Biology subjects

Szweykowska-Kulinska, Z.

Publications and source records attributed to Szweykowska-Kulinska, Z..

15 recordsLinked to original sources

bioRxiv2024

Barley miRNAs and their targets regulation in response to heat stress at the early stage of development

MiRNAs are key regulators of gene expression controlling plant development and response to environmental stresses. In this work we studied global dynamics of accumulation of conserved and identified novel barley miRNAs at early stage of plant development during heat stress (1h, 3h and 6h of heat stress). The majority of miRNAs responds to heat stress after 3h and 6h of heat stress duration (124 and 155, respectively). The comparison of heat-induced changes in mature miRNA accumulation to their cognate precursor levels allowed to indicate a smaller group of miRNAs that are controlled at transcriptional level and a larger group that is controlled posttranscriptionally in response to heat stress. For miRNAs with the significant accumulation changes during heat treatment, target mRNAs were identified. Moreover, novel targets have been experimentally assigned for selected miRNAs. mRNA of the effector protein of miRNA activity, AGO1B was found to be downregulated by increased miR168 during heat stress. Importantly, miRNA/mRNA target module miR399c/PHO2 responsible for the phosphorus uptake exhibits dynamic changes under heat stress conditions suggesting adaptation of plant development to stress conditions. This study provides new data for developing miRNA and their mRNA target-based strategies in barley breeding in response to heat stress. Keypoints- Involvement of small RNAs in response to the heat stress conditions have been studied in young barley plants. - The largest number of heat responsive miRNAs was found after 3h and 6h of heat duration. - Heat-induced mature miRNA accumulation and their precursor levels showed complex transcriptional or posttranscriptional regulation. - MiRNA-target modules responsive to heat stress were identified

molecular biology↗

bioRxiv2024

Plant Cleavage Factor I complex is essential for precise cleavage and polyadenylation site determination

Cleavage factor I (CFI) is a four-subunit protein complex of the pre-mRNA 3 end processing machinery in eukaryotes. In Arabidopsis, AtCFI25a, AtCFI25b, AtCFI59, and AtCFI68 have been identified as potential components of AtCFI, in silico. Here, we show that the AtCFI25a, AtCFI59, and AtCFI68 proteins each pulled down all components of the CFI, confirming that these subunits form the plant CFI complex. Furthermore, either AtCFI59 or AtCFI68 was essential for nuclear localization of the smallest subunit, AtCFI25a. Mutants with single loss-of-function for AtCFI59 or AtCFI68 showed no obvious morphological defects compared to wild-type plants, while the double mutant displayed pleiotropic morphological defects, identical to those previously reported for AtCFI25a loss-of-function plants. Moreover, these morphological defects correlated with alterations in the usage of 3 UTR cleavage and polyadenylation sites. atcfi25a, atcfi25a atcfi25b and atcfi59 atcfi68 double mutants showed widespread changes in the choice of cleavage and polyadenylation sites. In most cases, more proximal cleavage and polyadenylation sites were used, leading to shorter 3 UTRs. In particular, genes involved in light intensity, light harvesting, photosynthesis and cold responses showed significant dependence on AtCFI function. Furthermore, transcripts coding for AtCFI subunits showed altered 3 end processing in these mutants, suggesting self-regulation function of AtCFI in plants.

molecular biology↗

bioRxiv2023

Interplay between m6A modification and overall transcripts quantity: Impacts on mRNA composition in plant stress granules

Stress granules (SGs) are cytoplasmic structures that emerge in response to unfavorable environmental conditions. They contain a rich pool of RNA, including non-translated mRNA. The mechanisms governing transcripts accumulation in SGs is only partially understood. Despite the recognized role of m6A in plant transcriptome regulation, its impact on SGs composition and assembly remains elusive. We examined the formation of SGs, the presence of m6A, and the transcription-level-dependent localization of selected mRNAs within these granules during hypoxia in the roots of Lupinus angustifolius and Arabidopsis thaliana. In lupine, SGs exhibit a distinctive bi-zonal structure, comprising of a ring and a central area with differences in ultrastructure and composition. Following the transcriptome analysis, mRNAs were selected for examination of their localization in SGs and m6A levels. Transcripts from genes responsive to hypoxia (ADH1 and HUP7) exhibited significant lower levels of m6A compared to housekeeping genes but only ADH1 was not present in SGs. HUP7 mRNA with low quantity of m6A, is present both in the SGs and cytoplasm probably due to extremely high expression level. It was also shown that the amount of m6A in SGs was higher than in the cytoplasm only in the first hours of hypoxia and then decreased. In mutants of A. thaliana with reduced level of m6A, formation and quantity of SGs were studied. In this line, ECT2 was not observed and poly(A) RNA levels were slightly reduced in SGs. Additionally the number of SGs was lower than that of the wild type. In summary, our findings demonstrate the limited impact of m6A modification on SGs assembly. However the interplay between m6A modification and the overall transcript quantity in the cytoplasm plays a regulatory role in mRNA partitioning into SGs.

cell biology↗

bioRxiv2023

MiRNAs differentially expressed in vegetative and reproductive organs of Marchantia polymorpha, insights into their expression pattern, gene structures and function

MicroRNAs (miRNAs) regulate gene expression affecting a variety of plant developmental processes. The evolutionary position of Marchantia polymorpha makes it a significant model to understand miRNA-mediated gene regulatory pathways in plants. Previous studies focused on conserved miRNA-target mRNA modules showed their critical role in Marchantia development. Here, we demonstrate that differential expression of conserved miRNAs and their targets in selected organs of Marchantia additionally underlines their role in regulating fundamental developmental processes. The main aim of this study was to characterize selected liverwort-specific miRNAs, as there is a limited knowledge on their biogenesis, accumulation, targets, and function in Marchantia. We demonstrate their differential accumulation in vegetative and generative organs. We reveal that all liverwort-specific miRNAs examined are encoded by independent transcriptional units. MpmiR11737a, MpmiR11887 and MpmiR11796, annotated as being encoded within protein-encoding genes, have their own independent transcription start sites. The analysis of selected liverwort-specific miRNAs and their pri-miRNAs often reveal correlation in their levels, suggesting transcriptional regulation. However, MpmiR11796 shows a reverse correlation to its pri-miRNA level, suggesting post-transcriptional regulation. Moreover, we identify novel targets for selected liverwort-specific miRNAs and demonstrate an inverse correlation between their expression and miRNA accumulation. In the case of one miRNA precursor, we provide evidence that it encodes two functional miRNAs with two independent targets. Overall, our research sheds light on liverwort-specific miRNA gene structure, provides new data on their biogenesis and expression regulation. Furthermore, identifying their targets, we hypothesize the potential role of these miRNAs in early land plant development and functioning.

molecular biology↗

bioRxiv2023

PEP444c encoded within the MIR444c gene regulates microRNA444c accumulation in barley

MicroRNAs are small, non-coding RNA molecules that regulate expression of their target genes. The MIR444 gene family is present exclusively in monocotyledons, and microRNAs444 from this family have been shown to target certain MADS-box transcription factors in rice and barley. We identified three barley MIR444 (MIR444a/b/c) genes and comprehensively characterized their structure and the processing pattern of the primary transcripts (pri-miRNAs444). Pri-microRNAs444 undergo extensive alternative splicing, by which functional and non-functional pri-miRNA444 isoforms are generated. We show that barley pri-miRNAs444 contain numerous open reading frames (ORFs) whose transcripts associate with ribosomes. Using specific antibodies, we provide evidence that selected ORFs encoding PEP444a within MIR444a and PEP444c within MIR444c are expressed in barley plants. Moreover, we demonstrate that CRISPR-associated endonuclease 9 (Cas9)-mediated mutagenesis of the PEP444c encoding sequence results in a decreased level of PEP444 transcript in barley shoots and roots, and a 5-fold reduced level of mature microRNA444c in roots. Taken together, our observations suggest that PEP444c encoded by the MIR444c gene is involved in microRNA444c biogenesis in barley.

plant biology↗

bioRxiv2023

Comparative analysis of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family between bryophytes and seed plants

SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes encode plant-specific transcription factors which have been found to be conserved in green plants lineage. SPL proteins are important regulators of diverse plant developmental processes in bryophytes and vascular plants. In our study, we took advantage of available genome sequences of representatives of each bryophyte clade to investigate the relationships of SPL genes between bryophytes and model angiosperm Arabidopsis thaliana. We have identified four SPL genes in each of the two hornworts species, Anthoceros agrestis and Anthoceros punctatus, what is similar to the set of SPL genes present in the liverwort Marchantia polymorpha. Thus, the analyzed hornworts and liverwort genomes encode a minimal set of SPL genes in comparison to other land plants that may resemble an archetype of SPL genes present in the ancestor of land plants. The phylogenetic analysis revealed the presence of four SPL groups. Comparative gene structure analysis showed that SPLs share similar exon-intron organization within the same phylogenetic group with some exceptions in hornworts. While we have identified conserved protein motifs between bryophytes and Arabidopsis in three out of four phylogenetic groups, the motif content differed explicitly in the fourth group. Since current understanding of SPL genes mostly arises from seed plants, the presented comparative and phylogenetic analysis will provide better understanding of SPL gene family from the representatives of the oldest living land plants.

plant biology↗

bioRxiv2022

Chromatin-associated microprocessor assembly is regulated by PRP40, the U1 snRNP auxiliary protein

Cotranscriptional processing of RNA polymerase II-generated primary transcripts is a well-documented phenomenon. We recently showed that in plants, miRNA biogenesis is also a cotranscriptional event. Here, we report that Arabidopsis PRP40, the U1 snRNP auxiliary protein, positively regulates the recruitment of SE, the core component of the plant microprocessor, to miRNA genes. The association of DCL1, the microprocessor endoribonuclease, with chromatin was altered in prp40ab mutant plants. Impaired cotranscriptional microprocessor assembly was accompanied by RNA polymerase II accumulation at miRNA genes and retention of miRNA precursors at their transcription sites in the prp40ab mutant plants. We show that cotranscriptional microprocessor assembly, regulated by AtPRP40, positively affects RNAPII transcription of miRNA genes and is important to reach the correct levels of produced miRNAs.

molecular biology↗

bioRxiv2021

R-loops between nascent pri-miRNAs and the encoding loci promote co-transcriptional processing of miRNAs in plants

In most organisms, the maturation of nascent RNAs is coupled to transcription, undergoing many processing steps co-transcriptionally. Unlike in animals, the RNA polymerase II (RNAPII) transcribes microRNAs (miRNAs) as long and structurally variable pri-miRNAs in plants. Current evidence suggests that the miRNA biogenesis complex assembly initiates early during the transcription of pri-miRNAs in plants. However, it is unknown whether miRNA processing occurs co-transcriptionally. Here, we show that plant miRNA biogenesis is coupled to transcription in a process that relies on the formation of DNA:RNA hybrids (R-loops) between the nascent transcript and the encoding loci. We used native elongating transcript sequencing data and imaging techniques to demonstrate that plant miRNA biogenesis occurs co-transcriptionally. We found that the entire biogenesis occurs coupled to transcription for pri-miRNAs processed from the loop but requires a second nucleoplasmic step for those processed from the base of the hairpin. Furthermore, we found that co- and post-transcriptional miRNA processing mechanisms co-exist for most miRNAs in a dynamic balance. Notably, we discovered that R-loops between the 5-end single-stranded arm of the pri-miRNAs and the encoding loci anchor the transcript, promoting co-transcriptional processing. Our data demonstrate the coupling of transcription and miRNA processing in plants and discovered an unexpected function for R-loops promoting RNA processing. Furthermore, our results suggest the neo-functionalization of co-transcriptionally processed miRNAs, boosting countless regulatory scenarios.

plant biology↗

bioRxiv2021

A high resolution single molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis

BackgroundAccurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single molecule long read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation or incomplete cDNA synthesis. ResultsWe present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 160k transcripts - twice that of the best current Arabidopsis transcriptome and including over 1,500 novel genes. 79% of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We developed novel methods to determine splice junctions and transcription start and end sites accurately. Mis- match profiles around splice junctions provided a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identified high confidence transcription start/end sites and removed fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provided higher resolution of transcript expression profiling and identified cold- and light-induced differential transcription start and polyadenylation site usage. ConclusionsAtRTD3 is the most comprehensive Arabidopsis transcriptome currently available. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single molecule sequencing analysis from any species.

plant biology↗

bioRxiv2021

Arabidopsis spliceosome factor SmD3 modulates immunity to Pseudomonas syringae infection

SmD3 is a core component of the small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. The role of Arabidopsis SmD3 in plant immunity was assessed by testing sensitivity of smd3a and smd3b mutants to Pseudomonas syringae pv. tomato (Pst) DC3000 infection and its pathogenesis effectors flagellin (flg22), EF-Tu (elf18) and coronatine (COR). Both smd3 mutants exhibited enhanced susceptibility to Pst accompanied by marked changes in the expression of key pathogenesis markers. mRNA levels of these factors were also altered upon treatment with Pseudomonas effectors. We showed that SmD3-b dysfunction impairs mainly stomatal immunity as a result of defects in stomatal development. Our genome-wide transcriptome analysis of the smd3b-1 mutant infected with Pst revealed that lack of SmD3-b deregulates defense against Pst infection at the transcriptional and posttranscriptional levels including defects in splicing and an altered pattern of alternative splicing. Other changes in the smd3b-1 mutant involved enhanced elf18- and flg22-induced callose deposition, reduction of flg22-triggered production of early ROS and boost of secondary ROS caused by Pst infection. Together, our data indicate that SmD3 contributes to the plant immune response possibly via regulation of mRNA splicing of key pathogenesis factors.

molecular biology↗

bioRxiv2021

The MYB33, MYB65, and MYB101 transcription factors affect Arabidopsis and potatoresponses to drought by regulating the ABA signaling pathway

AimsDrought is a climate threat limiting crop production. Potato is one of the four most important food crops worldwide and is sensitive to water shortage. The CBP80 gene was shown to affect plant response to drought by regulating the level of microRNA159, and, consequently, the levels of the MYB33 and MYB101 transcription factors. (TF) Our studies aimed to show whether indeed the level of MYB33, MYB65, and MYB101 TFs affects plant response to water shortage. MethodsArabidopsis transgenic plants exhibiting downregulation and Arabidopsis and potato transgenic plants exhibiting overexpression of selected MYB TFs were obtained. Plants response to drought was mainly measured using relative water content (RWC) and stomata closure upon exogenous ABA. ResultsThree MYB TFs studied are involved in plant response to drought. When downregulated in Arabidopsis, the MYB33, MYB65 and MYB101 genes cause stomatal hyposensitivity to ABA, leading to reduced tolerance to drought. Transgenic Arabidopsis and potato plants overexpressing a mutated version of these genes with changed miR159 recognition site, show hypersensitivity to ABA and relatively high tolerance to drought conditions. ConclusionsThe MYB33, MYB65, and MYB101 genes are good be potential targets for innovative breeding to obtain crops with relatively high tolerance to drought.

molecular biology↗

bioRxiv2020

SERRATE interacts with the nuclear exosome targeting (NEXT) complex to degrade primary miRNA precursors in Arabidopsis

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the Nuclear Exosome Targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.

plant biology↗

bioRxiv2019

DRB1 as a mediator between transcription and microRNA processing

DRB1 (HYL1) is a double-stranded RNA binding protein involved in miRNA processing in plants. It is a core component of the Microprocessor complex and enhances the efficiency and precision of miRNA processing by DCL1 protein. In this work, we report a novel function of DRB1 protein in the transcription of MIR genes. DRB1 co-localizes with RNA Polymerase II and affects its distribution along MIR genes. Moreover, proteomic experiments revealed that DRB1 protein interacts with many transcription factors. Finally, we show that the action of DRB1 is not limited to MIR genes as it impacts expression of many other genes, majority of which are involved in plant response to light. These discoveries add DRB1 as another player of gene regulation at transcriptional level, independent of its role in miRNA biogenesis.

plant biology↗

bioRxiv2019

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

m6A, one of the most abundant mRNA modifications, has been associated with various metabolic processes in plants. Here we show that m6A also plays a role in miRNA biogenesis in Arabidopsis thaliana. Significant reductions in plant m6A/MTA levels results in lower accumulation of miRNAs whereas pri-miRNA levels tend to be higher in such plants. m6A-IP Seq and MTA-GFP RIP were used to show that many pri-miRNAs are m6A methylated and are bound by MTA, further demonstrating that pri-miRNAs can also be substrates for m6A methylation by MTA. We report that MTA interacts with RNA Pol II, supporting the assumption that m6A methylation is a co-transcriptional process, and also identify TGH, a known miRNA biogenesis related protein, as a novel protein that interacts with MTA. Finally, reduced levels of miR393b may partially explain the strong auxin insensitivity seen in Arabidopsis plants with reduced m6A levels.

plant biology↗

bioRxiv2017

The plastid-nucleus located DNA/RNA binding protein WHIRLY1 regulates microRNA-levels during stress

In this article a novel mechanism of retrograde signaling by chloroplasts during stress is described. This mechanism involves the DNA/RNA binding protein WHIRLY1 as a regulator of microRNA levels. By virtue of its dual localization in chloroplasts and the nucleus of the same cell, WHIRLY1 was proposed as an excellent candidate coordinator of chloroplast function and nuclear gene expression (Grabowski et al., 2008; Foyer et al., 2014). In this study the putative involvement of WHIRLY1 in stress dependent retrograde signaling was investigated by comparison of barley (Hordeum vulgare L., cv. Golden Promise) wild-type and transgenic plants with an RNAi-mediated knockdown of WHIRLY1. In contrast to the wild type, the transgenic plants were unable to cope with continuous high light conditions. They were impaired in production of several microRNAs mediating post-transcriptional responses during stress (Kruszka et al., 2012, Sunkar et al., 2012). The results support a central role of WHIRLY1 in retrograde signaling and underpin a so far underestimated role of microRNAs in this process.

plant biology↗