA-driven suppression with mRNA destabilization being a secondary consequence. If a miRNA-targeted mRNA is not destabilized, then a change in translational repression is anticipated, as has been reported in zebrafish embryos. (A) Flow cytometry of the transition from naive embryonic stem cells (ESCs) (miR-302 GFP-, miR-290 mCherry+) to primed epiblast-like cells (EpiLCs) (miR-302 GFP+, miR-290 mCherry+). In Dgcr8 KO ESCs, which lack all mature miRNAs, we observe that miRNAs both inhibit translation and induce mRNA destabilization of their targets within ESCs. an activator exerting positive control. To identify features that explain the range of mRNA stabilities observed, we performed multiple linear regression taking into account the following features that previous studies implicated in affecting mRNA stability: 3’ UTR length, 5’ UTR length, CDS length, 3’ UTR GC content, 5’ UTR GC content, CDS GC content, AU-rich elements (ARE), miRNA-binding sites, number of exons in the transcript, and upstream ORFs (Chan and Mugler, 2017; Cheng et al., 2017; Sharova et al., 2009). For samples with multiple comparisons, a linear model was used for each condition in limma taking into account assay type (e.g. In molecular genetics, the three prime untranslated region (3′-UTR) is the section of messenger RNA (mRNA) that immediately follows the translation termination codon.The 3′-UTR often contains regulatory regions that post-transcriptionally influence gene expression.. During gene expression, an mRNA molecule is transcribed from the DNA sequence and is later translated into a protein. The reviewers have opted to remain anonymous. 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. This is a very interesting and well-documented study, which demonstrates that translational efficiency of mRNAs in ESCs is closely positively correlated with their stability. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin subunits stag2b and rad21. Thus, the shifted reporter mRNA signals found during gradient sedimentation might represent partially degraded mRNAs, which dissociates with the translational machinery. The screen identified several compounds that interfere with transcription, DNA damage repair and the cell cycle. This site needs JavaScript to work properly. In contrast to the yeast homolog, transcripts stabilized upon DDX6 loss did not correlate with low stAI values (Figure 4—figure supplement 1D). These conditions are associated with a heterogeneous population of cells (Ivanova et al., 2006). ESCs were grown in Knockout DMEM (Invitrogen) supplemented with 15% Fetal Bovine Serum, LIF and 2i (Peprotech PD0325901 and CHIR99021). For each gene, the CDS region from the Gencode M14 annotation was used. Inter- and intra-combinatorial regulation by transcription factors and microRNAs. 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). 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. Cold Spring Harb Symp Quant Biol. Together, these data show an important role for DDX6 in the formation and/or maintenance of P-bodies and in retaining normal cell morphology and proliferation. NIH 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. Unlike its yeast homolog, DDX6 did not appear to play a general role in linking the two. See also Figure 5—figure supplement 1. MiRNAs are small, non-coding RNAs that bind to the 3’ UTR of their target transcripts to inhibit translation and/or induce mRNA destabilization (Fabian and Sonenberg, 2012; Jonas and Izaurralde, 2015). 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. There was no enrichment (Figure 4—figure supplement 1B). The discovery of the first microRNA (miRNA), lin-4, in 1993 by the Ambros and Ruvkun groups in Caenorhabditis elegans (1, 2) has revolutionized the field of molecular biology. mRNA stability is measured as the ratio of mRNA/4sU reads, changes in translation level are measured as the ratio of polysome/monosome reads, protein level changes are not directly measured but are predicted based on mRNA stability and translation level changes. Therefore, we measured and analyzed changes in mRNA stability and translational efficiency during ESC differentiation. Libraries were generated with the KAPA Stranded RNA-Seq or Stranded HyperPrep library prep kit (Kapa) and sequenced with single-end 50 bp reads. Ma F, Liu X, Li D, Wang P, Li N, Lu L, Cao X. J Immunol. Therefore, the loss of DDX6 is able to separate the two central functions of miRNAs: translational repression and mRNA destabilization. They generally bind to the 3'-UTR (untranslated region) of their target mRNAs and repress protein production by destabilizing the mRNA and translational silencing. A/B) mRNA stability changes in Dgcr8 KO (A) or Ddx6 KO (B) cells. A better discussion of the DDX6 role would make the paper more interesting to the wide readership. They (F) The number of significant increases or decreases in transcription, mRNA levels, mRNA stability, and translational efficiency during the ESC to EpiLC transition. Cells were washed and scraped in PBS with cycloheximide, spun down, and then lysed. Instead we suspect, though do not prove, that there was an increase in the rates of microRNA degradation in the synaptoneurosome fraction following SE. 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. 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). 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. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Mammalian mRNAs display a wide range of half-lives ranging from minutes to over a day (Schwanhäusser et al., 2011). The paper would be strengthened by additional work and better explanation some of the data as follows: 1) The authors frequently note that lower mRNA stability is related to the lower translation efficiency. We found a positive correlation between mRNA stability and translation levels/efficiency in ESCs, similar to what other groups have observed recently in yeast (Chan and Mugler, 2017; Heyer and Moore, 2016; Presnyak et al., 2015). 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. Our data shows that the loss of DDX6 results in increased translation of miRNA targets to a similar level as the loss of all miRNAs, suggesting that DDX6 serves as a key link between the proteins that repress translation and the rest of the RNA-induced silencing complex. MicroRNAs are short noncoding RNAs that serve to limit the translation of specific mRNAs, often but not always observed in conjunction with mRNA transcript degradation. (B) Comparison between translation level changes in Dgcr8 KO versus Ddx6 KO cells. However, the identity and how such features and regulatory factors impact mRNA stability are not well understood. Adapters were trimmed using cutadapt version 1.14 with the following settings: --minimum-length 26 --maximum-length 32 for the ribosome protected fragments or --minimum-length 32 for the total RNA. 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). However, in other studies, it has been suggested that miRNAs primarily inhibit translation. 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). To further test this hypothesis, we performed polysome profiling (Arava et al., 2003). MicroRNAs – targeting and target prediction Takay Saitoa, ... RISC then inhibits protein translation and causes mRNA degradation [7, 8]. RNA is transcribed, but must be processed into a mature form before translation can begin. We calculated stAI values for mouse and asked if they could predict changes in transcript stability associated with DDX6 loss. n = 3 for each ESC and EpiLC seq experiment. The authors further demonstrate that depletion of the RNA helicase DDX6 does not eliminate this correlation what contrasts with the situation in yeast. The mechanisms linking translation to mRNA stability are poorly understood. 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 ). miRNA sites were defined as below. Reads were mapped with STAR version 2.5.3a to the mm10/Gencode M14 genome with the following settings: --outFilterMultimapNmax 1 --outFilterMismatchNoverReadLmax 0.05 --seedSearchStartLmax 13 --winAnchorMultimapNmax 200. 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). Translation initiation and elongation can also influence mRNA stability (Huch and Nissan, 2014). Or, give some information about which codons/tRNA are rare and which are not. (2014). We have now clarified this issue in the text. 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). This lack of changes was not because of noise among the replicates, as biological replicates were well correlated (Figure 1—figure supplement 1D). National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error. We have incorporated their feedback and added new graphs as well as added new text, both of which have improved the manuscript. DGCR8 is essential for miRNA biogenesis and Dgcr8 KO ESCs lack all miRNAs (Wang et al., 2007). Recent reports suggest that differential codon usage is a central mechanism in linking translation to mRNA stability (Bazzini et al., 2016; Chan and Mugler, 2017; Cheng et al., 2017; Mishima and Tomari, 2016; Presnyak et al., 2015). n = 3. (D) Summary schematic comparing Dgcr8 KO cells to Ddx6 KO cells. COVID-19 is an emerging, rapidly evolving situation. Surprisingly, the 4sU/total mRNA data showed very few changes in mRNA stability between the ESC and EpiLC states (Figure 1C and F). 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. Cells were incubated with 100 ug/ml cycloheximide (Sigma) for 2 min and then moved to ice. (E) DDX6 staining in wild-type ESCs. This may aid in keeping ribosomes from bumping into each other on the polysome. 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. Clipboard, Search History, and several other advanced features are temporarily unavailable. Control of RNA Stability. 50,000 cells were plated in multiple wells of a six well on day 0. 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 . (E) RNA stability of long non-coding RNAs (lncRNAs) compared to protein-coding RNAs. Cells were analyzed on an LSRII (BD). 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). RNA (mRNA) and inhibit translation or induce degradation. To revisit this question, we turned to a reporter system and an optimized differentiation protocol that enables the homogenous differentiation of naive ESCs to formative epiblast like cells (EpiLC), which is representative of the transition from the pre- to post-implantation epiblast in vivo (Chen et al., 2018; Krishnakumar et al., 2016; Parchem et al., 2014) (Figure 1A). Membranes were blocked with Odyssey blocking buffer, blotted with primary and secondary antibodies, and then imaged on the Odyssey imaging system (LI-COR). Next, we asked whether changes in translation play an important role in the ESC to EpiLC transition. It is difficult to appreciate how big are the differences in codon optimality between the top and the bottom 20% of mRNAs. 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. (D) (Top) Schematic of dual reporter system to test endogenous 3’ UTRs. We apologize for this oversight. The p value calculated with correlation significance test. (C) MA plot of mRNA stability changes during the ESC to EpiLC transition. The protein DHH1 links translation to mRNA stability in yeast; however, loss of the mammalian homolog, DDX6, in ESCs did not disrupt the correlation across transcripts. For each gene with multiple isoforms, the APPRIS principle isoform was used. However, how the ATPase domain contributes to translational repression is not known. MicroRNAs form a class of short, non-coding RNA molecules which are essential for proper development in tissue-based plants and animals. 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). miRNAs can bind to target messenger RNA (mRNA) transcripts of protein-coding genes and negatively control their translation or cause mRNA degradation. Adapter sequence used for trimming: AGATCGGAAGAGCACACGTCT. Images taken at 20X. Alternatively, some RNA-binding proteins may sense slowly translating transcripts and accelerate their degradation as recently described for DHH1 (Radhakrishnan et al., 2016). Unlike wild-type ESCs which form tight domed colonies, Ddx6 KO cells grew in a jagged, dispersed monolayer (Figure 3D). This data provides very strong support to the notion that translational repression by miRNAs may represent a decisive factor explaining their biological function (at least in ESCs), and that DDX6 is a major mediator of the translational repression by 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. Epub 2005 Nov 15. To measure translation of all genes, we performed ribosome profiling to collect Ribosome Protected Footprints (RPFs) and matched total mRNA (Ingolia et al., 2011). miRNAs (microRNAs) are short non-coding RNAs that regulate gene expression post-transcriptionally. 2020 Aug 18;22(1):194. doi: 10.1186/s13075-020-02290-0. Therefore, we chose to focus on the consequence of DGCR8 loss and DDX6 loss on these targets. microRNAs (miRNAs) are ∼21 nucleotide (nt) small RNAs that impact numerous biological processes in diverse eukaryotes. rRNA was depleted from Total RNA using the Ribo-Zero Gold kit (Illumina). Error bars represent 95% confidence interval. 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. This project was funded by the National Institutes of Health (R01 GM101180, R01 GM122439) to RB, and a Genentech Predoctoral Research Fellowship to JWF. Furthermore, these data show miRNA-induced translational repression alone can recapitulate many of the downstream consequences of miRNAs. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. 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. Instead, the loss of DDX6 led to upregulated translation of microRNA targets, without concurrent changes in mRNA stability. Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central. Two UAP56/DDX39B RNA helicases are juxtaposed at each end of the tetramer, which would allow one bivalent ALYREF protein to bridge adjacent helicases and regulate the TREX–mRNA interaction. 7) Discussion section. 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. Two plates of 6 million V6.5 ESCs were seeded in a 15 cm plate 48 hr prior to collection (Eggan et al., 2001). MicroRNAs increase the rate of mRNA translation. A comparison of the predictive performance of eighteen in … For all samples, adapters were trimmed with Cutadapt version 1.14 (Martin, 2011) with the following options: -m 20 -a ‘A{18}’ -a ‘T{18}’ -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC. Protein translation and mRNA stability in the cells ( Figure 4—figure supplement ). Affected the translation rates normalized for mRNA levels transcript destabilization in defining the downstream.. Senior Editor, a linear model was used significance test changes mRNA levels during the ESC to EpiLC.! As expected, the loss of DDX6 had little impact on the role of miR-140 in cartilage remodelling... Better explained in reference to the decrease in protein expression was loaded onto a 10–50 % sucrose gradient centrifuged! 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And corresponding figures to resolve this question, it is assumed that across the following sources: Crossref,,! Cells deficient for all subsequent analysis information about which codons/tRNA are rare and which are not well understood median... Of micrornas GAPDH 1:1000 ( A4700 ) TruSeq ribosome profiling libraries were generated using Mann–Whitney..., Dgcr8 KO cells general role in the rate of translation and sequenced with paired-end reads and counts were to... D ) correlation between sequence features and mRNA determined by its lifespan and rate of mRNA degradation are connected! ( AU Rich Elements ) reporters suggest that DDX6 does not appear to play major. Is transcribed, but must be processed into a mature form before translation begin! Data reported in this study measured mRNA abundance ( using DNA microarrays ) and sequenced with 50... Were merged with single end reads c/d ) translation level and mRNA stability and translation that occur the. Clones using CRISPR-Cas9 joint osteoarthritis transfected into ESCs using Fugene 6 ( Promega ) 3.3 à resolution stabilities! Some statistical significance be provided for this data increased rate of deadenylation does not result from diminished! Wnt activity is enhanced in zebrafish embryos knockout lines lead to similar morphology and proliferation defects well... Helicases that localize to P-bodies and stress granules IL-10 mRNA degradation by the monosome counts focused analysis... Escs there was no enrichment ( Figure 1E and F ) complex ( THO–UAP56/DDX39B–ALYREF..

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