Biology subjects

Arribas-Hernandez, L.

Publications and source records attributed to Arribas-Hernandez, L..

7 recordsLinked to original sources

bioRxiv2023

Sensing of viral RNA in plants via a DICER-LIKE Ribonuclease

Sensors of intracellular double-stranded RNA are central components of metazoan innate antiviral immunity, but such sensors have not been identified in plants. RNA interference (RNAi) constitutes a potent plant antiviral defense mechanism that relies on conversion of viral RNA into small interfering RNAs by two DICER-LIKE (DCL) ribonucleases, DCL4 and DCL2. Here, we show that while plant DCL4 is dedicated to RNAi, cytoplasmic dicing by DCL2 also triggers RNAi-independent defense gene expression via at least two intracellular nucleotide-binding domain/leucine-rich repeat (NLR) immune receptors. Combined DCL4/NLR inactivation abrogates basal resistance to a positive strand RNA virus. Our results redefine the basis of plant antiviral immunity, including autoimmunity as an explanation for DCL2-dependent growth arrest in dcl and RNA decay mutants in several plant species. One sentence summaryThe plant immune system uses Dicer-like ribonucleases for both antiviral RNA interference and double-stranded RNA sensing.

plant biology↗

bioRxiv2023

Evidence for an RNAi-independent role of DICER-LIKE2 in conferring growth inhibition and basal antiviral resistance

Higher plants encode four DICER-LIKE (DCL) enzymes responsible for the production of small non-coding RNAs which function in RNA interference (RNAi). Different RNAi pathways in plants effect transposon silencing, antiviral defense and endogenous gene regulation. DCL2 acts genetically redundantly with DCL4 to confer basal antiviral defense, but in other settings, DCL2 has the opposite function of DCL4, at least in formal genetic terms. For example, knockout of DCL4 causes growth defects that are suppressed by inactivation of DCL2. Current models maintain that the biochemical basis of both of these effects is RNAi via DCL2-dependent small interfering RNAs (siRNAs). Here, we report that neither DCL2-mediated antiviral resistance nor growth defects can be explained by silencing effects of DCL2-dependent siRNAs. Both functions are defective in genetic backgrounds that maintain high levels of DCL2-dependent siRNAs, either through specific point mutations in DCL2 or simply by reducing DCL2 dosage in plants heterozygous for dcl2 knockout alleles. Intriguingly, however, all functions of DCL2 depend on it having some level of catalytic activity. We discuss this requirement for catalytic activity, but not for the resulting siRNAs, in the light of recent findings that reveal a function of DCL2 in activation of innate immunity in response to cytoplasmic double-stranded RNA.

plant biology↗

bioRxiv2022

Plant YTHDF proteins are direct effectors of antiviral immunity against an m6A-containing RNA virus

In virus-host interactions, nucleic acid-directed first lines of defense that allow viral clearance without compromising growth are of paramount importance. Plants use the RNA interference pathway as such a basal antiviral immune system, but additional RNA-based mechanisms of defense also exist. The infectivity of the plant positive strand RNA virus alfalfa mosaic virus (AMV) relies on demethylation of viral RNA by recruitment of the cellular N6-methyladenosine (m6A) demethylase ALKBH9B, but how demethylation of viral RNA promotes AMV replication remains unknown. Here, we show that inactivation of the cytoplasmic YT521-B homology domain (YTH)-containing m6A-binding proteins, ECT2, ECT3, and ECT5 is sufficient to restore AMV infectivity in partially resistant alkbh9b mutants. We also show that the antiviral function of ECT2 is distinct from its previously demonstrated function in promotion of proliferation of primordial cells, because an ECT2 mutant carrying a small deletion in its intrinsically disordered region is partially compromised for antiviral defense, but not for developmental functions. These results indicate that the m6A-YTH axis constitutes a novel branch of basal antiviral immunity in plants.

plant biology↗

bioRxiv2022

Systematic Analysis of the Functional Overlap among Plant YTHDF proteins

YT521-B homology (YTH) domain proteins act as readers of N6-methyladenosine (m6A) in mRNA. Members of the YTHDF clade determine properties of m6A-containing mRNAs in the cytoplasm. Vertebrates encode three YTHDF proteins whose possible functional specialization is debated. In land plants, the YTHDF clade has expanded from one member in basal lineages to eleven so-called EVOLUTIONARILY CONSERVED C-TERMINAL REGION1-11 (ECT1-11) proteins in Arabidopsis thaliana, named after the conserved YTH domain placed behind a long N-terminal intrinsically disordered region (IDR). ECT2, ECT3 and ECT4 show genetic redundancy in stimulation of primed stem cell division, but the origin and implications of YTHDF expansion in higher plants are unknown, as it is unclear whether it involves acquisition of fundamentally different molecular properties, in particular of their divergent IDRs. Here, we use functional complementation of ect2/ect3/ect4 mutants to test whether different YTHDF proteins can perform the same function when similarly expressed in leaf primordia. We show that stimulation of primordial cell division relies on an ancestral molecular function of the m6A-YTHDF axis in land plants that is present in bryophytes and is conserved over YTHDF diversification, as it appears in all major clades of YTHDF proteins in flowering plants. Importantly, although our results indicate that the YTH domains of all arabidopsis ECT proteins have m6A-binding capacity, lineage-specific neo-functionalization of ECT1, ECT9 and ECT11 happened after late duplication events, and involves altered properties of both the YTH domains, and, especially, of the IDRs. We also identify two biophysical properties recurrent in IDRs of YTHDF proteins able to complement ect2 ect3 ect4 mutants, a clear phase separation propensity and a charge distribution that creates electric dipoles. Human and fly YTHDFs do not have IDRs with this combination of properties and cannot replace ECT2/3/4 function in arabidopsis, perhaps suggesting different molecular activities of YTHDF proteins between major taxa. Author SummaryRegulation of gene expression is essential to life. It ensures correct balancing of cellular activities and the controlled proliferation and differentiation necessary for the development of multicellular organisms. Methylation of adenosines in mRNA (m6A) contributes to genetic control, and absence of m6A impairs embryo development in plants and vertebrates. m6A-dependent regulation can be exerted by a group of cytoplasmic proteins called YTHDFs. Higher plants have many more YTHDFs than animals, but it is unknown whether these many YTHDF proteins carry out fundamentally different or roughly the same molecular functions, as only a small fraction of them have been studied thus far. This work addresses the origin and reasons behind YTHDF expansion during land plant evolution and reveals that most YTHDFs in flowering plants have the same molecular functions that facilitate rapid division of differentiating stem cells. Remarkably, this molecular activity is present in the most basal lineage of land plants and functions similarly across 450 million years of land plant evolution. We also identified a few plant YTHDF proteins with divergent molecular function despite their ability to bind to m6A. Our work provides a firm basis for further advances on understanding molecular properties and biological contexts underlying YTHDF diversification.

molecular biology↗

bioRxiv2021

The Arabidopsis m6A-binding proteins ECT2 and ECT3 bind largely overlapping mRNA target sets and influence target mRNA abundance, not alternative polyadenylation

Gene regulation via N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2, ECT3 and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.

molecular 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↗

bioRxiv2021

Principles of mRNA targeting and regulation via the Arabidopsis m6A-binding proteins ECT2 and ECT3

Specific recognition of N6-methyladenosine (m6A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH-domain protein ECT2 is thought to bind to mRNA at URU(m6A)Y sites, yet RR(m6A)CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A binding activity. Here, we apply iCLIP (individual-nucleotide resolution cross-linking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to define high-quality target sets of ECT2, and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(m6A)CH. Pyrimidine-rich motifs are enriched around, but not at m6A-sites, reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m6A sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of m6A-YTH function in plants, and reveals new insight into the mode of RNA recognition by YTH-domain-containing proteins.

molecular biology↗