The role of the P1BS element containing promoter-driven genes in Pi transport and homeostasis in plants

Inorganic phosphate (Pi) is an easily accessible form of phosphorus for plants. Plant Pi uptake is usually limited by slow Pi diffusion through the soil which adsorps the Pi quite strong. That is why plants have developed mechanisms to increase Pi availability. There are abiotic (phosphate level) and biotic (mycorrhiza) factors regulating the expression of Pi-responsive genes. Transcription factors binding to the promoters of Pi-responsive genes activate different pathways of Pi transport, distribution and homeostasis maintenance. Pi metabolism involves proteins, as well as microRNAs and other noncoding RNAs

Barley primary microRNA expression pattern is affected by soil water availability

MicroRNAs are short molecules of 21–24 nt in length. They are present in all eukaryotic organisms and regulate gene expression by guiding posttranscriptional silencing of mRNAs. In plants, they are key players in signal transduction, growth and development, and in response to abiotic and biotic stresses. Barley (Hordeum vulgare) is an economically important monocotyledonous crop plant. Drought is the world’s main cause of loss in cereal production. We have constructed a highthroughput Real-Time RT-qPCR platform for parallel determination of 159 barley primary microRNAs’ levels. The platform was tested for two drought-and-rehydrationtreated barley genotypes (Rolap and Sebastian). We have determined changes in the expression of primary microRNAs responding to mild drought, severe drought, and rehydration. Based on the results obtained, we conclude that alteration in the primary microRNA expression is relative to the stress’s intensity. Mild drought and rehydration mostly decrease the pri-miRNA levels in both of the tested genotypes. Severe drought mainly induces the primary microRNA expression. The main difference between the genotypes tested was a much-stronger induction of pri-miRNAs in Rolap encountering severe drought. The primary microRNAs respond dynamically to mild drought, severe drought, and rehydration treatments. We propose that some of the individual primiRNAs could be used as drought stress or rehydration markers. The usage of the platform in biotechnology is also postulated

Heat Stress Affects Pi-related Genes Expression and Inorganic Phosphate Deposition/Accumulation in Barley

Phosphorus (P) in plants is taken from soil as an inorganic phosphate (Pi) and is one of the most important macroelements in growth and development. Plants actively react to Pi starvation by the induced expression of Pi transporters, MIR399, MIR827, and miR399 molecular sponge – IPS1 genes and by the decreased expression of the ubiquitin-conjugating enzyme E2 (PHOSPHATE2 – PHO2) and Pi sensing and transport SPX-MFS genes. The PHO2 protein is involved in the degradation of Pi transporters PHT1;1 (from soil to roots) and PHO1 (from roots to shoots). The decreased expression of PHO2 leads to Pi accumulation in shoots. In contrast, the pho1 mutant shows a decreased level of Pi concentration in shoots. Finally, Pi starvation leads to decreased Pi concentration in all plant tissues. Little is known about plant Pi homeostasis in other abiotic stress conditions. We found that, during the first hour of heat stress, Pi accumulated in barley shoots but not in the roots, and transcriptomic data analysis as well as RT-qPCR led us to propose an explanation for this phenomenon. Pi transport inhibition from soil to roots is balanced by lower Pi efflux from roots to shoots directed by the PHO1 transporter. In shoots, the PHO2 mRNA level is decreased, leading to an increased Pi level. We concluded that Pi homeostasis in barley during heat stress is maintained by dynamic changes in Pi-related genes expression

Promoter-based identification of novel non-coding RNAs reveals the presence of dicistronic snoRNA-miRNA genes in Arabidopsis thaliana

List of Arabidopsis mRNA genes containing Telo-box and Site II elements in 1kb upstream of TSS. (XLSX 134 kb

FUS/TLS contributes to replication-dependent histone gene expression by interaction with U7 snRNPs and histone-specific transcription factors

Replication-dependent histone genes are up-regulated during the G1/S phase transition to meet the requirement for histones to package the newly synthesized DNA. In mammalian cells, this increment is achieved by enhanced transcription and 3′ end processing. The non-polyadenylated histone mRNA 3′ ends are generated by a unique mechanism involving the U7 small ribonucleoprotein (U7 snRNP). By using affinity purification methods to enrich U7 snRNA, we identified FUS/TLS as a novel U7 snRNP interacting protein. Both U7 snRNA and histone transcripts can be precipitated by FUS antibodies predominantly in the S phase of the cell cycle. Moreover, FUS depletion leads to decreased levels of correctly processed histone mRNAs and increased levels of extended transcripts. Interestingly, FUS antibodies also co-immunoprecipitate histone transcriptional activator NPAT and transcriptional repressor hnRNP UL1 in different phases of the cell cycle. We further show that FUS binds to histone genes in S phase, promotes the recruitment of RNA polymerase II and is important for the activity of histone gene promoters. Thus, FUS may serve as a linking factor that positively regulates histone gene transcription and 3′ end processing by interacting with the U7 snRNP and other factors involved in replication-dependent histone gene expressio

Mutation in HvCBP20 (Cap binding protein 20) adapts barley to drought stress at phenotypic and transcriptomic levels

This work was supported by the European Regional Development Fund through the Innovative Economy for Poland 2007–2013, project WND-POIG.01.03.01-00-101/08 POLAPGEN-BD “Biotechnological tools for breeding cereals with increased resistance to drought,” task 22; National Science Centre, Poland, project SONATA 2015/19/D/NZ9/03573 “Translational genomics approach to identify the mechanisms of CBP20 signalosome in Arabidopsis and barley under drought stress.”CBP20 (Cap-Binding Protein 20) encodes a small subunit of the cap-binding complex (CBC), which is involved in the conserved cell processes related to RNA metabolism in plants and, simultaneously, engaged in the signaling network of drought response, which is dependent on ABA. Here, we report the enhanced tolerance to drought stress of barley mutant in the HvCBP20 gene manifested at the morphological, physiological, and transcriptomic levels. Physiological analyses revealed differences between the hvcbp20.ab mutant and its WT in response to a water deficiency. The mutant exhibited a higher relative water content (RWC), a lower stomatal conductance and changed epidermal pattern compared to the WT after drought stress. Transcriptome analysis using the Agilent Barley Microarray integrated with observed phenotypic traits allowed to conclude that the hvcbp20.ab mutant exhibited better fitness to stress conditions by its much more efficient and earlier activation of stress-preventing mechanisms. The network hubs involved in the adjustment of hvcbp20.ab mutant to the drought conditions were proposed. These results enabled to make a significant progress in understanding the role of CBP20 in the drought stress response.European Regional Development Fund; National Science Centre, Polan

Identification of human tRNA:m(5)C methyltransferase catalysing intron-dependent m(5)C formation in the first position of the anticodon of the [Formula: see text]

We identified a human orthologue of tRNA:m(5)C methyltransferase from Saccharomyces cerevisiae, which has been previously shown to catalyse the specific modification of C(34) in the intron-containing yeast [Formula: see text]. Using transcripts of intron-less and intron-containing human [Formula: see text] genes as substrates, we have shown that m(5)C(34) is introduced only in the intron-containing tRNA precursors when the substrates were incubated in the HeLa extract. m(5)C(34) formation depends on the nucleotide sequence surrounding the wobble cytidine and on the structure of the prolongated anticodon stem. Expression of the human Trm4 (hTrm4) cDNA in yeast partially complements the lack of the endogenous Trm4p enzyme. The yeast extract prepared from the strain deprived of the endogenous TRM4 gene and transformed with hTrm4 cDNA exhibits the same activity and substrate specificity toward human pre-tRNA(Leu) transcripts as the HeLa extract. The hTrm4 MTase has a much narrower specificity against the yeast substrates than its yeast orthologue: human enzyme is not able to form m(5)C at positions 48 and 49 of human and yeast tRNA precursors. To our knowledge, this is the first report showing intron-dependent methylation of human [Formula: see text] and identification of human gene encoding tRNA methylase responsible for this reaction

FUS/TLS contributes to replication-dependent histone gene expression by interaction with U7 snRNPs and histone-specific transcription factors.

Replication-dependent histone genes are up-regulated during the G1/S phase transition to meet the requirement for histones to package the newly synthesized DNA. In mammalian cells, this increment is achieved by enhanced transcription and 3' end processing. The non-polyadenylated histone mRNA 3' ends are generated by a unique mechanism involving the U7 small ribonucleoprotein (U7 snRNP). By using affinity purification methods to enrich U7 snRNA, we identified FUS/TLS as a novel U7 snRNP interacting protein. Both U7 snRNA and histone transcripts can be precipitated by FUS antibodies predominantly in the S phase of the cell cycle. Moreover, FUS depletion leads to decreased levels of correctly processed histone mRNAs and increased levels of extended transcripts. Interestingly, FUS antibodies also co-immunoprecipitate histone transcriptional activator NPAT and transcriptional repressor hnRNP UL1 in different phases of the cell cycle. We further show that FUS binds to histone genes in S phase, promotes the recruitment of RNA polymerase II and is important for the activity of histone gene promoters. Thus, FUS may serve as a linking factor that positively regulates histone gene transcription and 3' end processing by interacting with the U7 snRNP and other factors involved in replication-dependent histone gene expression

mRNA adenosine methylase (MTA) deposits m6A on pri-miRNAs to modulate miRNA biogenesis in Arabidopsis thaliana

Copyright © 2020 the Author(s). Published by PNAS. In Arabidopsis thaliana, the METTL3 homolog, mRNA adenosine methylase (MTA) introduces N6-methyladenosine (m6A) into various coding and noncoding RNAs of the plant transcriptome. Here, we show that an MTA-deficient mutant (mta) has decreased levels of microRNAs (miRNAs) but accumulates primary miRNA transcripts (pri-miRNAs). Moreover, pri-miRNAs are methylated by MTA, and RNA structure probing analysis reveals a decrease in secondary structure within stem-loop regions of these transcripts in mta mutant plants. We demonstrate interaction between MTA and both RNA Polymerase II and TOUGH (TGH), a plant protein needed for early steps of miRNA biogenesis. Both MTA and TGH are necessary for efficient colocalization of the Microprocessor components Dicer-like 1 (DCL1) and Hyponastic Leaves 1 (HYL1) with RNA Polymerase II. We propose that secondary structure of miRNA precursors induced by their MTA-dependent m6A methylation status, together with direct interactions between MTA and TGH, influence the recruitment of Microprocessor to plant pri-miRNAs. Therefore, the lack of MTA in mta mutant plants disturbs pri-miRNA processing and leads to the decrease in miRNA accumulation. Furthermore, our findings reveal that reduced miR393b levels likely contributes to the impaired auxin response phenotypes of mta mutant plants