N6-methyladenosine (m6A) is the most abundant mRNA nucleotide modification and regulates critical aspects of cellular physiology and differentiation. m6A is thought to mediate its effects through a ...complex network of interactions between different m6A sites and three functionally distinct cytoplasmic YTHDF m6A-binding proteins (DF1, DF2, and DF3). In contrast to the prevailing model, we show that DF proteins bind the same m6A-modified mRNAs rather than different mRNAs. Furthermore, we find that DF proteins do not induce translation in HeLa cells. Instead, the DF paralogs act redundantly to mediate mRNA degradation and cellular differentiation. The ability of DF proteins to regulate stability and differentiation becomes evident only when all three DF paralogs are depleted simultaneously. Our study reveals a unified model of m6A function in which all m6A-modified mRNAs are subjected to the combined action of YTHDF proteins in proportion to the number of m6A sites.
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•YTHDF proteins function together to mediate degradation of m6A-mRNAs•YTHDF proteins show identical binding to all m6A sites in mRNAs•Each YTHDF paralog can compensate for the function of the other YTHDF paralogs•Depletion of all three YTHDF proteins promotes differentiation of leukemia cells
Analysis of the transcriptome-wide effects of m6A-mRNA effectors, known as YTHDF proteins, demonstrates that they act redundantly to induce degradation of the same subset of mRNAs, with no evidence of a direct role in promoting translation.
Many recent studies using ChIP-seq approaches cross-referenced to trascriptome data and also to potentially unbiased in vitro DNA binding selection experiments are detailing with increasing precision ...the p53-directed gene regulatory network that, nevertheless, is still expanding. However, most experiments have been conducted in established cell lines subjected to specific p53-inducing stimuli, both factors potentially biasing the results.
We developed p53retriever, a pattern search algorithm that maps p53 response elements (REs) and ranks them according to predicted transactivation potentials in five classes. Besides canonical, full site REs, we developed specific pattern searches for non-canonical half sites and 3/4 sites and show that they can mediate p53-dependent responsiveness of associated coding sequences. Using ENCODE data, we also mapped p53 REs in about 44,000 distant enhancers and identified a 16-fold enrichment for high activity REs within those sites in the comparison with genomic regions near transcriptional start sites (TSS). Predictions from our pattern search were cross-referenced to ChIP-seq, ChIP-exo, expression, and various literature data sources. Based on the mapping of predicted functional REs near TSS, we examined expression changes of thirteen genes as a function of different p53-inducing conditions, providing further evidence for PDE2A, GAS6, E2F7, APOBEC3H, KCTD1, TRIM32, DICER, HRAS, KITLG and TGFA p53-dependent regulation, while MAP2K3, DNAJA1 and potentially YAP1 were identified as new direct p53 target genes.
We provide a comprehensive annotation of canonical and non-canonical p53 REs in the human genome, ranked on predicted transactivation potential. We also establish or corroborate direct p53 transcriptional control of thirteen genes. The entire list of identified and functionally classified p53 REs near all UCSC-annotated genes and within ENCODE mapped enhancer elements is provided. Our approach is distinct from, and complementary to, existing methods designed to identify p53 response elements. p53retriever is available as an R package at: http://tomateba.github.io/p53retriever .
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Activation of p53 by the small molecule Nutlin can result in a combination of cell cycle arrest and apoptosis. The relative strength of these events is difficult to predict by classical gene ...expression analysis, leaving uncertainty as to the therapeutic benefits. In this study, we report a translational control mechanism shaping p53-dependent apoptosis. Using polysome profiling, we establish Nutlin-induced apoptosis to associate with the enhanced translation of mRNAs carrying multiple copies of an identified 3′ UTR CG-rich motif mediating p53-dependent death (CGPD-motif). We identify PCBP2 and DHX30 as CGPD-motif interactors. We find that in cells undergoing persistent cell cycle arrest in response to Nutlin, CGPD-motif mRNAs are repressed by the PCBP2-dependent binding of DHX30 to the motif. Upon DHX30 depletion in these cells, the translation of CGPD-motif mRNAs increases, and the response to Nutlin shifts toward apoptosis. Instead, DHX30 inducible overexpression in SJSA1 cells leads to decreased translation of CGPD-motif mRNAs.
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•p53-dependent transcriptomes are intrinsically diverse and do not predict cell outcome•Nutlin-induced translatomes correlate with cell outcomes•Translational control mediated by DHX30 and PCBP2 inhibits p53-dependent apoptosis
Rizzotto et al. establish the role of PCBP2 and DHX30 in modulating the induction of p53-dependent apoptosis by controlling the translation of mRNAs acting via the 3′ UTR CGPD-motif.
DNA integrity in blood is an emerging biomarker in cancer. Here we report a real time PCR approach for the absolute quantification of four amplicons of 67, 180, 306 and 476bp in cutaneous melanoma. ...Three different integrity indexes (180/67, 306/67 and 476/67 ratios) were tested for their ability to reflect differences in plasma cell-free DNA (cfDNA) fragmentation in 79 patients affected by cutaneous melanoma and 34 healthy subjects. All the three integrity indexes showed higher values in melanoma patients in comparison with healthy subjects.
According to ROC curve analysis, the ratio 180/67 is the most suitable index to be used in cancer patient selection, even if the combination of the 3 indexes gives the best performance in terms of clinical sensitivity.
The most represented fragments in plasma of melanoma patients are those comprised between 181 and 307bp, while in healthy subjects there is a prevalence of shorter fragments (67–180bp).
In conclusion, DNA integrity indexes can be considered suitable parameters for monitoring cfDNA fragmentation in melanoma patients.
► cfDNA integrity is investigated for the first time in cutaneous melanoma ► Three integrity indexes 180/67, 306/67 and 476/67 were determined by qPCR. ► DNA integrity indexes were higher in melanoma patients than healthy controls. ► The integrity index 180/67 showed the highest clinical specificity and sensitivity
Reading, writing and erasing mRNA methylation Zaccara, Sara; Ries, Ryan J; Jaffrey, Samie R
Nature reviews. Molecular cell biology,
10/2019, Letnik:
20, Številka:
10
Journal Article
Recenzirano
RNA methylation to form N
-methyladenosine (m
A) in mRNA accounts for the most abundant mRNA internal modification and has emerged as a widespread regulatory mechanism that controls gene expression ...in diverse physiological processes. Transcriptome-wide m
A mapping has revealed the distribution and pattern of m
A in cellular RNAs, referred to as the epitranscriptome. These maps have revealed the specific mRNAs that are regulated by m
A, providing mechanistic links connecting m
A to cellular differentiation, cancer progression and other processes. The effects of m
A on mRNA are mediated by an expanding list of m
A readers and m
A writer-complex components, as well as potential erasers that currently have unclear relevance to m
A prevalence in the transcriptome. Here we review new and emerging methods to characterize and quantify the epitranscriptome, and we discuss new concepts - in some cases, controversies - regarding our understanding of the mechanisms and functions of m
A readers, writers and erasers.
N6-methyladenosine (m6A) is the most prevalent modified nucleotide in mRNA, and it has important functions in mRNA regulation. However, our understanding of the specific functions of m6A along with ...its cytosolic readers, the YTHDF proteins, has changed substantially in recent years. The original view was that different m6A sites within an mRNA could have different functions depending on which YTHDF paralog was bound to it, with bound YTHDF1 inducing translation, while bound YTHDF2 induced mRNA degradation. As a result, each YTHDF was proposed to have unique physiologic roles that arise from their unique binding properties and regulatory effects on mRNA. More recent data have called much of this into question, showing that all m6A sites bind all YTHDF proteins with equal ability, with a single primary function of all three YTHDF proteins to mediate mRNA degradation. Here, we describe the diverse technical concerns that led to the original model being questioned and the newer data that overturned this model and led to the new understanding of m6A and YTHDF function. We also discuss how any remaining questions about the functions of the YTHDF proteins can be readily resolved.
mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N6-methyladenosine (m6A), found within mRNAs, and ...N6,2′-O-dimethyladenosine (m6Am), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates m6Am. We find that PCIF1 binds and is dependent on the m7G cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish m6Am and m6A. We find that m6A and m6Am misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain m6Am that maps to “internal” sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of m6Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m6Am’s function.
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•PCIF1 is the N6-adenosine methylase that produces m6Am in an m7G cap-dependent manner•PCIF1 depletion allows transcriptome-wide mapping of m6A and m6Am•m6Am mapping identifies alternative “internal” transcription start sites•m6Am increases stability of a subset of mRNAs and has no effect on translation
m6Am is a prevalent mRNA modification occurring adjacent to the m7G cap. Boulias, Toczydlowska-Socha, Hawley et al. identify PCIF1 as the m6Am methyltransferase and perform transcriptome-wide mapping to distinguish m6Am from m6A and identify “internal” TSSs. m6Am increases stability of a subset of mRNAs but has minimal effects on translation.
Few studies have explored the association between SNPs and alterations in mRNA translation potential. We developed an approach to identify SNPs that can mark allele-specific protein expression levels ...and could represent sources of inter-individual variation in disease risk. Using MCF7 cells under different treatments, we performed polysomal profiling followed by RNA sequencing of total or polysome-associated mRNA fractions and designed a computational approach to identify SNPs showing a significant change in the allelic balance between total and polysomal mRNA fractions. We identified 147 SNPs, 39 of which located in UTRs. Allele-specific differences at the translation level were confirmed in transfected MCF7 cells by reporter assays. Exploiting breast cancer data from TCGA we identified UTR SNPs demonstrating distinct prognosis features and altering binding sites of RNA-binding proteins. Our approach produced a catalog of tranSNPs, a class of functional SNPs associated with allele-specific translation and potentially endowed with prognostic value for disease risk.
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•SNPs can alter mRNA translation potential•Polysomal imbalance of heterozygous SNPs can mark allele-specific protein expression•UTR SNPs associated with allele-specific translation demonstrate prognostic value
Molecular mechanism of gene regulation; Computational bioinformatics; Transcriptomics
The search for mechanisms underlying different cellular responses to the treatment with Nutlin-3, an MDM2 inhibitor that unleashes p53, revealed a translational control mechanism involving the RNA ...binding proteins PCBP2 and, particularly, DHX30. Sifting through a multi-functional p53-dependent transcriptional output, this translational control can modulate the activation of cell death pathways.
N6-methyladenosine (m6A) is the most prevalent modified nucleotide in mRNA, and it has important functions in mRNA regulation. However, our understanding of the specific functions of m6A along with ...its cytosolic readers, the YTHDF proteins, has changed substantially in recent years. The original view was that different m6A sites within an mRNA could have different functions depending on which YTHDF paralog was bound to it, with bound YTHDF1 inducing translation, while bound YTHDF2 induced mRNA degradation. As a result, each YTHDF was proposed to have unique physiologic roles that arise from their unique binding properties and regulatory effects on mRNA. More recent data have called much of this into question, showing that all m6A sites bind all YTHDF proteins with equal ability, with a single primary function of all three YTHDF proteins to mediate mRNA degradation. Here, we describe the diverse technical concerns that led to the original model being questioned and the newer data that overturned this model and led to the new understanding of m6A and YTHDF function. We also discuss how any remaining questions about the functions of the YTHDF proteins can be readily resolved.
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