Genome-wide CRISPR screens identify noncanonical translation factor eIF2A as an enhancer of SARS-CoV-2 programmed -1 ribosomal frameshifting.

Many positive-strand RNA viruses, including all known coronaviruses, employ programmed -1 ribosomal frameshifting (-1 PRF) to regulate the translation of polycistronic viral RNAs. However, only a few host factors have been shown to regulate -1 PRF. Through a genome-wide CRISPR-Cas9 knockout screen, we have identified host factors that either suppress or enhance severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) -1 PRF. Among them, eukaryotic translation initiation factor 2A (eIF2A) specifically and directly enhances -1 PRF independent of changes in initiation. Consistent with the crucial role of efficient -1 PRF in transcriptase/replicase expression, loss of eIF2A reduces SARS-CoV-2 replication in cells. Furthermore, transcriptome-wide analysis shows that eIF2A preferentially binds CG-rich RNA motifs, including a region within 18S ribosomal RNA near the contacts between the SARS-CoV-2 frameshift-stimulatory element (FSE) and the ribosome. Thus, our results indicate a role for eIF2A in modulating the translation of specific RNAs independent of its role during initiation.

RNA demethylation increases the yield and biomass of rice and potato plants in field trials.

RNA N6-methyladenosine (m6A) modifications are essential in plants. Here, we show that transgenic expression of the human RNA demethylase FTO in rice caused a more than threefold increase in grain yield under greenhouse conditions. In field trials, transgenic expression of FTO in rice and potato caused ~50% increases in yield and biomass. We demonstrate that the presence of FTO stimulates root meristem cell proliferation and tiller bud formation and promotes photosynthetic efficiency and drought tolerance but has no effect on mature cell size, shoot meristem cell proliferation, root diameter, plant height or ploidy. FTO mediates substantial m6A demethylation (around 7% of demethylation in poly(A) RNA and around 35% decrease of m6A in non-ribosomal nuclear RNA) in plant RNA, inducing chromatin openness and transcriptional activation. Therefore, modulation of plant RNA m6A methylation is a promising strategy to dramatically improve plant growth and crop yield.

Structural insights into FTO's catalytic mechanism for the demethylation of multiple RNA substrates.

FTO demethylates internal N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am; at the cap +1 position) in mRNA, m6A and m6Am in snRNA, and N1-methyladenosine (m1A) in tRNA in vivo, and in vitro evidence supports that it can also demethylate N6-methyldeoxyadenosine (6mA), 3-methylthymine (3mT), and 3-methyluracil (m3U). However, it remains unclear how FTO variously recognizes and catalyzes these diverse substrates. Here we demonstrate-in vitro and in vivo-that FTO has extensive demethylation enzymatic activity on both internal m6A and cap m6Am Considering that 6mA, m6A, and m6Am all share the same nucleobase, we present a crystal structure of human FTO bound to 6mA-modified ssDNA, revealing the molecular basis of the catalytic demethylation of FTO toward multiple RNA substrates. We discovered that (i) N6-methyladenine is the most favorable nucleobase substrate of FTO, (ii) FTO displays the same demethylation activity toward internal m6A and m6Am in the same RNA sequence, suggesting that the substrate specificity of FTO primarily results from the interaction of residues in the catalytic pocket with the nucleobase (rather than the ribose ring), and (iii) the sequence and the tertiary structure of RNA can affect the catalytic activity of FTO. Our findings provide a structural basis for understanding the catalytic mechanism through which FTO demethylates its multiple substrates and pave the way forward for the structure-guided design of selective chemicals for functional studies and potential therapeutic applications.

The m6A Reader ECT2 Controls Trichome Morphology by Affecting mRNA Stability in Arabidopsis.

The epitranscriptomic mark N6-methyladenosine (m6A) can be written, read, and erased via the action of a complex network of proteins. m6A binding proteins read m6A marks and transduce their downstream regulatory effects by altering RNA metabolic processes. The characterization of m6A readers is an essential prerequisite for understanding the roles of m6A in plants, but the identities of m6A readers have been unclear. Here, we characterized the YTH-domain family protein ECT2 as an Arabidopsis thaliana m6A reader whose m6A binding function is required for normal trichome morphology. We developed the formaldehyde cross-linking and immunoprecipitation method to identify ECT2-RNA interaction sites at the transcriptome-wide level. This analysis demonstrated that ECT2 binding sites are strongly enriched in the 3' untranslated regions (3' UTRs) of target genes and led to the identification of a plant-specific m6A motif. Sequencing analysis suggested that ECT2 plays dual roles in regulating 3' UTR processing in the nucleus and facilitating mRNA stability in the cytoplasm. Disruption of ECT2 accelerated the degradation of three ECT2 binding transcripts related to trichome morphogenesis, thereby affecting trichome branching. The results shed light on the underlying mechanisms of the roles of m6A in RNA metabolism, as well as plant development and physiology.

ALKBH10B Is an RNA N6-Methyladenosine Demethylase Affecting Arabidopsis Floral Transition.

N6-methyladenosine (m6A) is the most abundant, internal, posttranscriptional modification in mRNA among all higher eukaryotes. In mammals, this modification is reversible and plays broad roles in the regulation of mRNA metabolism and processing. Despite its importance, previous studies on the role and mechanism of m6A methylation in Arabidopsis thaliana have been limited. Here, we report that ALKBH10B is a demethylase that oxidatively reverses m6A methylation in mRNA in vitro and in vivo. Depletion of ALKBH10B in the alkbh10b mutant delays flowering and represses vegetative growth. Complementation with wild-type ALKBH10B, but not a catalytically inactive mutant (ALKBH10B H366A/E368A), rescues these effects in alkbh10b-1 mutant plants, suggesting the observed phenotypes are controlled by the catalytic action of ALKBH10B We show that ALKBH10B-mediated mRNA demethylation affects the stability of target transcripts, thereby influencing floral transition. We identified 1190 m6A hypermethylated transcripts in the alkbh10b-1 mutant involved in plant development. The discovery and characterization of the archetypical RNA demethylase in Arabidopsis sheds light on the occurrence and functional role(s) of reversible mRNA methylation in plants and defines the role of m6A RNA modification in Arabidopsis floral transition.