scholarly journals Regulation of RNA stability at the 3′ end

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.

scholarly journals Loss of Cnot6l impairs inosine RNA modifications in mouse oocytes

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.

scholarly journals Loss of Cnot6l Impairs Inosine RNA Modifications in Mouse Oocytes

2021 ◽  
Vol 22 (3) ◽  
pp. 1191
Author(s):  
Pavla Brachova ◽  
Nehemiah S. Alvarez ◽  
Lane K. Christenson
Keyword(s):  
Mrna Decay ◽  
Rna Stability ◽  
Adaptor Protein ◽  
Rna Seq ◽  
Rna Modifications ◽  
Oocyte Competence ◽  
Maternal Mrna ◽  
Maternal Transcripts ◽  
Translation Mechanism ◽  
Gene 4

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.

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

Genes ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 26 ◽  
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.

scholarly journals 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.

1995 ◽  
Vol 15 (10) ◽  
pp. 5777-5788 ◽  
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.

scholarly journals 5-Methylcytosine RNA Modifications Promote Retrovirus Replication in an ALYREF Reader Protein-Dependent Manner

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Matthew Eckwahl ◽  
Ruyi Xu ◽  
Julia Michalkiewicz ◽  
Wen Zhang ◽  
Pooja Patel ◽  
...  
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rna Stability ◽  
Translation Efficiency ◽  
Dependent Manner ◽  
Rna Modifications ◽  
Nucleotide Resolution ◽  
And Function ◽  
Murine Leukemia ◽  
Retrovirus Replication

ABSTRACT RNA modifications play diverse roles in regulating RNA function, and viruses co-opt these pathways for their own benefit. While recent studies have highlighted the importance of N6-methyladenosine (m6A)—the most abundant mRNA modification—in regulating retrovirus replication, the identification and function of other RNA modifications in viral biology have been largely unexplored. Here, we characterized the RNA modifications present in a model retrovirus, murine leukemia virus (MLV), using mass spectrometry and sequencing. We found that 5-methylcytosine (m5C) is highly enriched in viral genomic RNA relative to uninfected cellular mRNAs, and we mapped at single-nucleotide resolution the m5C sites, which are located in multiple clusters throughout the MLV genome. Further, we showed that the m5C reader protein ALYREF plays an important role in regulating MLV replication. Together, our results provide a complete m5C profile in a virus and its function in a eukaryotic mRNA. IMPORTANCE Over 130 modifications have been identified in cellular RNAs, which play critical roles in many cellular processes, from modulating RNA stability to altering translation efficiency. One such modification, 5-methylcytosine, is relatively abundant in mammalian mRNAs, but its precise location and function are not well understood. In this study, we identified unexpectedly high levels of m5C in the murine leukemia virus RNA, precisely mapped its location, and showed that ALYREF, a reader protein that specifically recognizes m5C, regulates viral production. Together, our findings provide a high-resolution atlas of m5C in murine leukemia virus and reveal a functional role of m5C in viral replication.

Attack from both ends: mRNA degradation in the crenarchaeon Sulfolobus solfataricus

2013 ◽  
Vol 41 (1) ◽  
pp. 379-383 ◽  
Author(s):  
Elena Evguenieva-Hackenberg ◽  
Udo Bläsi
Keyword(s):  
Mrna Decay ◽  
Sulfolobus Solfataricus ◽  
Rna Stability ◽  
Mrna Degradation ◽  
Stability Control ◽  
Control Gene ◽  
Control Gene Expression ◽  
Domains Of Life ◽  
Enzyme Complexes ◽  
Exosome Complex

RNA stability control and degradation are employed by cells to control gene expression and to adjust the level of protein synthesis in response to physiological needs. In all domains of life, mRNA decay can commence in the 5′–3′ as well as in the 3′–5′-direction. Consequently, mechanisms are in place conferring protection on mRNAs at both ends. Upon deprotection, dedicated enzymes/enzyme complexes access either end and trigger 5′–3′ or 3′–5′-directional decay. In the present paper, we first briefly review the general mRNA decay pathways in Bacteria and Eukarya, and then focus on 5′–3′ and 3′–5′-directional decay in the crenarchaeon Sulfolobus solfataricus, which is executed by a RNase J-like ribonuclease and the exosome complex respectively. In addition, we describe mechanisms that stabilize mRNAs at the 5′- as well as at the 3′-end.

scholarly journals Dual roles for MEF2A and MEF2D during human macrophage terminal differentiation and c-Jun expression

2010 ◽  
Vol 430 (2) ◽  
pp. 237-244 ◽  
Author(s):  
Catherine Aude-Garcia ◽  
Véronique Collin-Faure ◽  
Huguette Bausinger ◽  
Daniel Hanau ◽  
Thierry Rabilloud ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Potential Interaction ◽  
Human Macrophage ◽  
Dual Roles ◽  
Differentiated Cells ◽  
Deacetylase Inhibitor ◽  
Undifferentiated Cells ◽  
Low Levels ◽  
Chromatin Acetylation ◽  
First Time

Recent reports have evidenced a role for MEF2C (myocyte enhancer factor 2C) in myelopoiesis, although the precise functions of this transcription factor are still unclear. We show in the present study that MEF2A and MEF2D, two other MEF2 family members, are expressed in human primary monocytes and in higher amounts in monocyte-derived macrophages. High levels of MEF2A–MEF2D heterodimers are found in macrophage-differentiated HL60 cells. Chromatin immunoprecipitations demonstrate that MEF2A is present on the c-Jun promoter, both in undifferentiated and in macrophage-differentiated cells. Moreover, c-Jun expression is derepressed in undifferentiated cells in the presence of HDAC (histone deacetylase) inhibitor, indicating the importance of chromatin acetylation in this process. We show that MEF2A/D dimers strongly interact with HDAC1, and to a lesser extent with HDAC7 in macrophages, whereas low levels of MEF2A/D–HDAC1 complexes are found in undifferentiated cells or in monocytes. Since trichostatin A does not disrupt MEF2A/D–HDAC1 complexes, we analysed the potential interaction of MEF2A with p300 histone acetyltransferase, whose expression is up-regulated in macrophages. Interestingly, endogenous p300 only associates with MEF2A in differentiated macrophages, indicating that MEF2A/D could activate c-Jun expression in macrophages through a MEF2A/D–p300 activator complex. The targets of MEF2A/D–HDAC1–HDAC7 multimers remain to be identified. Nevertheless, these data highlight for the first time the possible dual roles of MEF2A and MEF2D in human macrophages, as activators or as repressors of gene transcription.

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

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.

scholarly journals Deep and accurate detection of m6A RNA modifications using miCLIP2 and m6Aboost machine learning

2020 ◽  
Author(s):  
Nadine Körtel ◽  
Cornelia Rücklé ◽  
You Zhou ◽  
Anke Busch ◽  
FX Reymond Sutandy ◽  
...  
Keyword(s):  
Machine Learning ◽  
Rna Stability ◽  
Rna Modification ◽  
List Type ◽  
Rna Modifications ◽  
Uv Crosslinking ◽  
Input Material ◽  
Machine Learning Model ◽  
Eukaryotic Mrnas ◽  
Nucleotide Resolution

AbstractN6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing, such as RNA stability and translation. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites in the transcriptome with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present several experimental and computational innovations, that significantly improve transcriptome-wide detection of m6A sites. Based on the recently developed iCLIP2 protocol, the optimised miCLIP2 results in high-complexity libraries from less input material, which yields a more comprehensive representation of m6A sites. Next, we established a robust computational pipeline to identify true m6A sites from our miCLIP2 data. The analyses are calibrated with data from Mettl3 knockout cells to learn the characteristics of m6A deposition, including a significant number of m6A sites outside of DRACH motifs. In order to make these results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification.HighlightsmiCLIP2 produces complex libraries to map m6A RNA modificationsMettl3 KO miCLIP2 allows to identify Mettl3-dependent RNA modification sitesMachine learning predicts genuine m6A sites from human and mouse miCLIP2 data without Mettl3 KOm6A modifications frequently occur outside of DRACH motifs and associates with alternative splicing

Sign in / Sign up

Export Citation Format

Share Document