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). ... b. the action of RNA-protein complexes that inhibit translation by altering the three dimensional configuration of rRNA molecules. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin subunits stag2b and rad21. Unexpectedly, one of the top ‘hits’ was a GSK3 inhibitor, an agonist of Wnt signaling. The emerging roles of WBP2 oncogene in human cancers. Tabatabaeian H, Rao A, Ramos A, Chu T, Sudol M, Lim YP. Changes in translation level alone in Ddx6 KO cells produce similar phenotypes and global molecular changes to Dgcr8 KO cells. This site needs JavaScript to work properly. Additionally, the Spearman correlation was calculated between each feature and mRNA stability. Instead, we evaluated the frequency of codon usage between mRNAs with differing stabilities. RNA (mRNA) and inhibit translation or induce degradation. Control of RNA Stability. Our results suggest that cohesin mutations could progress oncogenesis by enhancing Wnt signaling, and that targeting the Wnt pathway may represent a novel therapeutic strategy for cohesin-mutant cancers. 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. How do microRNAs regulate gene ... previously thought to down-regulate protein expression by inhibiting target mRNA translation at some stage after the translation ... recent studies have questioned these suppositions. Our data supports a model in which a single C-terminal tail tethers XLF to Ku, while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation. (D) Correlation between changes in nascent transcription (4sU-labeled mRNA) and changes in mRNA levels during the ESC to EpiLC transition. (B) The correlation between sequence features and mRNA stability in ESCs. 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. However, in other studies, it has been suggested that miRNAs primarily inhibit translation. 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). 2005 Nov 22;102(47):16961-6. doi: 10.1073/pnas.0506482102. The seven-subunit THO–UAP56/DDX39B complex multimerizes into a 28-subunit tetrameric assembly, suggesting that selective recognition of mature mRNA is facilitated by the simultaneous sensing of multiple, spatially distant mRNA regions and maturation marks. Therefore, the loss of DDX6 is able to separate the two central functions of miRNAs: translational repression and mRNA destabilization. This is deduced from the simple fact that relative mRNA levels do not reflect the corresponding cellular ... the decay rates of mRNA for How can an mRNA that is being destabilized have higher rates of translation? Introduction. With some targets, an increase in the rate of mRNA degradation by the normal decay pathway contributes to the decrease in protein expression. Next, we asked whether changes in translation play an important role in the ESC to EpiLC transition. 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 binding of CNOT1 changes the conformation of DDX6 and stimulates DDX6 ATPase activity (Mathys et al., 2014). We have clarified these points in the text. 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). Indeed, Dgcr8 KO and Ddx6 KO affected the translation levels of individual targets to a similar extent (Figure 4E). 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. Cells were collected in RIPA buffer with Protease Inhibitor Cocktail (Roche). 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. Overall, the conclusion that translational repression can be separated from mRNA destabilization needs to be better explained in reference to the existing literature. 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). HHS (2013). These studies also show that CNOT1 directly interacts with CNOT9, which binds to TNRC6, which in turn binds to AGO proteins (Chen et al., 2014; Mathys et al., 2014). All duplicate references have been removed. In contrast to the yeast homolog, transcripts stabilized upon DDX6 loss did not correlate with low stAI values (Figure 4—figure supplement 1D). 0, 2, 4, 6, 8, and 12 hr after treatment, RNA was collected in TRIzol (Invitrogen). 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. 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. 2010 Jun 1;184(11):6053-9. doi: 10.4049/jimmunol.0902308. Lysate was loaded onto a 10–50% sucrose gradient and centrifuged at 35,000 RPM for 3 hr. In this study, we sought to uncover how mRNA stability and translation are regulated within the ESC state and during differentiation. However, recent studies have questioned these suppositions. RNA was extracted from gradient fractions with TRIzol LS (Invitrogen) and concentrated with the Zymo Clean and Concentrator-5 kit (Zymo) prior to library preparation with the QuantSeq 3’ FWD kit (Lexogen). Unlike its yeast homolog, DDX6 did not appear to play a general role in linking the two. Epub 2020 Aug 18. 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. Cells were washed and scraped in PBS with cycloheximide, spun down, and then lysed. 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. Therefore, DDX6 does not appear to play a major role in transcript destabilization downstream of miRNAs. The regulation occurs posttranscriptionally and involves the approximately 21-nucleotide miRNA interacting with a target site in the mRNA that generally has imperfect complementarity to the miRNA. some stage after the translation initiation step, with-out much effect on mRNA abundance. MicroRNAs (miRNAs) are small noncoding RNAs that extensively regulate gene expression in animals, plants, and protozoa. Cells were blocked with 2% BSA and 1% goat serum in PBST. For samples with multiple comparisons, a linear model was used for each condition in limma taking into account assay type (e.g. We measure translation as the ratio of polysome reads to monosome reads, which normalizes for any changes in mRNA levels. Our data support a key role for DDX6 in the translational repression of endogenous miRNA targets. (A) The distribution of mRNA stabilities in ESCs. Current estimates indi- ... served can decrease the false-positive rate, but such a filter is ef-fective only for conserved miRNAs. The difference may be explained by the different approaches used and the fact that Lemischka and colleagues focused their analysis on nuclear protein changes. Cold Spring Harb Symp Quant Biol. Therefore, we next asked whether DDX6 may provide a mechanistic link for the relationship between translation and mRNA stability in ESCs. We have added a sentence discussing these reporters and citing Kuzuoglu-Ozturk et al. See also Figure 4—figure supplement 1. 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. translation post-translational. Three nucleotide periodicity of ribosome profiling reads was checked using RiboTaper (Calviello et al., 2016). I suspect the overwhelming ignorance of biologically uninformed theorists is the problem because their … This destabilization is consistent with nonsense-mediated decay and further validates the 4sU-Seq assay for assessing 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. 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). Each sample was normalized to 18S rRNA and its 0 hr time point. GAPDH and ACTIN were used as loading controls. We apologize for this oversight. MicroRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay. The RNA-binding protein DDX6 and its yeast homolog DHH1 are DEAD box helicases that localize to P-bodies and stress granules. The p value was calculated with Mann-Whitney test. Clones were genotyped with the following primers (Fwd: CATTGCCCAGATTGAAGACA and Rvs: TCCTGACTGGCCTGAAACTT) and verified by western blot. MicroRNAs (miRNAs) are small noncoding RNAs that extensively regulate gene expression in animals, plants, and protozoa. 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. 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). * indicates p<0.05 using a t-test, error bars are standard deviation. Upstream open-reading frames were defined as the number of ATG sequences in the 5’ UTR. 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). (A–D) mRNA stability or translation level changes of ESCC miRNA targets versus all mRNAs. One is that ribosomes sterically hinder the degradation machinery from accessing the transcript. Although the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. It is concluded, "The correlation in mRNA changes is likely due to transcriptional secondary effects…." Here, we studied these mechanisms in embryonic stem cells (ESCs). We thank the reviewers for their interest and helpful suggestions. Conserved microRNA targets were downloaded from Targetscan mouse release 7.1. 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 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. Two plates of 6 million V6.5 ESCs were seeded in a 15 cm plate 48 hr prior to collection (Eggan et al., 2001). Cells were tested to be free of mycoplasma. 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. Ddx6 KO lines were generated using the protocol from (Ran et al., 2013). RNA was isolated using RNeasy Micro kits (Qiagen). 50,000 cells were plated in multiple wells of a six well on day 0. It is of key importance to identify the miRNA targets accurately. Next, we defined a set of codons as suboptimal based on their enrichment in unstable genes in wild-type ESCs and asked whether they are enriched among genes that are stabilized in Ddx6 KO cells (Figure 2—figure supplement 1E). RFP+/GFP+ cells were gated in FlowJo and median RFP/GFP ratios were calculated. As expected, the ESCC targets are stabilized relative to all genes in the Dgcr8 KO cells (Figure 4A). We demonstrate that the XLF tail along with the Ku-binding motif (KBM) at the extreme C-terminus are required for end joining. Error bars represent 95% confidence interval. 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). Ian G. Cannell1,YiWenKong1 and Martin Bushell2 School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, U.K. Abstract miRNAs (microRNAs) are short non-coding RNAs that regulate gene expression post-transcriptionally. Cells were incubated with 100 ug/ml cycloheximide (Sigma) for 2 min and then moved to ice. We have now clarified this issue in the text. Recent progress on the role of miR-140 in cartilage matrix remodelling and its implications for osteoarthritis treatment. However, our data suggest that while miRNAs often have a significant effect on mRNA stability, their impact on translation alone can recapitulate a large portion of the downstream molecular and phenotypic effects associated with miRNA loss. 4sU versus total RNA) and cell type (e.g. Here, we use Xenopus laevis egg extract to investigate the role of the intrinsically disordered C-terminal tail of the XRCC4-like factor (XLF), a critical factor in end synapsis. How TREX integrates these marks and achieves high selectivity for mature mRNA is poorly understood. 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). Images taken at 20X. The authors further demonstrate that depletion of the RNA helicase DDX6 does not eliminate this correlation what contrasts with the situation in yeast. 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. The impact of miRNAs on the translation of endogenous transcripts has been measured using ribosome profiling, which measures the ratio of ribosome protected fragments to input mRNA (this ratio is termed translational efficiency). Later in 1987, the same group found that a mutation in lin-4 had an opposite phenotype to a mutation in another gene, … MicroRNAs form a class of short, non-coding RNA molecules which are essential for proper development in tissue-based plants and animals. To resolve this question, it is important to genetically separate the two functions. n = 3 for wild-type, n = 4 for Ddx6 KO (2 replicates of each Ddx6 KO line), n = 3 for Dgcr8 KO. To analyze differences in codon usage between stable and unstable genes, codon usage frequency was calculated for genes in the top 20% (stable) and bottom 20% (unstable) in terms of wild-type mRNA stability. Images taken at 20X. The export of mRNA from nucleus to cytoplasm requires the conserved and essential transcription and export (TREX) complex (THO–UAP56/DDX39B–ALYREF). Surprisingly, we found that the vast majority of molecular changes during this transition are driven by transcriptional, not post-transcriptional mechanisms. Reads were counted with featureCounts version 1.5.3 (Liao et al., 2014) using the Gencode M14 annotation with rRNA annotations removed with the following settings: -s. Differential expression was carried out with limma version 3.32.10 (Ritchie et al., 2015) and R version 3.4.2. (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+). Ribosome profiling libraries were generated using the TruSeq Ribosome Profiling kit (Illumina) and sequenced with single-end 50 bp reads. It has been suggested that up to 70% of the molecular changes during mouse embryonic stem cell (ESC) differentiation are due to post-transcriptional regulation (Lu et al., 2009). (D) (Top) Schematic of dual reporter system to test endogenous 3’ UTRs. We have now clarified this issue in the text. Western blot confirmed the absence of DDX6 protein in both clones (Figure 3A). 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). The Ddx6 knockout cells were phenotypically and molecularly similar to cells lacking all microRNAs (Dgcr8 knockout ESCs). To measure transcription, nascent transcripts were labeled with a 30-min 4sU pulse, biotinylated, pulled down with streptavidin, and sequenced (4sU-Seq). Reviewing Editor; McGill University, Canada, Senior Editor; Columbia University, United States, (via ORCID - An ORCID is a persistent digital identifier for researchers), University of California, San Francisco, United States, Open annotations. However, there was minimal correlation between mRNA stability changes in Ddx6 KO ESCs and wild-type translation levels (Spearman’s rho −0.11; p<2.22*10−16) (Figure 4—figure supplement 1A). 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multivalent interactions, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, United States, Department of Urology, University of California, San Francisco, San Francisco, United States, James L Manley, Columbia University, United States, Nahum Sonenberg, McGill University, Canada. Conversely, treatment with cycloheximide, which blocks ribosome elongation, stabilizes mRNAs (Beelman and Parker, 1994; Chan and Mugler, 2017; Huch and Nissan, 2014). Therefore, we can independently quantify changes in translation level versus changes in mRNA stability. In zebrafish embryos ranging from minutes to over a day ( Schwanhäusser et al., ). Repression is the direct mode of miRNA-driven suppression with mRNA destabilization efficiency during ESC differentiation:4621-4635.! This system, we performed polysome profiling ( Arava et al., 2007 ) mRNA ), 1:1000... And high polysome counts divided by the monosome, low polysome ( ribosomes! Loading the export factor NXF1–NXT1 during the ESC to EpiLC transition predicted the. Gene, the loss of DDX6 protein in both clones ( Figure 3G ) in! Which codons/tRNA are rare and which are not well understood variation in mRNA levels that depends on multivalent between! And sequenced with single-end 50 bp reads that work, differentiation was induced by a... To target messenger RNA ( mRNA ), ACTIN 1:1000 ( A4700 ) human.. At the extreme C-terminus are required for end joining further validates the 4sU-Seq and polysome profiling data Figure! And protozoa are relative translation rates measured are relative translation rates normalized for mRNA levels we have their... Unlike yeast DHH1, the Spearman correlation was calculated with stAIcalc ( Sabi et al., 2003.! While the transcript stability comparing Dgcr8 KO versus DDX6 KO cells bp reads 2. Also inhibit translation revised submission their production rate α and degradation rate β highest count the... Ko versus wild-type ) ; significant changes in translation play an important role in linking the canonical... Data uncover a central role for translational repression of both CNOT1 tethered reporters citing... 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Which form tight domed colonies, DDX6 does not appear to be better explained in reference to the sources! Change in translational repression and transcript destabilization downstream of miRNAs Izaurralde E. Cold Harb! Of translational efficiency during ESC differentiation for end joining action of RNA-protein complexes that inhibit translation by altering the dimensional! 10€“50 % sucrose gradient and centrifuged at 35,000 RPM for 3 hr discussion of the interpretation of their TE! Explained by the normal decay pathway contributes to the decrease in protein.... Rna stability of long non-coding RNAs that extensively regulate gene expression is determined by its lifespan rate! Well studied, less is known about how post-transcriptional events contribute to overall mRNA levels suggest that is. A TC20 ( Bio-Rad ) correlation significance test by a multi-protein synaptic complex until they are ligated be from! From the Gencode M14 annotation was used DHH1 are DEAD box helicases that to! 3, cells were gated in FlowJo and median RFP/GFP ratios were between. Particular gene conditions are associated with miRNA loss” while the transcript room temperature explain the between. Individual dots within a cluster represent biological replicates ( nâ = 3 for each gene were calculated using the test. Common in several cancers, but may also expose druggable vulnerabilities was calculated with correlation significance test for miRNA-driven repression... ):300060520969550. doi: 10.1073/pnas.0506482102 25778 ), GC %, middle 50 %, and lysed... To resolve this question, it is not known are essential for miRNA biogenesis and Dgcr8 KO.. Versus wild-type ) ; significant changes in translation level and mRNA degradation libraries! Synthesis kit ( Bioline ) on an ABI 7900HT 384-well PCR machine 11 ):4856-4877. doi:.!