Biology subjects

Duque, P.

Publications and source records attributed to Duque, P..

9 recordsLinked to original sources

bioRxiv2026

Identification and binding-site mapping of RNA-binding proteins interacting with microRNA precursors in Arabidopsis

BackgroundMicroRNAs (miRNAs) play key roles in modulating gene expression. Upon transcription, primary miRNA transcripts (pri-miRNAs or MIRNAs) fold into stem-loop structures. Endonucleolytic cleavage releases a miRNA/miRNA* duplex from the stem, which is subsequently matured. In higher plants, the pri-miRNA hairpins vary widely in length and structure. Pri-miRNA processing is extensively regulated by RNA-binding proteins (RBPs) and the range of underlying mechanisms continues to expand. ResultsHere, we performed an unbiased screen to identify Arabidopisis thaliana RBPs that interact with pri-miRNAs, using pri-miRNA159a and pri-miRNA398b as paradigms. Nucleoplasmic proteins were significantly enriched in pulldowns with bead-immobilized in vitro transcripts of the stem-loop regions of these pri-miRNAs compared with pulldowns using empty beads. The enriched proteins included several members of the family of the serine/arginine-rich (SR) splicing factor family, including RS31 and SR34a. Mutant analysis indicated that selected candidate interactors affect pri-miRNA and/or mature miRNA accumulation. To validate interactions with pri-miRNAs in vivo, we performed individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). Whereas our conventional plant iCLIP procedure detected only a few crosslink sites on pri-miRNAs, likely because of the low abundance of these transcripts, our improved plant iCLIP2 protocol enabled the identification of binding sites on numerous pri-miRNAs. RS31 and SR34a bound overlapping sets of pri-miRNAs and contacted both stem-loop regions and flanking regions upstream and downstream of the stem-loop. RS31 and SR34 bound both overlapping and distinct regions of pri-miRNAs. ConclusionIn vitro pulldowns of nucleoplasmic proteins identified RBPs associated with pri-miRNA stem-loops, and the improved plant iCLIP2 protocol revealed in vivo binding of RS31 and SR34a to a subset of pri-miRNAs. Together, these findings expand the repertoire of RBPs implicated in plant miRNA biogenesis and support a role for SR proteins as candidate regulators of pri-miRNA processing.

plant biology↗

bioRxiv2026

Upstream ORFs regulate translation of the Arabidopsis ZIF2 transporter and affect ER stress tolerance

Translational regulation mediated by upstream open reading frames (uORFs) is an important mechanism controlling gene expression in eukaryotes, yet the functional characterization of uORFs in plant transporter genes remains limited. ZINC-INDUCED FACILITATOR 2 (ZIF2) is an Arabidopsis thaliana tonoplast-localized zinc transporter whose expression is regulated at the translational level via alternative splicing of its 5 untranslated region (UTR), but the role of additional cis-regulatory elements within this region has remained unexplored. Here, we characterize the functional significance of three uORFs present in the ZIF2 5UTR and elucidate their contribution to translational control and stress responses. We demonstrate that two of the three uORFs (uORF2 and uORF3) act as negative regulators of ZIF2 translation. uORF2 is constitutively translated and plays the primary role in uORF-mediated translational inhibition, whereas uORF3 functions as a fail-safe mechanism that represses translation only when uORF2 is inactive. We further show that uORF2-mediated repression depends on the amino acid sequence of the encoded peptide and identify a short motif critical for its function. Importantly, uORF-mediated regulation of ZIF2 is physiologically relevant in planta: plants carrying mutations that abolish uORF2 translation accumulate higher levels of ZIF2 protein and display increased tolerance to endoplasmic reticulum stress. Together, our findings reveal a uORF-based fail-safe regulatory mechanism that modulates ZIF2 expression, contributes to plant stress adaptation, and is consistent with translational inhibition mediated by peptide-dependent ribosome stalling.

plant biology↗

bioRxiv2025

Alternative splicing of PIF4 regulates plant development under heat stress

The Phytochrome-Interacting Factor 4 (PIF4) is a key player in the integration of multiple internal and external stimuli to optimize different aspects of plant development. While both the DNA encoding this transcription factor and its protein are known to be under tight control, no regulation at the RNA level has been previously reported. Our genomic analysis revealed that the exon/intron structure of the basic Helix-Loop-Helix (bHLH) DNA binding domain of PIF4 is conserved and pointed to skipping of an exon in this region specifically in response to heat stress. We then showed that this alternative splicing event downregulates PIF4 function under heat, which in etiolated seedlings induces photomorphogenic-related traits. Our results disclose a role for PIFs in plant responses to heat and reveal a new regulatory layer for the control of PIF4 function, underscoring the critical role of posttranscriptional regulatory processes in the molecular integration of environmental cues.

plant biology↗

bioRxiv2025

Cotyledon opening during seedling deetiolation is determined by ABA-mediated splicing regulation

During seedling deetiolation, plants adjust their development to expose photosynthetic tissues to sunlight, enabling the transition from heterotrophic to autotrophic growth. While various plant hormones are known to influence this process, the role of abscisic acid (ABA) remains unclear. Here, we reveal that ABA plays a major role in controlling the dynamics of cotyledon aperture during seedling deetiolation. In darkness, ABA accumulates in the cotyledons to effectively repress their opening. However, light exposure reverses this effect, allowing the cotyledons to open. Our findings indicate that the ABA-mediated regulation of cotyledon dynamics is accompanied by genome-wide rearrangements in both transcriptional and splicing patterns. We demonstrate that the ABA-dependent adjustments of cotyledon and splicing dynamics in response to light depend on the positive role of two splicing factors, RS40 and RS41. Moreover, we identify transcriptional and posttranscriptional mechanisms that repress the activity of these proteins in the dark. Altogether, this work sheds light on the interplay between light and ABA, highlighting a new developmental outcome: cotyledon opening, and identifying a novel layer of gene regulation: alternative splicing.

plant biology↗

bioRxiv2023

An Arabidopsis SR protein relieving ABA inhibition of seedling establishment represses ABA-responsive alternative splicing

The phytohormone abscisic acid (ABA) inhibits postgerminative growth under unfavorable conditions to delay the transition to the autotrophic stage and promote plant survival. While stress-induced ABA accumulation is well established to trigger extensive transcriptional changes, it is becoming clear that it also relies on alternative splicing to enhance stress tolerance. However, the upstream components modulating posttranscriptional regulation of the ABA response remain largely unknown. Here, we show that loss of function of the Arabidopsis SR34a protein enhances sensitivity to ABA during seedling establishment. Individual-nucleotide crosslinking and immunoprecipitation (iCLIP) combined with RNA-sequencing revealed that SR34a is an alternative splicing regulator that binds predominantly GCU-rich exonic sequences near splice sites. We find that SR34a targets all alternative splicing event types, including in RNAs encoding known determinants of ABA sensitivity, to prevent ABA-responsive splicing in germinated seeds. Our study sheds mechanistic light on how plant SR proteins regulate alternative splicing and counteract ABA inhibition of early plant growth.

plant biology↗

bioRxiv2022

ABA signaling prevents phosphodegradation of the Arabidopsis SR45 splicing factor to negatively autoregulate inhibition of early seedling development

Alternative splicing is a key posttranscriptional mechanism to expand the coding capacity of eukaryotic genomes. Although the functional relevance of this process remains poorly understood in plant systems, major modulators of alternative splicing called serine/arginine-rich (SR) proteins have been implicated in plant stress responses mediated by the abscisic acid (ABA) hormone. Loss of function of the Arabidopsis thaliana SR-like protein SR45, a bona fide splicing factor, has been shown to cause plant hypersensitivity to environmental cues and activation of the ABA pathway. Also, consistent with both animal and plant SR proteins being extensively and reversibly phosphorylated at their C-termini, ABA-induced changes in the phosphorylation status of SR45 have been reported. Here, we show that SR45 overexpression reduces Arabidopsis sensitivity to ABA during early seedling development. Moreover, exposure to ABA dephosphorylates SR45 at multiple amino acid residues and leads to accumulation of the protein via reduction of SR45 ubiquitination and proteasomal degradation. Using phosphomutant and phosphomimetic transgenic Arabidopsis lines, we demonstrate the functional relevance of ABA-mediated dephosphorylation of a single SR45 residue, T264, in antagonizing SR45 ubiquitination and degradation to promote its function as a repressor of seedling ABA sensitivity. Taken together, our results reveal a mechanism in which ABA signaling negatively autoregulates during early plant development via posttranslational control of the SR45 splicing factor.

plant biology↗

bioRxiv2021

The plant-specific SCL30a SR protein regulates ABA-dependent seed traits and salt stress tolerance during germination

SR (serine/arginine-rich) proteins are conserved RNA-binding proteins best known as key regulators of splicing, which have also been implicated in other steps of gene expression. Despite mounting evidence for their role in plant development and stress responses, the molecular pathways underlying SR protein regulation of these processes remain elusive. Here we show that the plant-specific SCL30a SR protein negatively regulates abscisic acid (ABA) signaling to control important seed traits and salt stress responses during germination in Arabidopsis. The SCL30a gene is upregulated during seed imbibition and germination, and its loss of function results in smaller seeds displaying enhanced dormancy and elevated expression of ABA-responsive genes as well as of genes repressed during the germination process. Moreover, the knockout mutant is hypersensitive to ABA and high salinity, while transgenic plants overexpressing SCL30a exhibit reduced ABA sensitivity and enhanced tolerance to salt stress during seed germination. An ABA biosynthesis inhibitor rescues the mutants enhanced sensitivity to stress, and epistatic analyses confirm that this hypersensitivity requires a functional ABA pathway. Finally, seed ABA levels are unchanged by altered SCL30a expression, indicating that the SR protein positively regulates stress tolerance during seed germination by reducing sensitivity to the phytohormone. Our results reveal a new key player in ABA-mediated control of early development and stress response, and underscore the role of plant SR proteins as important regulators of the ABA signaling pathway. Author SummarySeed germination is a critical step in plant development determining the transition to aerial growth and exposure to a more challenging environment. As such, seeds have evolved mechanisms that prevent germination under adverse conditions, thereby increasing the chances of plant survival. As a general regulator of plant development and a key mediator of stress responses, the hormone abscisic acid (ABA) promotes a prolonged non-germinating state called dormancy, influences seed size and represses germination under environmental stress. Here, we show that an RNA-binding protein, SCL30a, controls seed size, dormancy, germination and tolerance to high salinity in the model plant Arabidopsis thaliana. Loss of SCL30a gene function results in smaller and more dormant seeds with reduced ability to germinate in a high-salt environment; by contrast, SCL30a overexpression produces larger seeds that germinate faster under salt stress. Using a large-scale gene expression analysis, we identify the ABA hormonal pathway as a putative target of SCL30a. We then use genetic and pharmacological tools to unequivocally demonstrate that the uncovered biological functions of SCL30a are achieved through modulation of the ABA pathway. Our study reveals a novel regulator of key seed traits and has biotechnological implications for crop improvement under adverse environments.

plant biology↗

bioRxiv2020

Expression of Castanea crenata Allene Oxide Synthase in Arabidopsis Improves the Defense to Phytophthora cinnamomi

Allene oxide synthase (AOS) is a key enzyme of the jasmonic acid (JA) signaling pathway. The AOS gene was previously found to be upregulated in an Asian chestnut species resistant to infection by the oomycete Phytophthora cinnamomi (Castanea crenata), while lower expression values were detected in the susceptible European chestnut (Castanea sativa). Here, we report a genetic and functional characterization of the C. crenata AOS (CcAOS) upon its heterologous gene expression in a susceptible ecotype of Arabidopsis thaliana, which contains a single AOS gene. It was found that Arabidopsis plants expressing CcAOS delay pathogen progression and exhibit more vigorous growth in its presence. They also show upregulation of jasmonic acid and salicylic acid-related genes. As in its native species, heterologous CcAOS localized to plastids, as revealed by confocal imaging of the CcAOS-eGFP fusion protein in transgenic Arabidopsis roots. This observation was confirmed upon transient expression in Nicotiana benthamiana leaf epidermal cells. To further confirm a specific role of CcAOS in the defense mechanism against the pathogen, we performed crosses between transgenic CcAOS plants and an infertile Arabidopsis AOS knockout mutant line. It was found that plants expressing CcAOS exhibit normal growth, remain infertile but are significantly more tolerant to the pathogen than wild type plants. Together, our results indicate that CcAOS is an important player in plant defense responses against oomycete infection and that its expression in susceptible varieties may be a valuable tool to mitigate biotic stress responses. One-sentence summaryHeterologous expression of the Castanea crenata allene oxide synthase gene in Arabidopsis thaliana improves the defense response to the pathogen Phytophthora cinnamomi.

plant biology↗

bioRxiv2020

A missense in HSF2BP causing Primary Ovarian Insufficiency affects meiotic recombination by its novel interactor C19ORF57/MIDAP

Primary Ovarian Insufficiency (POI) is a major cause of infertility, but its etiology remains poorly understood. Using whole-exome sequencing in a family with 3 cases of POI, we identified the candidate missense variant S167L in HSF2BP, an essential meiotic gene. Functional analysis of the HSF2BP-S167L variant in mouse, compared to a new HSF2BP knock-out mouse showed that it behaves as a hypomorphic allele. HSF2BP-S167L females show reduced fertility with small litter sizes. To obtain mechanistic insights, we identified C19ORF57/MIDAP as a strong interactor and stabilizer of HSF2BP by forming a higher-order macromolecular structure involving BRCA2, RAD51, RPA and PALB2. Meiocytes bearing the HSF2BP-S167L mutation showed a strongly decreased expression of both MIDAP and HSF2BP at the recombination nodules. Although HSF2BP-S167L does not affect heterodimerization between HSF2BP and MIDAP, it promotes a lower expression of both proteins and a less proficient activity in replacing RPA by the recombinases RAD51/DMC1, thus leading to a lower frequency of cross-overs. Our results provide insights into the molecular mechanism of two novel actors of meiosis underlying non-syndromic ovarian insufficiency. SummaryFelipe-Medina et al. describe a missense variant in the meiotic gene HSF2BP in a consanguineous family with Premature Ovarian Insufficiency, and characterize it as an hypormorphic allele, that in vivo impairs its dimerization with a novel meiotic actor, MIDAP/ C19ORF57, and affect recombination at double-strand DNA breaks.

genetics↗