Biology subjects

Kalyna, M.

Publications and source records attributed to Kalyna, M..

6 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

ALTERNATIVE SPLICING OF A CODING GENE PRODUCES A NUCLEAR REGULATORY LONG NON-CODING RNA

Alternative splicing (AS) is traditionally understood to increase transcript and protein diversity by generating multiple coding isoforms from a single gene. In addition, many alternative isoforms are degraded through non-sense mediated decay (NMD). Furthermore, we show here that AS also produces nuclear long non-coding RNAs (lncRNAs) from protein-coding genes, which can serve regulatory functions rather than expanding proteomic complexity. Focusing on the Arabidopsis thaliana SR protein gene At-RS31, we found that a non-coding isoform (mRNA3) accumulates in the nucleus under dark conditions. This transcript binds the protein product of its own transcriptional unit, modulating splicing decisions and balancing gene activity in response to environmental cues. Overexpression of mRNA3 down-regulates the action of At-RS31 on its target genes in trans and restores the phenotype induced by intron-less At-RS31 accumulation. Further sub-cellular fractionation and RT-PCR analyses show that many SR genes generate nuclear-retained non-coding isoforms, especially under dark conditions, suggesting a widespread mechanism. These findings redefine the role of AS in plants, highlighting its capacity to generate regulatory lncRNAs to fine-tune gene expression beyond protein diversification. Highlights- Alternative splicing generates regulatory lncRNAs from protein-coding genes - A nuclear-retained isoform forms an autoregulatory RNA-protein feedback loop - Non-coding isoforms of SR genes accumulate in the nucleus under dark conditions - Co-expression of the non-coding isoform rescues splicing factor overexpression phenotypes

molecular biology↗

bioRxiv2024

At-RS31 orchestrates hierarchical cross-regulation of splicing factors and integrates alternative splicing with TOR-ABA pathways

O_LIAlternative splicing is essential for plants, enabling a single gene to produce multiple transcript variants to boost functional diversity and fine-tune responses to environmental and developmental cues. At-RS31, a plant-specific splicing factor in the Serine/Arginine (SR)-rich protein family, responds to light and the Target of Rapamycin (TOR) signaling pathway, yet its downstream targets and regulatory impact remain unknown. C_LIO_LITo identify At-RS31 targets, we applied individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) and RNAcompete assays. Transcriptomic analyses of At-RS31 mutant and overexpressing plants further revealed its effects on alternative splicing. C_LIO_LIiCLIP identified 4,034 At-RS31 binding sites across 1,421 genes, enriched in CU-rich and CAGA RNA motifs. Comparative iCLIP and RNAcompete data indicate that the RS domain of At-RS31 may influence its binding specificity in planta, underscoring the value of combining in vivo and in vitro approaches. Transcriptomic analysis showed that At-RS31 modulates diverse splicing events, particularly intron retention and exitron splicing, and influences other splicing modulators, acting as a hierarchical regulator. C_LIO_LIBy regulating stress-response genes and genes in both TOR and abscisic acid (ABA) signaling pathways, At-RS31 may help integrate these signals, balancing plant growth with environmental adaptability through alternative splicing. C_LI

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↗

bioRxiv2019

Auxin-dependent xyloglucan remodelling defines differential tissue expansion in Arabidopsis thaliana

Size control is a fundamental question in biology, showing incremental complexity in case of plants whose cells possess a rigid cell wall. The phytohormone auxin is a vital growth regulator with central importance for differential growth control. Here we show that growth inducing and repressing auxin conditions correlate with reduced and enhanced complexity of extracellular xyloglucans, respectively. In agreement, genetic interference with xyloglucan complexity distinctly modulates auxin-dependent differential growth rates. Our work proposes that an auxin-dependent, spatially defined effect on xyloglucan structure and its effect on cell wall mechanics specify differential, gravitropic hypocotyl growth.

plant biology↗

bioRxiv2018

Remote control of alternative splicing in roots through TOR kinase

For plants, light is the source of energy and the most relevant regulator of growth and adaptations to the environment by inducing changes in gene expression at various levels, including alternative splicing. Chloroplasts trigger retrograde signals that control alternative splicing in leaves and roots in response to light. Here we provide evidence suggesting that sugars, derived from photosynthesis, act as mobile signals controlling alternative splicing in roots. The inhibition of TOR kinase activity diminishes the alternative splicing response to light and/or sugars in roots, showing the relevance of the TOR pathway in this signaling mechanism. Furthermore, disrupting the function of the mitochondria abolishes alternative splicing changes supporting a key role for these organelles in this signaling axis. We conclude that sugars can act as mobile signals coordinating alternative splicing responses to light throughout the whole plant, exerting this function in roots by activating the TOR pathway.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC=\"FIGDIR/small/472126v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (76K):\norg.highwire.dtl.DTLVardef@a8c978org.highwire.dtl.DTLVardef@1430da0org.highwire.dtl.DTLVardef@1339990org.highwire.dtl.DTLVardef@a6dd5a_HPS_FORMAT_FIGEXP M_FIG C_FIG Art by Dr. Luciana Giono.

molecular biology↗