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N6-methyladenosine (m6A) is a common epitranscriptional mRNA modification in eukaryotes. Thirteen putative m6A readers, mostly annotated as EVOLUTIONARILY CONSERVED C-TERMINAL REGION (ECT) proteins, have been identified in Arabidopsis (Arabidopsis thaliana), but few have been characterized. Here, we show that the Arabidopsis m6A reader ECT1 modulates salicylic acid (SA)–mediated plant stress responses. ECT1 undergoes liquid–liquid phase separation in vitro, and its N-terminal prion-like domain is critical for forming in vivo cytosolic biomolecular condensates in response to SA or bacterial pathogens. Fluorescence-activated particle sorting coupled with quantitative PCR analyses unveiled that ECT1 sequesters SA-induced m6A modification-prone mRNAs through its conserved aromatic cage to facilitate their decay in cytosolic condensates, thereby dampening SA-mediated stress responses. Consistent with this finding, ECT1 overexpression promotes bacterial multiplication in plants. Collectively, our findings unequivocally link ECT1-associated cytosolic condensates to SA-dependent plant stress responses, advancing the current understanding of m6A readers and the SA signaling network. The m6A reader EVOLUTIONARILY CONSERVED C-TERMINAL REGION 1 modulates salicylic acid–dependent plant stress responses, such as growth inhibition and cell death, in cytosolic biomolecular condensates. [ABSTRACT FROM AUTHOR]

SUMMARY: Chloroplast pre‐ribosomal RNA (rRNA) undergoes maturation, which is critical for ribosome assembly. While the central and auxiliary factors in rRNA maturation have been elucidated in bacteria, their mode of action remains largely unexplored in chloroplasts. We now reveal chloroplast‐specific factors involved in 16S rRNA maturation, Arabidopsis thaliana orthologs of bacterial RsmD methyltransferase (AtRsmD) and ribosome maturation factor RimM (AtRimM). A forward genetic screen aimed to find suppressors of the Arabidopsis yellow variegated 2 (var2) mutant defective in photosystem II quality control found a causal nonsense mutation in AtRsmD. The substantially impaired 16S rRNA maturation and translation due to the mutation rescued the leaf variegation phenotype by lowering the levels of chloroplast‐encoded proteins, including photosystem II core proteins, in var2. The subsequent co‐immunoprecipitation coupled with mass spectrometry analyses and bimolecular fluorescence complementation assay found that AtRsmD interacts with AtRimM. Consistent with their interaction, loss of AtRimM also considerably impairs 16S rRNA maturation with decelerated m2G915 modification in 16S rRNA catalyzed by AtRsmD. The atrimM mutation also rescued var2 mutant phenotypes, corroborating the functional interplay between AtRsmD and AtRimM towards modification and maturation of 16S rRNA and chloroplast proteostasis. The maturation and post‐transcriptional modifications of rRNA are critical to assembling ribosomes responsible for protein translation. Here, we revealed that the cooperative regulation of 16S rRNA m2G915 modifications by AtRsmD methyltransferase and ribosome assembly factor AtRimM contributes to 16S rRNA maturation, ribosome assembly, and proteostasis in chloroplasts. Significance Statement: The maturation and post‐transcriptional modifications of ribosomal RNA (rRNA) are critical to assembling ribosomes responsible for protein translation. Here, we revealed that the cooperative regulation of 16S rRNA m2G915 modifications by AtRsmD methyltransferase and ribosome assembly factor AtRimM contributes to 16S rRNA maturation, ribosome assembly, and proteostasis in chloroplasts. [ABSTRACT FROM AUTHOR]

Programmable A > I RNA editing is a valuable tool for basic research and medicine. A variety of editors have been created, but a genetically encoded editor that is both precise and efficient has not been described to date. The trade‐off between precision and efficiency is exemplified in the state of the art editor REPAIR, which comprises the ADAR2 deaminase domain fused to dCas13b. REPAIR is highly efficient, but also causes significant off‐target effects. Mutations that weaken the deaminase domain can minimize the undesirable effects, but this comes at the expense of on‐target editing efficiency. We have now overcome this dilemma by using a multipronged approach: We have chosen an alternative Cas protein (CasRx), inserted the deaminase domain into the middle of CasRx, and redirected the editor to the nucleus. The new editor created, dubbed REPAIRx, is precise yet highly efficient, outperforming various previous versions on both mRNA and nuclear RNA targets. Thus, REPAIRx markedly expands the RNA editing toolkit and illustrates a novel strategy for base editor optimization. Synopsis: Development of CRISPR/Cas9 programmable RNA base editors that exhibit both high efficiency and high specificity has proven challenging. A multipronged approach overcomes this limitation and allowed engineering of an optimized A > I RNA base editor, REPAIRx. Previously developed A > I editors based on ADAR2 deaminase fusion to dCas13b (REPAIRv1) cause substantial off‐target effects in the transcriptome.Off‐target effect‐reducing mutations in the ADAR2 deaminase domain (REPAIRv2) also significantly decrease on‐target editing.Fusion to an alternative targeting moiety (dCasRx), deaminase domain insertion in its middle, and nuclear localization of the fusion protein, yields a precise yet highly specific A > I editor.Nuclear localization of dCasRx allows editing of nuclear DNA in addition to cytoplasmic targets. [ABSTRACT FROM AUTHOR]

Programmable RNA cytidine deamination has recently been achieved using a bifunctional editor (RESCUE‐S) capable of deaminating both adenine and cysteine. Here, we report the development of "CURE", the first cytidine‐specific C‐to‐U RNA Editor. CURE comprises the cytidine deaminase enzyme APOBEC3A fused to dCas13 and acts in conjunction with unconventional guide RNAs (gRNAs) designed to induce loops at the target sites. Importantly, CURE does not deaminate adenosine, enabling the high‐specificity versions of CURE to create fewer missense mutations than RESCUE‐S at the off‐targets transcriptome‐wide. The two editing approaches exhibit overlapping editing motif preferences, with CURE and RESCUE‐S being uniquely able to edit UCC and AC motifs, respectively, while they outperform each other at different subsets of the UC targets. Finally, a nuclear‐localized version of CURE, but not that of RESCUE‐S, can efficiently edit nuclear RNAs. Thus, CURE and RESCUE are distinct in design and complementary in utility. Synopsis: The current approach for programmable C‐to‐U RNA editing (RESCUE‐S) is limited by low editing efficiency at certain targets, the inability to edit nuclear RNAs, and non‐specific deamination of adenosines. Here, an alternative C‐to‐U RNA editing system—"CURE"—is introduced that complements RESCUE‐S in application. Fusion of the human cytidine‐specific deaminase APOBEC3A to dCas13 yields a distinct C>U RNA editor (CURE)The CURE system uses unconventional guide RNAs, which induce loops at target sites to imitate the natural substrate of APOBEC3ACURE and RESCUE‐S can complement each other in terms of editing motif preference, on‐target editing efficiency and off‐target effects. [ABSTRACT FROM AUTHOR]

Here, we report inducible mosaic animal for perturbation (iMAP), a transgenic platform enabling in situ CRISPR targeting of at least 100 genes in parallel throughout the mouse body. iMAP combines Cre-loxP and CRISPR-Cas9 technologies and utilizes a germline-transmitted transgene carrying a large array of individually floxed, tandemly linked gRNA-coding units. Cre-mediated recombination triggers expression of all the gRNAs in the array but only one of them per cell, converting the mice to mosaic organisms suitable for phenotypic characterization and also for high-throughput derivation of conventional single-gene perturbation lines via breeding. Using gRNA representation as a readout, we mapped a miniature Perturb-Atlas cataloging the perturbations of 90 genes across 39 tissues, which yields rich insights into context-dependent gene functions and provides a glimpse of the potential of iMAP in genome decoding. [Display omitted] • Inducible mosaic animal for perturbation (iMAP) combines Cre-LoxP and CRISPR-Cas • iMAP enables large-scale in situ multiplexed mosaic gene targeting and Perturb-Atlas mapping • iMAP enables rapid derivation of single-gene perturbation mouse lines via breeding • iMAP lines are permanent resources just as conventional transgenic lines iMAP is a multiplexed mosaic in situ gene-perturbation platform, which enables large-scale mapping of the Perturb-Atlases profiling genome function across the whole body and high-throughput derivation of single-gene perturbation mouse lines. [ABSTRACT FROM AUTHOR]

[Display omitted] • Pd single atoms supported over γ-AlOOH was created by a photoinduction method. • DFT calculations reveal the support can provide anchoring sites for Pd atoms. • This catalyst shows excellent catalytic efficiency in trans -stilbene epoxidation. • The high catalytic activity originates from the unique coordination environment. Exploring the interaction between metal and support is of great importance in enhancing the catalytic performance and deepening the mechanistic understanding of single atom catalysis. Here we describe a photoinduction method to construct atomically dispersed palladium atoms supported over defect-containing boehmite (γ-AlOOH) nanoleaf with a palladium loading of 0.32 wt%. The existence of singly dispersed palladium atoms is confirmed by spherical aberration correction electron microscopy, extended X-ray absorption fine structure measurement, and CO-absorbed diffuse reflectance infrared Fourier transform spectroscopy. This catalyst shows excellent catalytic efficiency in trans -stilbene epoxidation to yield trans -stilbene oxide (TOF: 416 h−1; conversion: 84 %; selectivity: 99 %), along with excellent recyclability and scalability. In addition, various aromatic olefins were successfully transformed into the corresponding epoxides with high selectivity and excellent yield. DFT study further reveals that the high catalytic activity originates from under-coordinated single palladium atoms in the defect-containing γ-AlOOH in a unique coordination environment. This lays the foundation for the facile creation of single atom catalysts and sheds light on the possibility for scale-up production. [ABSTRACT FROM AUTHOR]

Programmable C-to-U RNA editing using the human APOBEC3A deaminase GLO:1VT/03may21:embj2021108209-toc-0001.jpg PHOTO (COLOR): . gl The authors recently noticed that the affiliations for Xinxin Huang and Junjun Lv were incomplete. The missing affiliation, "CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China", is herewith added and corrected in the original article. [Extracted from the article]