Loss of Cnot6l Impairs Inosine RNA Modifications in Mouse Oocytes

Mammalian oocytes must degrade maternal transcripts through a process called translational mRNA decay, in which maternal mRNA undergoes translational activation, followed by deadenylation and mRNA decay. Once a transcript is translationally activated, it becomes deadenylated by the CCR4-NOT complex. Knockout of CCR4-NOT Transcription Complex Subunit 6 Like (Cnot6l), a deadenylase within the CCR4-NOT complex, results in mRNA decay defects during metaphase I (MI) entry. Knockout of B-cell translocation gene-4 (Btg4), an adaptor protein of the CCR4-NOT complex, results in mRNA decay defects following fertilization. Therefore, mechanisms controlling mRNA turnover have significant impacts on oocyte competence and early embryonic development. Post-transcriptional inosine RNA modifications can impact mRNA stability, possibly through a translation mechanism. Here, we assessed inosine RNA modifications in oocytes, eggs, and embryos from Cnot6l-/- and Btg4-/- mice, which display stabilization of mRNA and over-translation of the stabilized transcripts. If inosine modifications have a role in modulating RNA stability, we hypothesize that in these mutant backgrounds, we would observe changes or a disruption in inosine mRNA modifications. To test this, we used a computational approach to identify inosine RNA modifications in total and polysomal RNA-seq data during meiotic maturation (GV, MI, and MII stages). We observed pronounced depletion of inosine mRNA modifications in samples from Cnot6l-/-, but not in Btg4-/- mice. Additionally, analysis of ribosome-associated RNA revealed clearance of inosine modified mRNA. These observations suggest a novel mechanism of mRNA clearance during oocyte maturation, in which inosine-containing transcripts decay in an independent, but parallel mechanism to CCR4-NOT deadenylation.

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Loss of Cnot6l impairs inosine RNA modifications in mouse oocytes

10.1101/2020.11.04.358010 ◽

2020 ◽

Author(s):

Pavla Brachova ◽

Nehemiah S. Alvarez ◽

Lane K. Christenson

Keyword(s):

Mrna Decay ◽

Rna Stability ◽

Adaptor Protein ◽

Mrna Turnover ◽

Rna Seq ◽

Rna Modifications ◽

Oocyte Competence ◽

Maternal Mrna ◽

Maternal Transcripts ◽

Translation Mechanism

AbstractMammalian oocytes must degrade maternal transcripts through a process called translational mRNA decay, in which maternal mRNA undergoes translational activation, followed by deadenylation and mRNA decay. Once a transcript is translationally activated, it becomes deadenylated by the CCR4-NOT complex. Knockout of Cnot6l, a deadenylase within the CCR4-NOT complex, results in mRNA decay defects during MI entry. Knockout of Btg4, an adaptor protein of the CCR4-NOT complex, results in mRNA decay defects following fertilization. Therefore, mechanisms controlling mRNA turnover have significant impacts on oocyte competence and early embryonic development. Post-transcriptional inosine RNA modifications can impact mRNA stability, possibly through a translation mechanism. Here, we assessed inosine RNA modifications in oocytes from Cnot6l-/- and Btg4-/- mice, which display stabilization of mRNA and over-translation of the stabilized transcripts. If inosine modifications have a role in modulating RNA stability, we hypothesize that in these mutant backgrounds, we would observe changes or a disruption in inosine mRNA modifications. To test this, we used a computational approach to identify inosine RNA modifications in total and polysomal RNA-seq data during meiotic maturation (GV, MI, and MII stages). We observed pronounced depletion of inosine mRNA modifications in oocytes from Cnot6l-/-, but not in Btg4-/- mice. Additionally, analysis of ribosome-associated RNA revealed clearance of inosine modified mRNA. These observations suggest a novel mechanism of mRNA clearance during oocyte maturation, in which inosine-containing transcripts decay in an independent, but parallel mechanism to CCR4-NOT deadenylation.

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YBX1 Mediates Alternative Splicing and Maternal mRNA Decay During Pre-Implantation Development

10.21203/rs.3.rs-900476/v1 ◽

2021 ◽

Author(s):

Mingtian Deng ◽

Baobao Chen ◽

Zifei Liu ◽

Yongjie Wan ◽

Dongxu Li ◽

...

Keyword(s):

Alternative Splicing ◽

Transcriptional Activity ◽

Mrna Decay ◽

Molecular Mechanisms ◽

Rna Stability ◽

Differentially Expressed ◽

Rna Seq ◽

Cell Stage ◽

Aberrant Expression ◽

Maternal Mrna

Abstract Background: In mammals, maternal gene products decay and zygotic genome activation (ZGA) during maternal to zygotic transition (MZT) is critical for pre-implantation. Y-box binding protein YBX1 plays vital roles in RNA stabilization and transcriptional regulation, but its roles in pre-implantation development remain to be elucidated. The objective of this study is to investigate the role and the molecular mechanisms of YBX1 during MZT.Methods: RNA-seq datasets in mice, human, bovine, and goat embryos were re-analyzed. YBX1 was knocked down by siRNA microinjection. The 8-cell stage embryos were collected for RNA-seq. The differentially expressed genes and alternative splicing (AS) events were identified using DESeq2 and rMATs, respectively. GO/KEGG/GSEA enrichment analysis was performed using clusterProfiler and enrichplot. Furthermore, 5-EU staining was performed to confirm the effect of YBX1 knockdown on transcriptional activity.Results: The expression of YBX1 was increased during MZT in goat, bovine, human, and mice. By microinjection of siRNA against YBX1, we successfully knocked down YBX1, and the embryo development was impaired in YBX1 knockdown embryos. Using RNA-seq, we identified 1623 up-regulated and 3531 down-regulated genes in the 8-cell stage YBX1 knockdown embryos. Of note, the down-regulated genes were enriched in regulation of RNA/mRNA stability and spliceosome, suggesting that YBX1 might medicate RNA stability and AS. To this end, we identified 3284 differential AS events and 1322 differentially expressed maternal mRNAs at the 8-cell stage YBX1 knockdown embryos. Meanwhile, the splicing factors and mRNA decay related showed aberrant expression. Moreover, the transcriptional activity during ZGA in goat and mice was compromised when YBX1 was knocked down.Conclusion: Our results identify that YBX1 serves an important role in maternal mRNA decay, alternative splicing, and the transcriptional activity required for early embryogenesis, which will broaden the current understanding of YBX1 functions during the stochastic reprogramming events.

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Regulation of RNA stability at the 3′ end

Biological Chemistry ◽

10.1515/hsz-2020-0325 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Mallory I. Frederick ◽

Ilka U. Heinemann

Keyword(s):

Mrna Decay ◽

Rna Stability ◽

Rna Modifications ◽

Dual Roles ◽

Cellular Pathways ◽

Low Levels

AbstractRNA homeostasis is regulated by a multitude of cellular pathways. Although the addition of untemplated adenine residues to the 3′ end of mRNAs has long been known to affect RNA stability, newly developed techniques for 3′-end sequencing of RNAs have revealed various unexpected RNA modifications. Among these, uridylation is most recognized for its role in mRNA decay but is also a key regulator of numerous RNA species, including miRNAs and tRNAs, with dual roles in both stability and maturation of miRNAs. Additionally, low levels of untemplated guanidine and cytidine residues have been observed as parts of more complex tailing patterns.

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Dynamics and clinical relevance of maternal mRNA clearance during the oocyte-to-embryo transition in humans

Nature Communications ◽

10.1038/s41467-020-18680-6 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Qian-Qian Sha ◽

Wei Zheng ◽

Yun-Wen Wu ◽

Sen Li ◽

Lei Guo ◽

...

Keyword(s):

Mrna Decay ◽

Meiotic Spindle ◽

Preimplantation Embryos ◽

Human Embryos ◽

Developmental Potential ◽

Maternal Mrna ◽

Before And After ◽

Maternal Transcripts ◽

Human Preimplantation Embryos

Abstract Maternal mRNA clearance is an essential process that occurs during maternal-to-zygotic transition (MZT). However, the dynamics, functional importance, and pathological relevance of maternal mRNA decay in human preimplantation embryos have not yet been analyzed. Here we report the zygotic genome activation (ZGA)-dependent and -independent maternal mRNA clearance processes during human MZT and demonstrate that subgroups of human maternal transcripts are sequentially removed by maternal (M)- and zygotic (Z)-decay pathways before and after ZGA. Key factors regulating M-decay and Z-decay pathways in mouse have similar expression pattern during human MZT, suggesting that YAP1-TEAD4 transcription activators, TUT4/7-mediated mRNA 3ʹ-oligouridylation, and BTG4/CCR4-NOT-induced mRNA deadenylation may also be involved in the regulation of human maternal mRNA stability. Decreased expression of these factors and abnormal accumulation of maternal transcripts are observed in the development-arrested embryos of patients who seek assisted reproduction. Defects of M-decay and Z-decay are detected with high incidence in embryos that are arrested at the zygote and 8-cell stages, respectively. In addition, M-decay is not found to be affected by maternal TUBB8 mutations, although these mutations cause meiotic cell division defects and zygotic arrest, which indicates that mRNA decay is regulated independent of meiotic spindle assembly. Considering the correlations between maternal mRNA decay defects and early developmental arrest of in vitro fertilized human embryos, M-decay and Z-decay pathway activities may contribute to the developmental potential of human preimplantation embryos.

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RNA Modification Level Estimation with pulseR

Genes ◽

10.3390/genes9120619 ◽

2018 ◽

Vol 9 (12) ◽

pp. 619

Author(s):

Etienne Boileau ◽

Christoph Dieterich

Keyword(s):

Experimental Approach ◽

High Efficiency ◽

Rna Modification ◽

Model Parameters ◽

Metabolic Labeling ◽

Rna Seq ◽

Rna Modifications ◽

Log Odds ◽

Pros And Cons ◽

Wide Scale

RNA modifications regulate the complex life of transcripts. An experimental approach called LAIC-seq was developed to characterize modification levels on a transcriptome-wide scale. In this method, the modified and unmodified molecules are separated using antibodies specific for a given RNA modification (e.g., m6A). In essence, the procedure of biochemical separation yields three fractions: Input, eluate, and supernatent, which are subjected to RNA-seq. In this work, we present a bioinformatics workflow, which starts from RNA-seq data to infer gene-specific modification levels by a statistical model on a transcriptome-wide scale. Our workflow centers around the pulseR package, which was originally developed for the analysis of metabolic labeling experiments. We demonstrate how to analyze data without external normalization (i.e., in the absence of spike-ins), given high efficiency of separation, and how, alternatively, scaling factors can be derived from unmodified spike-ins. Importantly, our workflow provides an estimate of uncertainty of modification levels in terms of confidence intervals for model parameters, such as gene expression and RNA modification levels. We also compare alternative model parametrizations, log-odds, or the proportion of the modified molecules and discuss the pros and cons of each representation. In summary, our workflow is a versatile approach to RNA modification level estimation, which is open to any read-count-based experimental approach.

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Maternal factor PABPN1L is essential for maternal mRNA degradation during maternal-to-zygotic transition

10.1101/2020.08.20.258830 ◽

2020 ◽

Author(s):

Ying Wang ◽

Tianhao Feng ◽

Xiaodan Shi ◽

Siyu Liu ◽

Zerui Wang ◽

...

Keyword(s):

Mouse Model ◽

Large Scale ◽

Mrna Degradation ◽

Female Infertility ◽

Control Group ◽

Rna Seq ◽

Cell Stage ◽

Maternal Mrna ◽

Maternal To Zygotic Transition ◽

Group A

AbstractInfertility affects 10% - 15% of families worldwide. However, the pathogenesis of female infertility caused by abnormal early embryonic development is not clear. We constructed a mouse model (Pabpn1l -/-) simulating the splicing abnormality of human PABPN1L and found that the female was sterile and the male was fertile. The Pabpn1l -/- oocytes can be produced, ovulated and fertilized normally, but cannot develop beyond the 2-cell stage. Using RNA-Seq, we found a large-scale upregulation of RNA in Pabpn1l -/- MII oocytes. Of the 2401 transcripts upregulated in Pabpn1l-/- MII oocytes, 1523 transcripts (63.4%) were also upregulated in Btg4 -/- MII oocytes, while only 53 transcripts (2.2%) were upregulated in Ythdf2 -/- MII oocytes. We documented that transcripts in zygotes derived from Pabpn1l -/- oocytes have a longer poly(A) tail than the control group, a phenomenon similar to that in Btg4-/- mice. Surprisingly, the poly(A) tail of these mRNAs was significantly shorter in the Pabpn1l -/- MII oocytes than in the Pabpn1l +/+. These results suggest that PABPN1L is involved in BTG4-mediated maternal mRNA degradation, and may antagonize poly(A) tail shortening in oocytes independently of its involvement in maternal mRNA degradation. Thus, PABPN1L variants could be a genetic marker of female infertility.

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Temporal regulation of the Xenopus FGF receptor in development: a translation inhibitory element in the 3′ untranslated region

Development ◽

10.1242/dev.121.6.1775 ◽

1995 ◽

Vol 121 (6) ◽

pp. 1775-1785 ◽

Cited By ~ 3

Author(s):

E.P. Robbie ◽

M. Peterson ◽

E. Amaya ◽

T.J. Musci

Keyword(s):

Protein Interactions ◽

Translational Control ◽

Untranslated Region ◽

Rna Stability ◽

Immature Oocyte ◽

Translational Repression ◽

Meiotic Maturation ◽

Fgf Receptor ◽

Cis Acting ◽

Maternal Mrna

Early frog embryogenesis depends on a maternal pool of mRNA to execute critical intercellular signalling events. FGF receptor-1, which is required for normal development, is stored as a stable, untranslated maternal mRNA transcript in the fully grown immature oocyte, but is translationally activated at meiotic maturation. We have identified a short cis-acting element in the FGF receptor 3′ untranslated region that inhibits translation of synthetic mRNA. This inhibitory element is sufficient to inhibit translation of heterologous reporter mRNA in the immature oocyte without changing RNA stability. Deletion of the poly(A) tract or polyadenylation signal sequences does not affect translational inhibition by this element. At meiotic maturation, we observe the reversal of translational repression mediated by the inhibitory element, mimicking that seen with endogenous maternal FGF receptor mRNA at meiosis. In addition, the activation of synthetic transcripts at maturation does not appear to require poly(A) lengthening. We also show that an oocyte cytoplasmic protein specifically binds the 3′ inhibitory element, suggesting that translational repression of Xenopus FGF receptor-1 maternal mRNA in the oocytes is mediated by RNA-protein interactions. These data describe a mechanism of translational control that appears to be independent of poly(A) changes.

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Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis

Genes ◽

10.3390/genes10010026 ◽

2019 ◽

Vol 10 (1) ◽

pp. 26 ◽

Cited By ~ 18

Author(s):

Kayla Borland ◽

Jan Diesend ◽

Taku Ito-Kureha ◽

Vigo Heissmeyer ◽

Christian Hammann ◽

...

Keyword(s):

Messenger Rna ◽

Isotope Labeling ◽

Rna Stability ◽

Internal Standard ◽

Modified Nucleosides ◽

Rna Modification ◽

Mouse Tissue ◽

Rna Modifications ◽

Internal Standards ◽

Translation Fidelity

Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue.

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mRNA decay mediated by two distinct AU-rich elements from c-fos and granulocyte-macrophage colony-stimulating factor transcripts: different deadenylation kinetics and uncoupling from translation.

Molecular and Cellular Biology ◽

10.1128/mcb.15.10.5777 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5777-5788 ◽

Cited By ~ 188

Author(s):

C Y Chen ◽

N Xu ◽

A B Shyu

Keyword(s):

Mammalian Cells ◽

Mrna Decay ◽

Rna Stability ◽

Mrna Degradation ◽

Colony Stimulating Factor ◽

Turnover Rates ◽

Cis Acting ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Poly(A) tail removal is a critical first step in the decay pathway for many yeast and mammalian mRNAs. Poly(A) shortening rates can be regulated by cis-acting sequences within the transcribed portion of mRNA, which in turn control mRNA turnover rates. The AU-rich element (ARE), found in the 3' untranslated regions of many highly labile mammalian mRNAs, is a well-established example of this type of control. It represents the most widespread RNA stability determinant among those characterized in mammalian cells. Here, we report that two structurally different AREs, the c-fos ARE and the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE, both direct rapid deadenylation as the first step in mRNA degradation, but by different kinetics. For c-fos-ARE-mediated decay, the mRNA population undergoes synchronous poly(A) shortening and is deadenylated at the same rate, implying the action of distributive or nonprocessive ribonucleolytic digestion of poly(A) tails. In contrast, the population of granulocyte-macrophage colony-stimulating factor ARE-containing mRNAs is deadenylated asynchronously, with the formation of fully deadenylated intermediates, consistent with the action of processive ribonucleolytic digestion of poly(A) tails. An important general implication of this finding is that different RNA-destabilizing elements direct deadenylation either by modulating the processivity at which a single RNase functions or by recruiting kinetically distinct RNases. We have also employed targeted inhibition of translation initiation to demonstrate that the RNA-destabilizing function of both AREs can be uncoupled from translation by ribosomes. In addition, a blockade of ongoing transcription has been used to further probe the functional similarities and distinctions of these two AREs. Our data suggest that the two AREs are targets of two distinct mRNA decay pathways. A general model for ARE-mediated mRNA degradation involving a potential role for certain heterogeneous nuclear ribonucleoproteins and ARE-binding proteins is proposed.

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Analytical validation of an immune response assay for classifying solid tumors.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.7_suppl.68 ◽

2017 ◽

Vol 35 (7_suppl) ◽

pp. 68-68

Author(s):

Jeff Conroy ◽

Sean Glenn ◽

Blake Burgher ◽

Antonios Papanicolau-Sengos ◽

Jonathan Andreas ◽

...

Keyword(s):

Immune Response ◽

Nucleic Acids ◽

Solid Tumors ◽

Checkpoint Inhibitors ◽

Rna Stability ◽

Analytical Performance ◽

Rna Seq ◽

Diagnostic Laboratory ◽

Typical Sample ◽

Dna And Rna

68 Background: An advanced diagnostic laboratory test (Immune Advance) was developed that analyzes multiple DNA and RNA biomarkers to predict the likelihood of response to checkpoint inhibitors in patients with solid tumors. Using RNA-seq and DNA-seq, the NGS test measures gene expression of immune response genes and overall mutational burden. The studies described here were designed to validate the analytical performance of the test on the Ion Torrent System in our CLIA lab. Methods: Studies were designed to characterize the analytical performance of an immune response NGS assay using total nucleic acids from >100 FFPE tissues representing NSCLC, melanoma, renal cell carcinoma, and bladder cancer. Performance variables with respect to gene-specific amplicon specificity, linearity, and limits of detection were estimated with various sample dilutions and input nucleic acids. The effects of the tumor micro-environment (adjacent benign tissue, necrosis) was evaluated by including these potential interferents in the assay. Analytical precision including intra-assay, inter-assay, and inter-operator reproducibility was measured by testing replicate FFPE tissue sections. Accuracy was determined by comparing select transcript and DNA level variants with those from established IHC, RT-PCR, and NGS assays. Transcript stability in FFPE specimens was evaluated in serial sections from blocks with routine storage and compared to originating matched fresh frozen specimens. Results: RNA stability was demonstrated by high degree of correlation between matched frozen and FFPE samples. Analytic precision was demonstrated by high correlation between RNA-Seq and TaqMan results for genes evaluated. As compared to IHC the results for RNA-Seq were continuous rather than bimodal and allowed for a much more detailed analysis of the immune response. Immune signatures were maintained with variable RNA/DNA input amounts, altered tumor micro-environments, and potential interferents demonstrating tolerance to typical sample types tested. Reproducibility results show little variation between runs and operators. Conclusions: The analytical performance of the Immune Advance assay has been validated for clinical use using FFPE tumor specimens.

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