Biology subjects

Vigh, M. L.

Publications and source records attributed to Vigh, M. L..

4 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↗

bioRxiv2024

Arabidopsis terminal nucleotidyl transferases govern secondary siRNA production at distinct steps

In plants, RNA interference (RNAi) mediated by the endonucleolytic RNA-Induced Silencing Complex (RISC) defends against foreign RNA and regulates endogenous genes. Targeting of RISC to foreign RNA establishes amplification loops, wherein RNA-dependent RNA Polymerase 6 (RDR6) synthesizes double-stranded RNA (dsRNA) for secondary small interfering RNA (siRNA) biogenesis, using cleavage fragments of RNA targeted by RISC programmed with a primary siRNA as template. Secondary siRNA production from endogenous RISC targets requires a particular primary small RNA size or target site multiplicity. siRNA amplification in yeast and nematodes requires terminal nucleotidyl transferases (TNTases), but their roles in plants are unclear. Here, we demonstrate two functions of TNTases in siRNA amplification in Arabidopsis thaliana. URT1 prevents initiation of microRNA-induced secondary siRNA formation through uridylation of 5-cleavage fragments, sometimes redundantly with the exosome and the TNTase HESO1. Once initiated via RDR6 recruitment, HESO1 and other TNTases stimulate secondary siRNA formation by producing 2-nt 3overhangs on RDR6-synthesized dsRNA to yield substrates for processing into siRNAs by DICER-LIKE4. These results define molecular mechanisms by which TNTases control siRNA amplification in plants.

plant biology↗

bioRxiv2021

Nuclear and cytoplasmic RNA exosomes and PELOTA1 prevent miRNA-induced secondary siRNA production in Arabidopsis

Amplification of short interfering RNA (siRNAs) via RNA dependent RNA Polymerases (RdRPs) is of fundamental importance in RNA silencing. In plants, silencing by microRNAs (miRNAs) generally does not lead to engagement of RdRPs, in part thanks to an as yet poorly understood activity of the cytoplasmic exosome adaptor SKI2. Here, we show that mutation of the cytoplasmic exosome subunit RRP45B results in siRNA production very similar to what is observed in ski2 mutants. Furthermore, loss of the nuclear exosome adaptor HEN2 leads to secondary siRNA production from miRNA targets largely distinct from those producing siRNAs in ski2. Importantly, mutation of the Release Factor paralogue PELOTA1 required for subunit dissociation of stalled ribosomes causes siRNA production from miRNA targets overlapping with, but distinct from, those affected in ski2 and rrp45b mutants. We also show that miRNA-induced illicit secondary siRNA production correlates with miRNA levels rather than accumulation of stable 5-cleavage fragments. We propose that stalled RNA-induced Silencing Complex (RISC) and ribosomes, but not stable target mRNA cleavage fragments released from RISC, trigger secondary siRNA production, and that the exosome limits siRNA amplification by reducing RISC dwell time on miRNA target mRNAs while PELOTA1 does so by reducing ribosome stalling.

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↗