Changes in mRNA Degradation Efficiencies under Varying Conditions Are Regulated by Multiple Determinants in Arabidopsis thaliana

Abstract Multiple mechanisms are involved in gene expression, with mRNA degradation being critical for the control of mRNA accumulation. In plants, although some trans-acting factors and motif sequences have been identified in deadenylation-dependent mRNA degradation, endonucleolytic cleavage-dependent mRNA degradation has not been studied in detail. Previously, we developed truncated RNA-end sequencing (TREseq) in Arabidopsis thaliana and detected G-rich sequence motifs around 5′ degradation intermediates. However, it remained to be elucidated whether degradation efficiencies of 5′ degradation intermediates in A. thaliana vary among growth conditions and developmental stages. To address this issue, we conducted TREseq of cultured cells under heat stress and at three developmental stages (seedlings, expanding leaves and expanded leaves) and compared 5′ degradation intermediates data among the samples. Although some 5′ degradation intermediates had almost identical degradation efficiencies, others differed among conditions. We focused on the genes and sites whose degradation efficiencies differed. Changes in degradation efficiencies at the gene and site levels revealed an effect on mRNA accumulation in all comparisons. These changes in degradation efficiencies involved multiple determinants, including mRNA length and translation efficiency. These results suggest that several determinants govern the efficiency of mRNA degradation in plants, helping the organism to adapt to varying conditions by controlling mRNA accumulation.

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Degradation intermediates as an indicator of mRNA and cell metabolism

10.1101/2021.08.29.458069 ◽

2021 ◽

Author(s):

Yusheng Liu ◽

Yiwei Zhang ◽

Hu Nie ◽

Falong Lu ◽

Jiaqiang Wang

Keyword(s):

Degradation Rate ◽

Cultured Cells ◽

Cell Metabolism ◽

Single Cells ◽

Mrna Degradation ◽

Rate Analysis ◽

Mouse Tissues ◽

Degradation Intermediates ◽

Mrna Degradation Rate

Traditional mRNA degradation rate measurements involves complex experimental design with RNA labeling or transcription blocking together with sampling at multiple timepoints. These experimental requirements limit the application of transcriptome-wide mRNA degradation rate analysis mainly in cultured cells, but rarely in in vivo samples. Therefore, a direct and simple strategy needs to be developed to study mRNA degradation rate. Here, we defined mRNA degradation intermediates as transcripts where decay is about to occur or has partially occurred in the 3′-untranslated regions after poly(A) tail deadenylation, and found that the proportion of mRNA degradation intermediates is a very simple and convenient indicator for evaluating the degradation rate of mRNA in mouse and human cell lines. In addition, we showed that a higher proportion of mRNA degradation intermediates is correlated with faster cell cycle and higher turnover rate of mouse tissues. Further, we validated that in mouse maturing oocytes where transcription is silent, the proportion of mRNA degradation intermediates is positively correlated with the mRNA degradation rate. Together, these results demonstrate that degradation intermediates can function as a good indicator of mRNA, cell, and tissue metabolism, and can be easily assayed by total RNA 3′-end sequencing from a single bulk cell sample without the need for drug treatment or multi-timepoint sampling. This finding is of great potential for studies on mRNA degradation rate at the molecular, cellular, or organic level, including samples or systems that cannot be assayed with previous methods. In addition, further application of the findings into single cells will likely greatly aid the identification and study of rare cells with unique cellular metabolism dynamics such as tissue stem cells and tumor cells.

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Genomic structure of murine methylmalonyl-CoA mutase: evidence for genetic and epigenetic mechanisms determining enzyme activity

Biochemical Journal ◽

10.1042/bj2960663 ◽

1993 ◽

Vol 296 (3) ◽

pp. 663-670 ◽

Cited By ~ 13

Author(s):

M F Wilkemeyer ◽

E R Andrews ◽

F D Ledley

Keyword(s):

Enzyme Activity ◽

Steady State ◽

Transcription Initiation ◽

Cultured Cells ◽

Genomic Structure ◽

Genetic Regulation ◽

Regulatory Elements ◽

Transcription Unit ◽

Mrna Levels ◽

Sequence Motifs

Methylmalonyl-CoA mutase (MCM) is a nuclear-encoded mitochondrial matrix enzyme. We have reported characterization of murine MCM and cloning of a murine MCM cDNA and now describe the murine Mut locus, its promoter and evidence for tissue-specific variation in MCM mRNA, enzyme and holo-enzyme levels. The Mut locus spans 30 kb and contains 13 exons constituting a unique transcription unit. A B1 repeat element was found in the 3′ untranslated region (exon 13). The transcription initiation site was identified and upstream sequences were shown to direct expression of a reporter gene in cultured cells. The promoter contains sequence motifs characteristic of: (1) TATA-less housekeeping promoters; (2) enhancer elements purportedly involved in co-ordinating expression of nuclear-encoded mitochondrial proteins; and (3) regulatory elements including CCAAT boxes, cyclic AMP-response elements and potential AP-2-binding sites. Northern blots demonstrate a greater than 10-fold variation in steady-state mRNA levels, which correlate with tissue levels of enzyme activity. However, the ratio of holoenzyme to total enzyme varies among different tissues, and there is no correlation between steady-state mRNA levels and holoenzyme activity. These results suggest that, although there may be regulation of MCM activity at the level of mRNA, the significance of genetic regulation is unclear owning to the presence of epigenetic regulation of holoenzyme formation.

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Induction of metallothionein-I mRNA in cultured cells by heavy metals and iodoacetate: evidence for gratuitous inducers

Molecular and Cellular Biology ◽

10.1128/mcb.4.3.484-491.1984 ◽

1984 ◽

Vol 4 (3) ◽

pp. 484-491

Author(s):

D M Durnam ◽

R D Palmiter

Keyword(s):

Heavy Metals ◽

Cultured Cells ◽

Mrna Accumulation ◽

Mouse Hepatocyte ◽

Hepatocyte Cell Line ◽

Regulatory Molecule ◽

Heavy Metal Detoxification ◽

Double Minute ◽

Double Minute Chromosomes

A mouse hepatocyte cell line selected for growth in 80 microM CdSO4 (Cdr80 cells) was used to test the role of metallothioneins in heavy metal detoxification. The cadmium-resistant (Cdr80) cells have double minute chromosomes carrying amplified copies of the metallothionein-I gene and accumulate ca. 20-fold more metallothionein-I mRNA than unselected cadmium-sensitive (Cds) cells after optimal Cd stimulation. As a consequence, the amount of Cd which inhibits DNA synthesis by 50% is ca. 7.5-fold higher in Cdr80 cells than in Cds cells. Cds and Cdr80 cells were compared in terms of their resistance to other heavy metals. The results indicate that although Zn, Cu, Hg, Ag, Co, Ni, and Bi induce metallothionein-I mRNA accumulation in both Cdr80 and Cds cells, the Cdr80 cells show increased resistance to only a subset of these metals (Zn, Cu, Hg, and Bi). This suggests that not all metals which induce metallothionein mRNA are detoxified by metallothionein and argues against autoregulation of metallothionein genes. Metallothionein-I mRNA is also induced by iodoacetate, suggesting that the regulatory molecule has sensitive sulfhydryl groups.

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Capped mRNA Degradation Intermediates Accumulate in the Yeast spb8-2 Mutant

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.5062 ◽

1998 ◽

Vol 18 (9) ◽

pp. 5062-5072 ◽

Cited By ~ 82

Author(s):

Ronald Boeck ◽

Bruno Lapeyre ◽

Christine E. Brown ◽

Alan B. Sachs

Keyword(s):

Saccharomyces Cerevisiae ◽

Family Member ◽

Gene Deletion ◽

Binding Protein ◽

Mrna Degradation ◽

Protein Family ◽

Suppressor Mutation ◽

Mrna Decapping ◽

Mrna Species ◽

Degradation Intermediates

ABSTRACT mRNA in the yeast Saccharomyces cerevisiae is primarily degraded through a pathway that is stimulated by removal of the mRNA cap structure. Here we report that a mutation in the SPB8(YJL124c) gene, initially identified as a suppressor mutation of a poly(A)-binding protein (PAB1) gene deletion, stabilizes the mRNA cap structure. Specifically, we find that thespb8-2 mutation results in the accumulation of capped, poly(A)-deficient mRNAs. The presence of this mutation also allows for the detection of mRNA species trimmed from the 3′ end. These data show that this Sm-like protein family member is involved in the process of mRNA decapping, and they provide an example of 3′-5′ mRNA degradation intermediates in yeast.

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The THO/TREX Complex Component RAE2/TEX1 Is Involved in the Regulation of Aluminum Resistance and Low Phosphate Response in Arabidopsis

Frontiers in Plant Science ◽

10.3389/fpls.2021.698443 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yi-Fang Zhu ◽

Jinliang Guo ◽

Yang Zhang ◽

Chao-Feng Huang

Keyword(s):

Arabidopsis Thaliana ◽

Transcription Factor ◽

Mrna Export ◽

Critical Role ◽

Core Component ◽

Mrna Accumulation ◽

Zinc Finger Transcription Factor ◽

Phosphate Response ◽

Encoding Gene ◽

Malate Transporter

The C2H2-type zinc finger transcription factor SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) plays a critical role in aluminum (Al) resistance and low phosphate (Pi) response mainly through promoting the expression of the malate transporter-encoding gene ARABIDOPSIS THALIANA ALUMINUM ACTIVATED MALATE TRANSPORTER 1 (AtALMT1). We previously showed that REGULATION OF ATALMT1 EXPRESSION 3 (RAE3/HPR1), a core component of the THO/TREX complex, is involved in the regulation of nucleocytoplasmic STOP1 mRNA export to modulate Al resistance and low Pi response. Here, we report that RAE2/TEX1, another core component of the THO complex, is also involved in the regulation of Al resistance and low Pi response. Mutation of RAE2 reduced the expression of STOP1-downstream genes, including AtALMT1. rae2 was less sensitive to Al than rae3, which was consistent with less amount of malate secreted from rae3 roots than from rae2 roots. Nevertheless, low Pi response was impaired more in rae2 than in rae3, suggesting that RAE2 also regulates AtALMT1-independent pathway to modulate low Pi response. Furthermore, unlike RAE3 that regulates STOP1 mRNA export, mutating RAE2 did not affect STOP1 mRNA accumulation in the nucleus, although STOP1 protein level was reduced in rae2. Introduction of rae1 mutation into rae2 mutant background could partially recover the deficient phenotypes of rae2. Together, our results demonstrate that RAE2 and RAE3 play overlapping but distinct roles in the modulation of Al resistance and low Pi response.

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A new class of mutations in Arabidopsis thaliana, acaulis1, affecting the development of both inflorescences and leaves

Development ◽

10.1242/dev.118.3.751 ◽

1993 ◽

Vol 118 (3) ◽

pp. 751-764 ◽

Cited By ~ 2

Author(s):

H. Tsukaya ◽

S. Naito ◽

G. P. Redei ◽

Y. Komeda

Keyword(s):

Arabidopsis Thaliana ◽

Apical Meristem ◽

Developmental Stages ◽

Temperature Shift ◽

Wild Type ◽

New Class ◽

Group 4 ◽

Consequent Reduction ◽

In The Wild ◽

Specific Temperature

We isolated and analyzed mutants of Arabidopsis thaliana, acaulis, with flower stalks that are almost absent or are much reduced in length. The mutations are divided between two loci, acaulis1 (acl1) and acaulis2 (acl2). The acl1-1 mutation has been assigned to linkage group 4 in the vicinity of locus ap2. The acl1-1 mutant showed premature arrest of the inflorescence meristem after the onset of reproductive development, followed by consequent reduction in the number of flower-bearing phytomers and therefore flowers. The apical meristem of the inflorescences was morphologically normal but its radius was about half that of the wild type. The acl1 mutants are also defective in the development of foliage leaves. Both defects could be rescued by growth at a specific temperature (28°C). The length of the cells in acl1-3 mutant was less than that in the wild type but the numbers of cells in leaves and internodes of acl1 mutants were calculated to be the same as those of the wild type. Thus, the defects in inflorescences and leaves were attributed to defects in the process of elongation (maturation) of these cells. Temperature-shift experiments showed that the Acl1+ product was necessary at all developmental stages. A critical stage was shown to exist for recovery from the cessation of development of inflorescence meristems that was caused by the acl1-1 mutation. Grafting experiments showed that the acl1-1 mutation does not affect diffusible substances. An analysis of double mutants carrying both acl1-1 and one of developmental mutations, ap1, clv1, lfy, or tfl1, showed that ACL1 is a new class of gene.

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Evolutionary History and Activity of RNase H1-Like Proteins in Arabidopsis thaliana

Plant and Cell Physiology ◽

10.1093/pcp/pcaa040 ◽

2020 ◽

Vol 61 (6) ◽

pp. 1107-1119

Author(s):

Jan Kuciński ◽

Sebastian Chamera ◽

Aleksandra Kmera ◽

M Jordan Rowley ◽

Sho Fujii ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Embryonic Development ◽

Evolutionary History ◽

Common Ancestor ◽

Normal Growth ◽

Plastid Dna ◽

Growth Conditions ◽

Replicative Stress ◽

The Common ◽

Rna:Dna Hybrids

Abstract RNase H1 is an endonuclease specific toward the RNA strand of RNA:DNA hybrids. Members of this protein family are present in most living organisms and are essential for removing RNA that base pairs with DNA. It prevents detrimental effects of RNA:DNA hybrids and is involved in several biological processes. Arabidopsis thaliana has been previously shown to contain three genes encoding RNase H1 proteins that localize to three distinct cellular compartments. We show that these genes originate from two gene duplication events. One occurred in the common ancestor of dicots and produced nuclear and organellar RNase H1 paralogs. Second duplication occurred in the common ancestor of Brassicaceae and produced mitochondrial- and plastid-localized proteins. These proteins have the canonical RNase H1 activity, which requires at least four ribonucleotides for endonucleolytic digestion. Analysis of mutants in the RNase H1 genes revealed that the nuclear RNH1A and mitochondrial RNH1B are dispensable for development under normal growth conditions. However, the presence of at least one organellar RNase H1 (RNH1B or RNH1C) is required for embryonic development. The plastid-localized RNH1C affects plastid DNA copy number and sensitivity to replicative stress. Our results present the evolutionary history of RNH1 proteins in A. thaliana, demonstrate their canonical RNase H1 activity and indicate their role in early embryonic development.

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Plant Histone HTB (H2B) Variants in Regulating Chromatin Structure and Function

Plants ◽

10.3390/plants9111435 ◽

2020 ◽

Vol 9 (11) ◽

pp. 1435

Author(s):

Janardan Khadka ◽

Anat Pesok ◽

Gideon Grafi

Keyword(s):

Arabidopsis Thaliana ◽

Chromatin Structure ◽

Developmental Stages ◽

Histone Variants ◽

Structure And Function ◽

Core Histone ◽

Histone H2b ◽

Histone Proteins ◽

Genes Encoding ◽

And Function

Besides chemical modification of histone proteins, chromatin dynamics can be modulated by histone variants. Most organisms possess multiple genes encoding for core histone proteins, which are highly similar in amino acid sequence. The Arabidopsis thaliana genome contains 11 genes encoding for histone H2B (HTBs), 13 for H2A (HTAs), 15 for H3 (HTRs), and 8 genes encoding for histone H4 (HFOs). The finding that histone variants may be expressed in specific tissues and/or during specific developmental stages, often displaying specific nuclear localization and involvement in specific nuclear processes suggests that histone variants have evolved to carry out specific functions in regulating chromatin structure and function and might be important for better understanding of growth and development and particularly the response to stress. In this review, we will elaborate on a group of core histone proteins in Arabidopsis, namely histone H2B, summarize existing data, and illuminate the potential function of H2B variants in regulating chromatin structure and function in Arabidopsis thaliana.

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Nitric Oxide Overproduction by cue1 Mutants Differs on Developmental Stages and Growth Conditions

Plants ◽

10.3390/plants9111484 ◽

2020 ◽

Vol 9 (11) ◽

pp. 1484 ◽

Cited By ~ 1

Author(s):

Tamara Lechón ◽

Luis Sanz ◽

Inmaculada Sánchez-Vicente ◽

Oscar Lorenzo

Keyword(s):

Nitric Oxide ◽

Carbon Metabolism ◽

Root Elongation ◽

Developmental Stages ◽

Primary Root ◽

Carbon Sources ◽

Growth Conditions ◽

No Production

The cue1 nitric oxide (NO) overproducer mutants are impaired in a plastid phosphoenolpyruvate/phosphate translocator, mainly expressed in Arabidopsis thaliana roots. cue1 mutants present an increased content of arginine, a precursor of NO in oxidative synthesis processes. However, the pathways of plant NO biosynthesis and signaling have not yet been fully characterized, and the role of CUE1 in these processes is not clear. Here, in an attempt to advance our knowledge regarding NO homeostasis, we performed a deep characterization of the NO production of four different cue1 alleles (cue1-1, cue1-5, cue1-6 and nox1) during seed germination, primary root elongation, and salt stress resistance. Furthermore, we analyzed the production of NO in different carbon sources to improve our understanding of the interplay between carbon metabolism and NO homeostasis. After in vivo NO imaging and spectrofluorometric quantification of the endogenous NO levels of cue1 mutants, we demonstrate that CUE1 does not directly contribute to the rapid NO synthesis during seed imbibition. Although cue1 mutants do not overproduce NO during germination and early plant development, they are able to accumulate NO after the seedling is completely established. Thus, CUE1 regulates NO homeostasis during post-germinative growth to modulate root development in response to carbon metabolism, as different sugars modify root elongation and meristem organization in cue1 mutants. Therefore, cue1 mutants are a useful tool to study the physiological effects of NO in post-germinative growth.

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