21 results on '"Kruszka K"'
Search Results
2. Drought-regulated microRNAs in barley (Hordeum vulgare)
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Świda-Barteczka, A., Pacak, A., Kruszka, K., Karłowski, W., Jarmołowski, A., and Zofia Szweykowska-Kulińska
3. Developmentally regulated expression and complex processing of barley pri-microRNAs
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Kruszka Katarzyna, Pacak Andrzej, Swida-Barteczka Aleksandra, Stefaniak Agnieszka K, Kaja Elzbieta, Sierocka Izabela, Karlowski Wojciech, Jarmolowski Artur, and Szweykowska-Kulinska Zofia
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MicroRNA ,Pri-microRNA processing ,MicroRNA genes ,Splicing ,Alternative splicing ,Introns ,Barley ,Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background MicroRNAs (miRNAs) regulate gene expression via mRNA cleavage or translation inhibition. In spite of barley being a cereal of great economic importance, very little data is available concerning its miRNA biogenesis. There are 69 barley miRNA and 67 pre-miRNA sequences available in the miRBase (release 19). However, no barley pri-miRNA and MIR gene structures have been shown experimentally. In the present paper, we examine the biogenesis of selected barley miRNAs and the developmental regulation of their pri-miRNA processing to learn more about miRNA maturation in barely. Results To investigate the organization of barley microRNA genes, nine microRNAs - 156g, 159b, 166n, 168a-5p/168a-3p, 171e, 397b-3p, 1120, and 1126 - were selected. Two of the studied miRNAs originate from one MIR168a-5p/168a-3p gene. The presence of all miRNAs was confirmed using a Northern blot approach. The miRNAs are encoded by genes with diverse organizations, representing mostly independent transcription units with or without introns. The intron-containing miRNA transcripts undergo complex splicing events to generate various spliced isoforms. We identified miRNAs that were encoded within introns of the noncoding genes MIR156g and MIR1126. Interestingly, the intron that encodes miR156g is spliced less efficiently than the intron encoding miR1126 from their specific precursors. miR397b-3p was detected in barley as a most probable functional miRNA, in contrast to rice where it has been identified as a complementary partner miRNA*. In the case of miR168a-5p/168a-3p, we found the generation of stable, mature molecules from both pre-miRNA arms, confirming evolutionary conservation of the stability of both species, as shown in rice and maize. We suggest that miR1120, located within the 3′ UTR of a protein-coding gene and described as a functional miRNA in wheat, may represent a siRNA generated from a mariner-like transposable element. Conclusions Seven of the eight barley miRNA genes characterized in this study contain introns with their respective transcripts undergoing developmentally specific processing events prior to the dicing out of pre-miRNA species from their pri-miRNA precursors. The observed tendency to maintain the intron encoding miR156g within the transcript, and preferences in splicing the miR1126-harboring intron, may suggest the existence of specific regulation of the levels of intron-derived miRNAs in barley.
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- 2013
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4. MicroRNA172b-5p/trehalose-6-phosphate synthase module stimulates trehalose synthesis and microRNA172b-3p/AP2-like module accelerates flowering in barley upon drought stress.
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Swida-Barteczka A, Pacak A, Kruszka K, Nuc P, Karlowski WM, Jarmolowski A, and Szweykowska-Kulinska Z
- Abstract
MicroRNAs (miRNAs) are major regulators of gene expression during plant development under normal and stress conditions. In this study, we analyzed the expression of 150 conserved miRNAs during drought stress applied to barley ready to flower. The dynamics of miRNAs expression was also observed after rewatering. Target messenger RNA (mRNAs) were experimentally identified for all but two analyzed miRNAs, and 41 of the targets were not reported before. Drought stress applied to barley induced accelerated flowering coordinated by a pair of two differently expressed miRNAs originating from a single precursor: hvu-miR172b-3p and hvu-miR172b-5p. Increased expression of miRNA172b-3p during drought leads to the downregulation of four APETALA2(AP2)-like genes by their mRNA cleavage. In parallel, the downregulation of the miRNA172b-5p level results in an increased level of a newly identified target, trehalose-6-phosphate synthase, a key enzyme in the trehalose biosynthesis pathway. Therefore, drought-treated plants have higher trehalose content, a known osmoprotectant, whose level is rapidly dropping after watering. In addition, trehalose-6-phosphate, an intermediate of the trehalose synthesis pathway, is known to induce flowering. The hvu-miRNA172b-5p/trehalose-6-phosphate synthase and hvu-miRNA172b-3p/AP2-like create a module leading to osmoprotection and accelerated flowering induction during drought., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Swida-Barteczka, Pacak, Kruszka, Nuc, Karlowski, Jarmolowski and Szweykowska-Kulinska.)
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- 2023
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5. Pi-starvation induced transcriptional changes in barley revealed by a comprehensive RNA-Seq and degradome analyses.
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Sega P, Kruszka K, Bielewicz D, Karlowski W, Nuc P, Szweykowska-Kulinska Z, and Pacak A
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- Gene Expression Profiling, Gene Expression Regulation, Plant, RNA-Seq, Hordeum genetics, MicroRNAs genetics
- Abstract
Background: Small RNAs (sRNAs) are 20-30 nt regulatory elements which are responsible for plant development regulation and participate in many plant stress responses. Insufficient inorganic phosphate (Pi) concentration triggers plant responses to balance the internal Pi level., Results: In this study, we describe Pi-starvation-responsive small RNAs and transcriptome changes in barley (Hordeum vulgare L.) using Next-Generation Sequencing (NGS) RNA-Seq data derived from three different types of NGS libraries: (i) small RNAs, (ii) degraded RNAs, and (iii) functional mRNAs. We find that differentially and significantly expressed miRNAs (DEMs, Bonferroni adjusted p-value < 0.05) are represented by 15 molecules in shoot and 13 in root; mainly various miR399 and miR827 isomiRs. The remaining small RNAs (i.e., those without perfect match to reference sequences deposited in miRBase) are considered as differentially expressed other sRNAs (DESs, p-value Bonferroni correction < 0.05). In roots, a more abundant and diverse set of other sRNAs (DESs, 1796 unique sequences, 0.13% from the average of the unique small RNA expressed under low-Pi) contributes more to the compensation of low-Pi stress than that in shoots (DESs, 199 unique sequences, 0.01%). More than 80% of differentially expressed other sRNAs are up-regulated in both organs. Additionally, in barley shoots, up-regulation of small RNAs is accompanied by strong induction of two nucleases (S1/P1 endonuclease and 3'-5' exonuclease). This suggests that most small RNAs may be generated upon nucleolytic cleavage to increase the internal Pi pool. Transcriptomic profiling of Pi-starved barley shoots identifies 98 differentially expressed genes (DEGs). A majority of the DEGs possess characteristic Pi-responsive cis-regulatory elements (P1BS and/or PHO element), located mostly in the proximal promoter regions. GO analysis shows that the discovered DEGs primarily alter plant defense, plant stress response, nutrient mobilization, or pathways involved in the gathering and recycling of phosphorus from organic pools., Conclusions: Our results provide comprehensive data to demonstrate complex responses at the RNA level in barley to maintain Pi homeostasis and indicate that barley adapts to Pi-starvation through elicitation of RNA degradation. Novel P-responsive genes were selected as putative candidates to overcome low-Pi stress in barley plants.
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- 2021
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6. Quantitative Analysis of Plant miRNA Primary Transcripts.
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Dolata J, Zielezinski A, Stepien A, Kruszka K, Bielewicz D, Pacak A, Jarmolowski A, Karlowski W, and Szweykowska-Kulinska Z
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- Arabidopsis genetics, Gene Expression Regulation, Plant, Hordeum genetics, MicroRNAs chemistry, Arabidopsis metabolism, Hordeum metabolism, MicroRNAs metabolism
- Abstract
MicroRNAs control plant development and are key regulators of plant responses to biotic and abiotic stresses. Thus, their expression must be carefully controlled since both excess and deficiency of a given microRNA may be deleterious to plant cell. MicroRNA expression regulation can occur at several stages of their biogenesis pathway. One of the most important of these regulatory checkpoints is transcription efficiency. mirEX database is a tool for exploration and visualization of plant pri-miRNA expression profiles. It includes results obtained using high-throughput RT-qPCR platform designed to monitor pri-miRNA expression in different miRNA biogenesis mutants and developmental stages of Arabidopsis, barley, and Pellia plants. A step-by-step instruction for browsing the database and detailed protocol for high-throughput RT-qPCR experiments, including list of primers designed for the amplification of pri-miRNAs, are presented.
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- 2021
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7. A Functional Network of Novel Barley MicroRNAs and Their Targets in Response to Drought.
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Smoczynska A, Pacak AM, Nuc P, Swida-Barteczka A, Kruszka K, Karlowski WM, Jarmolowski A, and Szweykowska-Kulinska Z
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- Chromatin genetics, Droughts, Gene Expression Regulation, Plant genetics, Hordeum growth & development, Stress, Physiological genetics, Hordeum genetics, MicroRNAs genetics, Mitochondria genetics, RNA, Messenger genetics
- Abstract
The regulation of mRNA (messenger RNA) levels by microRNA-mediated activity is especially important in plant responses to environmental stresses. In this work, we report six novel barley microRNAs, including two processed from the same precursor that are severely downregulated under drought conditions. For all analyzed microRNAs, we found target genes that were upregulated under drought conditions and that were known to be involved in a plethora of processes from disease resistance to chromatin-protein complex formation and the regulation of transcription in mitochondria. Targets for novel barley microRNAs were confirmed through degradome data analysis and RT-qPCR using primers flanking microRNA-recognition site. Our results show a broad transcriptional response of barley to water deficiency conditions through microRNA-mediated gene regulation and facilitate further research on drought tolerance in crops.
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- 2020
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8. Identification of transcription factors that bind to the 5'-UTR of the barley PHO2 gene.
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Sega P, Kruszka K, Szewc Ł, Szweykowska-Kulińska Z, and Pacak A
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- Arabidopsis genetics, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Binding Sites, Gene Expression Regulation, Plant, Glucuronidase metabolism, MicroRNAs genetics, MicroRNAs metabolism, Phosphates metabolism, Plant Leaves metabolism, Plant Roots metabolism, RNA, Messenger metabolism, Nicotiana genetics, Nicotiana metabolism, 5' Untranslated Regions physiology, Hordeum genetics, Hordeum metabolism, Transcription Factors genetics, Transcription Factors isolation & purification, Transcription Factors metabolism, Ubiquitin-Conjugating Enzymes genetics, Ubiquitin-Conjugating Enzymes metabolism
- Abstract
In barley and other higher plants, phosphate homeostasis is maintained by a regulatory network involving the PHO2 (PHOSPHATE2) encoding ubiquitin-conjugating (UBC) E2 enzyme, the PHR1 (PHOSPHATE STARVATION RESPONSE 1) transcription factor (TF), IPS1 (INDUCED BYPHOSPHATESTARVATION1) RNA, and miR399. During phosphate ion (Pi) deprivation, PHR1 positively regulates MIR399 expression, after transcription and processing mature miR399 guides the Ago protein to the 5'-UTR of PHO2 transcripts. Non-coding IPS1 RNA is highly expressed during Pi starvation, and the sequestration of miR399 molecules protects PHO2 mRNA from complete degradation. Here, we reveal new cis- and trans-regulatory elements that are crucial for efficient PHO2 gene expression in barley. We found that the 5'-UTR of PHO2 contains two PHR1 binding sites (P1BSs) and one Pi-responsive PHO element. Using a yeast one-hybrid (Y1H) assay, we identified two candidate proteins that might mediate this transcriptional regulation: a barley PHR1 ortholog and a TF containing an uncharacterized MYB domain. Additional results classified this new potential TF as belonging to the APL (ALTERED PHLOEM DEVELOPMENT) protein family, and we observed its nuclear localization in barley protoplasts. Pi starvation induced the accumulation of barley APL transcripts in both the shoots and roots. Interestingly, the deletion of the P1BS motif from the first intron of the barley 5'-UTR led to a significant increase in the transcription of a downstream β-glucuronidase (GUS) reporter gene in tobacco leaves. Our work extends the current knowledge about putative cis- and trans-regulatory elements that may affect the expression of the barley PHO2 gene.
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- 2020
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9. Corrigendum: A Role of U12 Intron in Proper Pre-mRNA Splicing of Plant Cap Binding Protein 20 Genes.
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Pieczynski M, Kruszka K, Bielewicz D, Dolata J, Szczesniak M, Karlowski W, Jarmolowski A, and Szweykowska-Kulinska Z
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[This corrects the article DOI: 10.3389/fpls.2018.00475.]., (Copyright © 2019 Pieczynski, Kruszka, Bielewicz, Dolata, Szczesniak, Karlowski, Jarmolowski and Szweykowska-Kulinska.)
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- 2019
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10. A Role of U12 Intron in Proper Pre-mRNA Splicing of Plant Cap Binding Protein 20 Genes.
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Pieczynski M, Kruszka K, Bielewicz D, Dolata J, Szczesniak M, Karlowski W, Jarmolowski A, and Szweykowska-Kulinska Z
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The nuclear cap-binding complex (CBC) is composed of two cap-binding proteins: CBP20 and CBP80. The CBP20 gene structure is highly conserved across land plant species. All studied CBP20 genes contain eight exons and seven introns, with the fourth intron belonging to the U12 class. This highly conserved U12 intron always divides the plant CBP20 gene into two parts: one part encodes the core domain containing the RNA binding domain (RBD), and the second part encodes the tail domain with a nuclear localization signal (NLS). In this study, we investigate the importance of the U12 intron in the Arabidopsis thaliana CBP20 gene by moving it to different intron locations of the gene. Relocation of the U12 intron resulted in a significant decrease in the U12 intron splicing efficiency and the accumulation of wrongly processed transcripts. These results suggest that moving the U12 intron to any other position of the A. thaliana CBP20 gene disturbs splicing, leading to substantial downregulation of the level of properly spliced mRNA and CBP20 protein. Moreover, the replacement of the U12 intron with a U2 intron leads to undesired alternative splicing events, indicating that the proper localization of the U12 intron in the CBP20 gene secures correct CBP20 pre-mRNA maturation and CBP20 protein levels in a plant. Surprisingly, our results also show that the efficiency of U12 splicing depends on intron length. In conclusion, our study emphasizes the importance of proper U12 intron localization in plant CBP20 genes for correct pre-mRNA processing.
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- 2018
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11. Heat Stress Affects Pi-related Genes Expression and Inorganic Phosphate Deposition/Accumulation in Barley.
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Pacak A, Barciszewska-Pacak M, Swida-Barteczka A, Kruszka K, Sega P, Milanowska K, Jakobsen I, Jarmolowski A, and Szweykowska-Kulinska Z
- Abstract
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.
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- 2016
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12. Developmental changes in barley microRNA expression profiles coupled with miRNA target analysis.
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Pacak A, Kruszka K, Swida-Barteczka A, Nuc P, Karlowski W, Jarmolowski A, and Szweykowska-Kulinska Z
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- Base Sequence, Biomarkers metabolism, Gene Expression, Gene Expression Regulation, Plant, High-Throughput Nucleotide Sequencing, Hordeum growth & development, Hordeum metabolism, Inverted Repeat Sequences, MicroRNAs metabolism, Phylogeny, RNA Cleavage, RNA, Plant genetics, RNA, Plant metabolism, Seedlings growth & development, Seedlings metabolism, Hordeum genetics, MicroRNAs genetics, Seedlings genetics
- Abstract
MicroRNAs are 19- to 24-nt-long single-stranded RNAs that are crucial regulators of gene expression which control plant development and response to environmental cues. We have analyzed microtranscriptomes of five barley developmental stages. Generally, during the barley development, miR168-3p and miR1432-5p levels increase while the 5'U-miR156-5p level decreases (with exception for the 2-week-old barley). We have identified two miR156-5p izomiRs (called 5'U-miR156-5p [20 nt] and 5'UU-miR156-5p [21 nt]), which were expressed differently during barley development. The 5' U-miR156-5p level decreased in 3-week-, 6-week-, and 68-day-old barley, when compared to the 1-week-old plants. Meanwhile, the 5' UU-miR156-5p level increased significantly in the 68-day-old barley plants. Moreover, only the 5' U-miR156 isomiR recognizes and guides unique transcription factor mRNAs from the Squamosa Promoter Binding Protein-Like (SPL) family. We identified many non-canonical microRNAs with changed expression levels during the barley development. Here, we present the profiles of microRNA expression characteristics for particular barley developmental stages. These analyses are accompanied by the experimental degradome analysis of miRNA targets.
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- 2016
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13. Barley primary microRNA expression pattern is affected by soil water availability.
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Swida-Barteczka A, Kruszka K, Grabowska A, Pacak A, Jarmolowski A, Kurowska M, Szarejko I, and Szweykowska-Kulinska Z
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- Dehydration genetics, Dehydration metabolism, Droughts, Gene Expression, Gene Expression Regulation, Plant, Hordeum metabolism, MicroRNAs metabolism, RNA, Plant genetics, RNA, Plant metabolism, Soil, Stress, Physiological, Water physiology, Hordeum genetics, MicroRNAs genetics
- Abstract
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 high-throughput Real-Time RT-qPCR platform for parallel determination of 159 barley primary microRNAs' levels. The platform was tested for two drought-and-rehydration-treated 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 pri-miRNAs could be used as drought stress or rehydration markers. The usage of the platform in biotechnology is also postulated.
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- 2016
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14. Promoter-based identification of novel non-coding RNAs reveals the presence of dicistronic snoRNA-miRNA genes in Arabidopsis thaliana.
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Qu G, Kruszka K, Plewka P, Yang SY, Chiou TJ, Jarmolowski A, Szweykowska-Kulinska Z, Echeverria M, and Karlowski WM
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- Base Sequence, Binding Sites, Computational Biology methods, Gene Order, MicroRNAs chemistry, Nucleic Acid Conformation, Nucleotide Motifs, RNA Interference, RNA, Small Nucleolar chemistry, RNA, Untranslated chemistry, Regulatory Sequences, Nucleic Acid, Arabidopsis genetics, MicroRNAs genetics, Promoter Regions, Genetic, RNA, Small Nucleolar genetics, RNA, Untranslated genetics
- Abstract
Background: In the past few decades, non-coding RNAs (ncRNAs) have emerged as important regulators of gene expression in eukaryotes. Most studies of ncRNAs in plants have focused on the identification of silencing microRNAs (miRNAs) and small interfering RNAs (siRNAs). Another important family of ncRNAs that has been well characterized in plants is the small nucleolar RNAs (snoRNAs) and the related small Cajal body-specific RNAs (scaRNAs). Both target chemical modifications of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). In plants, the snoRNA genes are organized in clusters, transcribed by RNA Pol II from a common promoter and subsequently processed into mature molecules. The promoter regions of snoRNA polycistronic genes in plants are highly enriched in two conserved cis-regulatory elements (CREs), Telo-box and Site II, which coordinate the expression of snoRNAs and ribosomal protein coding genes throughout the cell cycle., Results: In order to identify novel ncRNA genes, we have used the snoRNA Telo-box/Site II motifs combination as a functional promoter indicator to screen the Arabidopsis genome. The predictions generated by this process were tested by detailed exploration of available RNA-Seq and expression data sets and experimental validation. As a result, we have identified several snoRNAs, scaRNAs and 'orphan' snoRNAs. We also show evidence for 16 novel ncRNAs that lack similarity to any reported RNA family. Finally, we have identified two dicistronic genes encoding precursors that are processed to mature snoRNA and miRNA molecules. We discuss the evolutionary consequences of this result in the context of a tight link between snoRNAs and miRNAs in eukaryotes., Conclusions: We present an alternative computational approach for non-coding RNA detection. Instead of depending on sequence or structure similarity in the whole genome screenings, we have explored the properties of promoter regions of well-characterized ncRNAs. Interestingly, besides expected ncRNAs predictions we were also able to recover single precursor arrangement for snoRNA-miRNA. Accompanied by analyses performed on rice sequences, we conclude that such arrangement might have interesting functional and evolutionary consequences and discuss this result in the context of a tight link between snoRNAs and miRNAs in eukaryotes.
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- 2015
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15. mirEX 2.0 - an integrated environment for expression profiling of plant microRNAs.
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Zielezinski A, Dolata J, Alaba S, Kruszka K, Pacak A, Swida-Barteczka A, Knop K, Stepien A, Bielewicz D, Pietrykowska H, Sierocka I, Sobkowiak L, Lakomiak A, Jarmolowski A, Szweykowska-Kulinska Z, and Karlowski WM
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- Arabidopsis metabolism, Gene Expression Profiling, Hepatophyta metabolism, Hordeum metabolism, MicroRNAs metabolism, RNA, Plant metabolism, Real-Time Polymerase Chain Reaction, Arabidopsis genetics, Databases, Nucleic Acid organization & administration, Hepatophyta genetics, Hordeum genetics, Internet, MicroRNAs genetics, RNA, Plant genetics
- Abstract
Background: MicroRNAs are the key post-transcriptional regulators of gene expression in development and stress responses. Thus, precisely quantifying the level of each particular microRNA is of utmost importance when studying the biology of any organism., Description: The mirEX 2.0 web portal ( http://www.combio.pl/mirex ) provides a comprehensive platform for the exploration of microRNA expression data based on quantitative Real Time PCR and NGS sequencing experiments, covering various developmental stages, from wild-type to mutant plants. The portal includes mature and pri-miRNA expression levels detected in three plant species (Arabidopsis thaliana, Hordeum vulgare and Pellia endiviifolia), and in A. thaliana miRNA biogenesis pathway mutants. In total, the database contains information about the expression of 461 miRNAs representing 268 families. The data can be explored through the use of advanced web tools, including (i) a graphical query builder system allowing a combination of any given species, developmental stages and tissues, (ii) a modular presentation of the results in the form of thematic windows, and (iii) a number of user-friendly utilities such as a community-building discussion system and extensive tutorial documentation (e.g., tooltips, exemplary videos and presentations). All data contained within the mirEX 2.0 database can be downloaded for use in further applications in a context-based way from the result windows or from a dedicated web page., Conclusions: The mirEX 2.0 portal provides the plant research community with easily accessible data and powerful tools for application in multi-conditioned analyses of miRNA expression from important plant species in different biological and developmental backgrounds.
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- 2015
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16. Transcriptionally and post-transcriptionally regulated microRNAs in heat stress response in barley.
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Kruszka K, Pacak A, Swida-Barteczka A, Nuc P, Alaba S, Wroblewska Z, Karlowski W, Jarmolowski A, and Szweykowska-Kulinska Z
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- Base Sequence, Down-Regulation, Gene Regulatory Networks, Hordeum metabolism, MicroRNAs genetics, Molecular Sequence Data, RNA Splicing, RNA, Plant genetics, RNA, Plant metabolism, Sequence Analysis, DNA, Gene Expression Regulation, Plant, Heat-Shock Response genetics, Hordeum genetics, MicroRNAs metabolism, RNA Processing, Post-Transcriptional
- Abstract
Heat stress is one of the major abiotic factors that can induce severe plant damage, leading to a decrease in crop plant productivity. Despite barley being a cereal of great economic importance, few data are available concerning its thermotolerance mechanisms. In this work microRNAs (miRNAs) involved in heat stress response in barley were investigated. The level of selected barley mature miRNAs was examined by hybridization. Quantitative real-time PCR (RT-qPCR) was used to monitor the changes in the expression profiles of primary miRNA (pri-miRNA) precursors, as well as novel and conserved target genes during heat stress. The miRNA-mediated cleavage sites in the target transcripts were confirmed by degradome analysis and the 5' RACE (rapid amplification of cDNA ends) approach. Four barley miRNAs (miR160a, 166a, 167h, and 5175a) were found which are heat stress up-regulated at the level of both mature miRNAs and precursor pri-miRNAs. Moreover, the splicing of introns hosting miR160a and miR5175a is also heat induced. The results demonstrate transcriptional and post-transcriptional regulation of heat-responsive miRNAs in barley. The observed induction of miRNA expression is correlated with the down-regulation of the expression level of their experimentally identified new and conservative target genes., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)
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- 2014
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17. Arabidopsis thaliana LSM proteins function in mRNA splicing and degradation.
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Golisz A, Sikorski PJ, Kruszka K, and Kufel J
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- Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins physiology, Cell Nucleus chemistry, Cytoplasm chemistry, Mutation, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, RNA-Binding Proteins physiology, Arabidopsis genetics, Arabidopsis Proteins metabolism, Gene Expression Regulation, Plant, RNA Splicing, RNA Stability, RNA, Messenger metabolism
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Sm-like (Lsm) proteins have been identified in all organisms and are related to RNA metabolism. Here, we report that Arabidopsis nuclear AtLSM8 protein, as well as AtLSM5, which localizes to both the cytoplasm and nucleus, function in pre-mRNA splicing, while AtLSM5 and the exclusively cytoplasmic AtLSM1 contribute to 5'-3' mRNA decay. In lsm8 and sad1/lsm5 mutants, U6 small nuclear RNA (snRNA) was reduced and unspliced mRNA precursors accumulated, whereas mRNA stability was mainly affected in plants lacking AtLSM1 and AtLSM5. Some of the mRNAs affected in lsm1a lsm1b and sad1/lsm5 plants were also substrates of the cytoplasmic 5'-3' exonuclease AtXRN4 and of the decapping enzyme AtDCP2. Surprisingly, a subset of substrates was also stabilized in the mutant lacking AtLSM8, which supports the notion that plant mRNAs are actively degraded in the nucleus. Localization of LSM components, purification of LSM-interacting proteins as well as functional analyses strongly suggest that at least two LSM complexes with conserved activities in RNA metabolism, AtLSM1-7 and AtLSM2-8, exist also in plants.
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- 2013
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18. Role of microRNAs and other sRNAs of plants in their changing environments.
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Kruszka K, Pieczynski M, Windels D, Bielewicz D, Jarmolowski A, Szweykowska-Kulinska Z, and Vazquez F
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- Gene Silencing, Plants genetics, RNA, Plant genetics, Signal Transduction, Stress, Physiological, Gene Expression Regulation, Plant genetics, MicroRNAs genetics, RNA, Small Interfering genetics
- Abstract
Plants constantly face a complex array of environmental biotic and abiotic stimuli. Recent studies in various plants have highlighted the key roles of microRNAs and of different siRNA classes in the post-transcriptional regulation of plant genes essential for conserved responses of plants to individual stress conditions. It is not yet clear how these different signals and responses are integrated in nature. In the present review, we summarize current knowledge on sRNA-mediated responses to stress, and highlight possible directions of future research., (Copyright © 2012 Elsevier GmbH. All rights reserved.)
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- 2012
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19. The ATP- and CoA-independent synthesis of arachidonoylethanolamide. A novel mechanism underlying the synthesis of the endogenous ligand of the cannabinoid receptor.
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Kruszka KK and Gross RW
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- Adenosine Triphosphate metabolism, Amides metabolism, Animals, Cell Fractionation, Coenzyme A metabolism, Cytosol metabolism, Endocannabinoids, Organ Specificity, Polyunsaturated Alkamides, Rabbits, Substrate Specificity, Amidohydrolases metabolism, Arachidonic Acids, Cerebral Cortex metabolism, Fatty Acids, Unsaturated biosynthesis, Microsomes metabolism
- Abstract
Recently, arachidonoylethanolamide was identified as the endogenous ligand of the delta 9 tetrahydrocannabinol receptor. Herein, we demonstrate that the synthesis of arachidonoylethanolamide is catalyzed by a novel CoA- and ATP-independent pathway, which is highly selective for arachidonic acid as the aliphatic constituent, is specific for ethanolamine as the polar moiety and occurs through a mechanism that utilizes a critical sulfhydryl residue. The production of arachidonoylethanolamide was calcium-independent and heat-labile and was selectively catalyzed by brain microsomal and cytosolic proteins among the tissues examined. Four independent lines of evidence demonstrate that the synthesis of arachidonoylethanolamide occurs through an ATP- and CoA-independent process. 1) The depletion of endogenous rabbit brain cytosolic or microsomal CoA and ATP by dialysis, or depletion of ATP by apyrase treatment, did not diminish the production of arachidonoylethanolamide; 2) the addition of exogenous ATP or CoA to cofactor depleted cytosol or microsomes did not stimulate arachidonoylethanolamide production; 3) the synthesis of arachidonoylethanolamide occurred in the absence of detectable arachidonoyl-CoA and did not correlate with either the amount of arachidonoyl-CoA in the incubation medium or the amount of arachidonoyl-CoA incorporated into polar and nonpolar lipids; 4) the addition of a 20-fold molar excess of unlabeled arachidonoyl-CoA to incubations containing [3H]arachidonic acid and ethanolamine did not attenuate the production of [3H]arachidonoylethanolamide by rabbit brain cytosolic or microsomal proteins. Collectively, these results demonstrate a novel chemical paradigm for the ATP- and CoA-independent conjugation of the carboxylate moiety of arachidonic acid to yield arachidonoylethanolamide, the first member of a new class of biologically active eicosanoid second messengers.
- Published
- 1994
20. Rat and human pancreatic islet cells contain a calcium ion independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by adenosine triphosphate and is specifically localized to islet beta-cells.
- Author
-
Gross RW, Ramanadham S, Kruszka KK, Han X, and Turk J
- Subjects
- Animals, Cell Membrane enzymology, Cytosol enzymology, Egtazic Acid pharmacology, Humans, Hydrolysis, Insulinoma enzymology, Male, Naphthalenes pharmacology, Pancreatic Neoplasms enzymology, Phospholipases A antagonists & inhibitors, Phospholipases A2, Pyrones pharmacology, Rats, Rats, Sprague-Dawley, Substrate Specificity, Tissue Distribution, Tumor Cells, Cultured, Adenosine Triphosphate pharmacology, Arachidonic Acid metabolism, Calcium pharmacology, Islets of Langerhans enzymology, Phospholipases A metabolism
- Abstract
The recent demonstration that myocardial Ca(2+)-independent phospholipase A2 exists as a complex of catalytic and regulatory polypeptides that is modulated by ATP has suggested a novel mechanisms through which alterations in glycolytic flux can be coupled to the generation of eicosanoids which facilitate insulin secretion. To determine the potential relevance of this mechanism, we examined the kinetic characteristics, substrate specificities, and cellular locus of phospholipase A2 activity in pancreatic islets. Rat pancreatic islets contain a Ca(2+)-independent phospholipase A2 activity which is optimal at physiologic pH, preferentially hydrolyzes phospholipid substrates containing a vinyl ether linkage at the sn-1 position, and prefers arachidonic acid compared to oleic acid in the sn-2 position. Rat islet Ca(2+)-independent phospholipase A2 activity is inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one and is stimulated by ATP. Purification of beta-cells from dispersed pancreatic islet cells by fluorescence-activated cell sorting demonstrated that beta-cells (but not non-beta-cells) contain Ca(2+)-independent, ATP-stimulated phospholipase A2 activity. Remarkably, clonal RIN-m5f insulinoma cells, which possess a defect in glucose-induced insulin secretion, contain a Ca(2+)-independent phospholipase A2 which is not modulated by alterations in ATP concentration. Collectively, these results and those of an accompanying paper [Ramanadham et al. (1993) Biochemistry (following paper in this issue)] implicate Ca(2+)-independent phospholipase A2 as a putative glucose sensor which can couple alterations in glycolytic metabolism to the generation of biologically active eicosanoids and thereby facilitate glucose-induced insulin secretion.
- Published
- 1993
- Full Text
- View/download PDF
21. Calcium is sufficient but not necessary for activation of sheep platelet cytosolic phospholipase A2.
- Author
-
Zupan LA, Kruszka KK, and Gross RW
- Subjects
- Animals, Chromatography, Gel, Enzyme Activation, Phospholipases A2, Phospholipids metabolism, Sheep, Sodium Chloride pharmacology, Blood Platelets enzymology, Calcium pharmacology, Phospholipases A blood
- Abstract
In this study we demonstrate that: (1) although the major phospholipase A2 present in sheep platelets is activated by calcium ions, it can effectively catalyze hydrolysis of the sn-2 ester linkage in phospholipids in the absence of calcium; (2) expression of calcium-independent phospholipase A2 activity can be induced by NaCl utilizing purified (but not crude) cytosolic enzyme; and (3) calcium-independent phospholipase A2 activity is regulated by a reconstitutable cytosolic protein. Collectively, these results underscore the fundamental catalytic differences between extracellular and intracellular calcium-dependent phospholipases A2 and demonstrate that calcium is sufficient, but not necessary, for the activation of this class of intracellular phospholipases A2.
- Published
- 1991
- Full Text
- View/download PDF
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