Biology subjects

Carbonell, A.

Publications and source records attributed to Carbonell, A..

8 recordsLinked to original sources

bioRxiv2025

The tryptophan-binding pockets of Arabidopsis AGO1 facilitate amplified RNA interference via SGS3

ARGONAUTE (AGO) proteins associate with small RNAs to form RNA-induced silencing complexes (RISCs). Arabidopsis AGO1 effects post-transcriptional silencing by microRNAs (miRNAs) and small interfering RNAs (siRNAs) and is necessary for siRNA amplification through conversion of RISC target RNAs into double-stranded RNA by the RNA-dependent RNA Polymerase RDR6 and its mandatory cofactors SGS3 and SDE5. Many AGO proteins harbor hydrophobic pockets that interact with tryptophan residues, often surrounded by glycine (GW/WG), in intrinsically disordered regions (IDRs) of RISC cofactors. Here, we show that GW/WG dipeptides in the IDR of SGS3 and the hydrophobic pockets in AGO1 are required for fully functional RDR6-dependent siRNA amplification. We also show that this mechanism requires AGO1-specific structural elements, including positively charged residues surrounding the binding pockets, and a conserved, negatively charged patch in the IDR of SGS3. Thus, the same, conserved protein-protein interaction site is used for different purposes in distinct eukaryotic AGO proteins: the GW/WG-mediated TNRC6-Ago2 interaction is crucial for miRNA-guided silencing in metazoans whereas the GW/WG-mediated SGS3-AGO1 interaction facilitates siRNA amplification via RDR6 in plants.

molecular biology↗

bioRxiv2025

Enhanced MicroRNA Accumulation and Gene Silencing Efficiency through Optimized Precursor Base Pairing

MicroRNAs (miRNAs) are endogenous 21-nucleotide small RNAs that direct sequence-specific silencing of complementary messenger RNAs to regulate a wide range of biological processes. In plants, miRNA precursors are processed from imperfect foldback structures by the RNase III enzyme DICER-LIKE1, in coordination with accessory proteins. While mismatches flanking the miRNA/miRNA* duplex in endogenous precursors can strongly influence miRNA accumulation, their impact has not been thoroughly examined in the context of artificial miRNAs (amiRNAs) used for targeted gene silencing in plants. Here, using silencing sensor systems in Nicotiana benthamiana, we systematically investigated how base-pairing at or near DCL1 cleavage sites affects amiRNA production from the recently described minimal shc precursor. Independent pairing of naturally mismatched positions revealed that introducing a G-C pair immediately upstream of the mature amiRNA remarkably enhances amiRNA accumulation and silencing efficiency. This effect was further validated in Arabidopsis transgenic lines targeting endogenous genes and confirmed by deep sequencing, which revealed highly accurate processing and predominant release of the intended amiRNAs, supporting the specificity of the approach. Our findings show that a single structural modification in an amiRNA precursor can significantly enhance the efficacy of amiRNA-mediated gene silencing. This optimized amiRNA platform is well suited for large-scale functional genomics screens and should facilitate the development of next-generation crops with enhanced resilience to environmental stresses. SIGNIFICANCE STATEMENTA large-scale mutational analysis of how base-pairing at or near DCL1 cleavage sites affects artificial microRNA (amiRNA) production from the minimal shc precursor revealed that introducing a single G-C base pair immediately upstream of the first DCL1 cleavage site significantly boosts amiRNA accumulation and gene-silencing efficiency. The incorporation of this structural tweak into a high-efficiency amiRNA platform provides an optimized, highly specific RNAi tool for functional genomics and crop engineering.

plant biology↗

bioRxiv2025

Precision RNAi in Tomato Using Synthetic Trans-Acting Small Interfering RNAs Derived From Minimal Precursors

RNA interference (RNAi) is a highly conserved gene silencing mechanism regulating gene expression at transcriptional and post-transcriptional levels in plants. Synthetic trans-acting small interfering RNAs (syn-tasiRNAs) have emerged as powerful tools for highly specific and efficient gene silencing. However, their application in crops has been constrained by the need for transgene integration and the relatively long length of TAS-derived precursors. Here, we developed a novel syn-tasiRNA platform for Solanum lycopersicum (tomato) based on minimal precursors targeted by endogenous SlmiR482b microRNA. These minimal precursors, comprising only a 22-nt miRNA target site, an 11-nt spacer and the syn-tasiRNA sequence(s), effectively produced functional syn-tasiRNAs in both transgenic and transient virus-induced gene silencing (syn-tasiR-VIGS) systems. To facilitate their broader application, we engineered a series of vectors for high-throughput cloning and efficient syn-tasiRNA expression from SlmiR482b-based minimal precursors in tomato. Our results show that minimal precursors induce robust gene silencing of endogenous tomato genes and confer antiviral resistance to the economically important tomato spotted wilt virus. Furthermore, we show that syn-tasiR-VIGS can be applied in a transgene-free manner through crude extract delivery, leading to efficient silencing of endogenous genes. This study establishes minimal syn-tasiRNA precursors as a versatile and efficient tool for precision RNAi in tomato, with applications in functional genomics and crop improvement.

plant biology↗

bioRxiv2025

Gene Silencing in Plants by Artificial Small RNAs Derived from Minimal Precursors and Expressed via Tobacco Rattle Virus

Highly specific, second-generation RNA interference tools are based on artificial small RNAs (art-sRNAs), such as artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs). Recent progress includes the use of minimal-length precursors to express art-sRNAs in plants. These minimal precursors retain the minimal structural elements for recognition and efficient processing by host enzymes. They yield high amounts of art-sRNAs and remain stable when incorporated into potato virus X-based viral vectors for art-sRNA-mediated virus-induced gene silencing (art-sRNA-VIGS). However, further adaptation to new viral vector systems with reduced symptomatology is needed to improve the versatility of art-sRNA-VIGS. Here, we developed a novel platform based on tobacco rattle virus (TRV) -a widely used viral vector inducing minimal or no symptoms- for the delivery of art-sRNAs into plants. TRV was engineered to express authentic amiRNAs and syn-tasiRNAs from minimal precursors in Nicotiana benthamiana, resulting in robust and highly specific silencing of endogenous genes. Notably, the expression of syn-tasiRNAs through TRV conferred strong resistance against tomato spotted wilt virus, an economically important pathogen. Furthermore, we established a transgene-free approach by applying TRV-containing crude extracts through foliar spraying, eliminating the need for stable genetic transformation. In summary, our results highlight the unique advantages of minimal precursors and extend the application of art-sRNA-VIGS beyond previously established viral vector systems, providing a scalable, rapid and highly specific tool for gene silencing. Key messageWe developed a novel tobacco rattle virus-based platform for the transgene-free expression of both artificial microRNAs and synthetic trans-acting small interfering RNAs for efficient gene silencing in plants.

plant biology↗

bioRxiv2025

Arabidopsis thaliana Iron Superoxide Dismutase FeSOD1 Protects ARGONAUTE 1 in a Copper-Dependent Manner

Copper (Cu) deficiency compromises plant growth and limits crop productivity. Plants respond to Cu scarcity by activating the expression of several microRNAs, known as Cu-miRNAs, which degrade mRNAs from various cuproproteins to conserve Cu. Cu-miRNAs, like most plant miRNAs, associate with ARGONAUTE 1 (AGO1), the primary effector protein of miRNA-mediated gene silencing pathways, whose function is typically modulated by interacting proteins acting as cofactors. However, how AGO1 is regulated and functions under Cu deficiency remains unknown. Here, we searched for AGO1 interactors in Arabidopsis thaliana plants expressing a functional AGO1 protein tagged with the Twin-Strep-tag (TST) polypeptide, grown under Cu-sufficient or Cu-deficient conditions. TST-AGO1 complexes were affinity-purified, and proteins were identified using tandem mass spectrometry. Interestingly, the iron superoxide dismutase 1 (FeSOD1) encoded by FSD1, was enriched in TST-AGO1 complexes purified from plants grown under Cu deficiency. Moreover, fsd1-2 mutant plants showed reduced levels of AGO1 compared to wild-type plants under Cu sufficiency, while both Cu-miRNA-specific and general AGO1 target mRNAs accumulated to higher levels in fsd1-2 plants under both Cu-deficient and Cu-sufficient conditions compared to wild-type plants. These findings suggest that FeSOD1 is essential for proper AGO1 function, and that its superoxide dismutase activity, which mitigates oxidative stress, enhances AGO1 stability, particularly under Cu deficiency. HIGHLIGHTAGO1 is essential for Cu-deficiency responses but is sensitive to oxidative stress. FeSOD1 interacts with AGO1 and protects it from superoxide radical-induced degradation, thereby preserving miRNA-mediated gene silencing pathways.

plant biology↗

bioRxiv2024

Syn-tasiR-VIGS: Virus-Based Targeted RNAi in Plants By Synthetic Trans Acting Small Interfering RNAs Derived from Minimal Precursors

Synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are 21-nucleotide (nt) small RNAs designed to silence plant transcripts with high specificity. Their use as biotechnological tools for functional genomics and crop improvement is limited by the need to transgenically express long TAS precursors to produce syn-tasiRNAs in vivo. Here, we show that authentic and highly effective syn-tasiRNAs can be produced from minimal, non-TAS precursors consisting of a 22-nt endogenous microRNA target site, an 11-nt spacer and the 21 nt syn-tasiRNA sequence(s). These minimal precursors, when transgenically expressed in Arabidopsis thaliana and Nicotiana benthamiana, generated highly phased syn-tasiRNAs that silenced one or multiple plant genes with high efficacy. Remarkably, minimal but not full-length TAS precursors produced authentic syn-tasiRNAs and induced widespread gene silencing in N. benthamiana when expressed from an RNA virus, which can be applied by spraying infectious crude extracts onto leaves in a GMO-free manner. This strategy, named syn-tasiRNA-based virus-induced gene silencing (syn-tasiR-VIGS), was further used to vaccinate plants against a pathogenic virus, resulting in complete plant immunization. Our results reveal that syn-tasiRNA precursors can be significantly shortened without compromising silencing efficacy, and that syn-tasiR-VIGS represents a versatile, scalable and non-transgenic platform for precision RNAi and antiviral vaccination in plants. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/629176v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@17b038org.highwire.dtl.DTLVardef@13230aeorg.highwire.dtl.DTLVardef@1a1613org.highwire.dtl.DTLVardef@9f606d_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2020

In response to Li et al.: Linker histones function in Drosophila embryogenesis

In an earlier paper (Perez-Montero et al., 2013), we reported that the embryonic linker histone of Drosophila dBigH1 was essential for early Drosophila embryogenesis since embryos homozygous for the bigH1100 mutation showed strong defects and did not survive beyond zygotic genome activation (ZGA) at cellularization. Recent results challenge these observations since null bigH1 mutations generated by CRISPR/Cas9 methodology turn out to be homozygous viable, as reported in Li et al. (2019) and here. In this regard, Li et al. described a novel mechanism by which lack of dBigH1 is compensated by the early expression of maternal dH1. Here, we confirm this observation and show that such compensatory mechanism is not activated in bigH1100 embryos.

molecular biology↗

bioRxiv2019

In vivo mRNA structure regulates miRNA cleavage in Arabidopsis

MicroRNA (miRNA)-mediated cleavage is involved in numerous essential cellular pathways. miRNAs recognize target RNAs via sequence complementarity. In addition to complementarity, in vitro and in silico studies have suggested that RNA structure may influence the accessibility of mRNAs to miRNA-Induced Silencing Complexes (miRISCs), thereby affecting RNA silencing. However, the regulatory mechanism of mRNA structure in miRNA cleavage remains elusive. Here, we investigated the role of in vivo RNA secondary structure in miRNA cleavage by developing the new CAP-STRUCTURE-seq method to capture the intact mRNA structurome in Arabidopsis thaliana. This approach revealed that miRNA target sites were not structurally accessible for miRISC binding prior to cleavage in vivo. Instead, the unfolding of the target site structure is the primary determinant for miRISC activity in vivo. Notably, we found that the single-strandedness of the two nucleotides immediately downstream of the target site, named Target Adjacent structure Motif (TAM), can promote miRNA cleavage but not miRNA binding, thus decoupling target site binding from cleavage. Our findings demonstrate that mRNA structure in vivo can regulate miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes.

molecular biology↗