Biology subjects

Gagliardi, D.

Publications and source records attributed to Gagliardi, D..

8 recordsLinked to original sources

bioRxiv2024

The TUTase URT1 regulates the transcriptome of seeds and their primary dormancy

RNA uridylation is a pervasive mechanism that regulates the degradation of eukaryotic mRNAs. In Arabidopsis, uridylation influences mRNA decay both by favoring 5 to 3 degradation and by preventing excessive deadenylation. Yet, the significance of mRNA uridylation during plant development remains largely unknown. Here, we adapted FLEP-seq2, a method based on nanopore sequencing, to generate a comprehensive inventory of mRNA uridylation events in different Arabidopsis tissues. We also evaluated the respective contribution of the two known Arabidopsis uridylyltransferases, URT1 and HESO1, in mRNA uridylation. Our transcriptome-wide analysis showed that, URT1 is the main enzyme responsible for mRNA uridylation, in all analyzed tissues, while HESO1 can marginally uridylate mRNAs. Importantly, our results revealed the singularity of mRNA uridylation pattern in seeds and the dual function of URT1-dependent uridylation in shaping the transcriptome during seed maturation. We propose that during the late stages of seed maturation, URT1-dependent uridylation facilitates the degradation of unnecessary mRNAs encoding translation-related proteins, while also promoting the accumulation of mRNAs associated with the maturation program, by hindering their deadenylation. In line with its important function in shaping the seed transcriptome, our study also identifies URT1 as a novel regulator of seed dormancy. Overall, our study reveals the biological relevance of mRNA uridylation during the late stages of seed maturation.

plant biology↗

bioRxiv2024

The Nuclear Pore Complex acts as a hub for pri-miRNA transcription and processing in plants

The regulation of miRNA biogenesis and movement is essential for plant development and environmental responses. HASTY (HST), a karyopherin protein, has been implicated in miRNA biogenesis and movement, though its role in non-cell-autonomous miRNA movement remains unclear. Through a genetic screen, we identified that mutations in the HAWAIIAN SKIRT (HWS) gene suppress the developmental defects of hst mutants by restoring miRNA movement. Our findings show that HWS interacts with nuclear transport factors and nuclear pore complex (NPC) components, including NUP1, positioning HWS as a regulator of miRNA nuclear export. Using microscopy and fluorescence in situ hybridization, we showed that pri-miRNA transcription, and likely their co-transcriptional processing, occurs at the nuclear pore. Notably, we uncovered an antagonistic relationship between HST and HWS in regulating MIRNA transcription at the NPC and AGO1 loading, which could explain the observed changes in miRNA movement. HST promotes the association of MIRNA loci with the NPC, likely facilitating co-transcriptional processing, while HWS negatively regulates this process by targeting MEDIATOR complex subunits to degradation detaching, consequently, the processing complex from the NPC. Furthermore, our data provides evidence of spatial coordination of miRNA transcription, biogenesis, and movement highlighting a novel role for the NPC in the miRNA pathway.

plant biology↗

bioRxiv2024

A multi-transcriptomics approach reveals the coordinated action of the endoribonuclease DNE1 and the decapping machinery in orchestrating mRNA decay

Decapping is a crucial step of mRNA degradation in eucaryotes and requires the formation of the holoenzyme complex between the decapping enzyme DCP2 and the decapping enhancer DCP1. In Arabidopsis, we recently identified DNE1, a NYN domain endoribonuclease, as a direct protein partner of DCP1. The function of both DNE1 and decapping are necessary to maintain phyllotaxis, the regularity of organ emergence in the apex. In this study we combined in vivo mRNA editing, RNA degradome, transcriptomics and small RNA-omics to identify targets of DNE1 and study how DNE1 and DCP2 cooperate in controlling mRNA fate. Our data reveal that DNE1 mainly contacts and cleaves mRNAs in the CDS and has sequence cleavage preferences. We found that DNE1 targets are also degraded through decapping, and that both RNA degradation pathways influence the production of mRNA-derived siRNAs. Finally, we detected mRNA features enriched in DNE1 targets including RNA G-quadruplexes and translated upstream-ORFs. Combining these four complementary high-throughput sequencing strategies greatly expands the range of DNE1 targets and allowed us to build a conceptual framework describing the influence of DNE1 and decapping on mRNA fate. These data will be crucial to unveil the specificity of DNE1 action and understand its importance for developmental patterning.

plant biology↗

bioRxiv2023

Cell type-specific control of secondary cell wall formation by Musashi-type translational regulators in Arabidopsis

Deciphering the mechanism of secondary cell wall/SCW formation in plants is key to understanding their development and the molecular basis of biomass recalcitrance. Although transcriptional regulation is essential for SCW formation, little is known about the implication of post-transcriptional mechanisms in this process. Here we report that two bonafide RNA-binding proteins homologous to the animal translational regulator Musashi, MSIL2 and MSIL4, function redundantly to control SCW formation in Arabidopsis. MSIL2/4 interactomes are similar and enriched in proteins involved in mRNA binding and translational regulation. MSIL2/4 mutations alter SCW formation in the fibers, leading to a reduction in lignin deposition, and an increase of 4-O-glucuronoxylan methylation. In accordance, quantitative proteomics of stems reveal an overaccumulation of glucuronoxylan biosynthetic machinery, including GXM3, in the msil2/4 mutant stem. We showed that MSIL4 immunoprecipitates GXM mRNAs, suggesting a novel aspect of SCW regulation, linking post-transcriptional control to the regulation of SCW biosynthesis genes.

plant biology↗

bioRxiv2021

A NYN domain protein directly interacts with DCP1 and is required for phyllotactic pattern in Arabidopsis.

In eukaryotes, general mRNA decay requires the decapping complex. The activity of this complex depends on its catalytic subunit, DCP2 and its interaction with decapping enhancers, including its main partner DCP1. Here, we report that in Arabidopsis, DCP1 also interacts with a NYN domain endoribonuclease, hence named DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1). Interestingly, we find DNE1 predominantly associated with DCP1 but not with DCP2 and reciprocally, suggesting the existence of two distinct protein complexes. We also show that the catalytic residues of DNE1 are required to repress the expression of mRNAs in planta upon transient expression. The overexpression of DNE1 in transgenic lines leads to growth defects and transcriptomic changes related to the one observed upon inactivation of the decapping complex. Finally, the combination of dne1 and dcp2 mutations, revealed a functional redundancy between DNE1 and DCP2 in controlling phyllotactic pattern formation in Arabidopsis. Our work identifies DNE1, a hitherto unknown DCP1 protein partner highly conserved in the plant kingdom and identifies its importance for developmental robustness. One-sentence summaryDNE1, a NYN domain protein interacts with the decapping activator DCP1 and, together with DCP2, specify phyllotactic patterns in Arabidopsis.

plant biology↗

bioRxiv2020

Molecular connection between the TUTase URT1 and decapping activators

Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3 terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development.

molecular biology↗

bioRxiv2020

SERRATE interacts with the nuclear exosome targeting (NEXT) complex to degrade primary miRNA precursors in Arabidopsis

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the Nuclear Exosome Targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.

plant biology↗

bioRxiv2019

RST1 and RIPR connect the cytosolic RNA exosome to the Ski complex in Arabidopsis

The RNA exosome is a key 3-5 exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known SKI subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans.

molecular biology↗