Biology subjects

Rodriguez, F. S.

Publications and source records attributed to Rodriguez, F. S..

4 recordsLinked to original sources

bioRxiv2026

Alarmone ((p)ppGpp) signalling tunes Arabidopsis thaliana nuclear gene expression to shape the balance of plant immune outcomes

RSH enzymes (RelA/SpoT homologs) synthesize ppGpp (guanosine tetra-/pentaphosphate) in plastids, regulating organelle function. More importantly, whether ppGpp acts as a simple rheostat on immune output or determines which mode of defence a plant deploys remains poorly understood. Using Arabidopsis thaliana lines with high (RSH3OX) or null (rshq) ppGpp levels, we show that ppGpp controls nuclear-encoded defence genes in salicylic acid (SA) and jasmonic acid (JA) metabolism, including hormone-inactivating enzymes in RSH3OX and MeSA-to-SA conversion genes in rshq. Conversely, pathogen infection induces the ppGpp synthases RSH2 and RSH3, with induction preceding defence gene activation and requiring SA biosynthesis and a functional Type III secretion system. RSH3OX plants are hypersusceptible to Pseudomonas syringae pv. tomato DC3000, show impaired pattern-triggered immunity, and exhibit an accelerated decline in photosynthetic efficiency, while rshq plants show enhanced resistance to P. syringae but increased susceptibility to the necrotrophic fungus Botrytis cinerea. Using an inducible line to acutely elevate ppGpp, we confirm this susceptibility is directly caused by ppGpp levels, not a chronic developmental consequence of RSH3OX. Together, these results indicate that ppGpp governs the mode of immune execution rather than simply scaling its magnitude, acting as a chloroplast-based checkpoint linking organellar status to the nuclear defence transcriptome.

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↗

bioRxiv2025

SuB3 a Simple Subcellular Fractionation Protocol for Localization Studies of Nucleic Acids and Proteins

Understanding the subcellular localization of RNA and proteins is critical to dissecting gene regulation in eukaryotic organisms. However, this task is elusive as existing fractionation methods often rely on protoplast isolation or commercial kits, that are labor-intensive, costly, and can introduce stress-induced transcriptomic and proteomic changes. Here, we present a simple, rapid, and cost-effective protocol for the fractionation of nuclear and cytoplasmic components directly from diverse plant tissues, that does not require protoplastization. This Subcellular-fractionation protocol in 3 steps (a.k.a. "bueno, bonito y barato"- spanish for "good, nice and cheap"-), referred to as "SuB3", yields nuclear- and cytoplasmic- enriched subcellular fractions suitable for downstream applications such as RT-PCR, RNA/cDNA sequencing, and Western blotting. The procedure is based on sequential detergent-assisted extraction and centrifugation and enables the simultaneous isolation of RNA and protein from the same biological material. Due to its simplicity, speed, and broad compatibility, this protocol is a valuable tool for plant molecular biology laboratories investigating subcellular dynamics of gene expression.

molecular biology↗

bioRxiv2022

Using deep learning predictions of inter-residue distances for model validation

Determination of protein structures typically entails building a model that satisfies the collected experimental observations and its deposition in the Protein Data Bank (PDB). Experimental limitations can lead to unavoidable uncertainties during the process of model building, which result in the introduction of errors into the deposited model. Many metrics are available for model validation, but most are limited to the consideration of the physico-chemical aspects of the model or its match to the map. The latest advances in the field of deep learning have enabled the increasingly accurate prediction of inter-residue distances, an advance which has played a pivotal role in the recent improvements observed in the field of protein ab initio modelling. Here we present new validation methods based on the use of these precise inter-residue distance predictions, which are compared with the distances observed in the protein model. Sequence register errors are particularly clearly detected, and the register shifts required for their correction can be reliably determined. The method is available in the package ConKit (www.conkit.org).

bioinformatics↗