Programmable RNA N-methyladenosine editing with CRISPR/dCas13a in plants.

N-methyladenonsine (mA) is the most prevalent internal modification of messenger RNA (mRNA) and plays critical roles in mRNA processing and metabolism. However, perturbation of individual mA modification to reveal its function and the phenotypic effects is still lacking in plants. Here, we describe the construction and characterization of programmable mA editing tools by fusing the mA writers, the core catalytic domain of the MTA and MTB complex, and the AlkB homologue 5 (ALKBH5) eraser, to catalytically dead Cas13a (dCas13a) to edit individual mA sites on mRNAs.

TurboID-mediated proximity labeling for screening interacting proteins of FIP37 in .

Proximity labeling was recently developed to detect protein-protein interactions and members of subcellular multiprotein structures in living cells. Proximity labeling is conducted by fusing an engineered enzyme with catalytic activity, such as biotin ligase, to a protein of interest (bait protein) to biotinylate adjacent proteins. The biotinylated protein can be purified by streptavidin beads, and identified by mass spectrometry (MS).

mRNA cleavage by 21-nucleotide phasiRNAs determines temperature-sensitive male sterility in rice.

Temperature-sensitive male sterility is one of the core components for hybrid rice (Oryza sativa) breeding based on the 2-line system. We previously found that knockout of ARGONAUTE 1d (AGO1d) causes temperature-sensitive male sterility in rice by influencing phased small interfering RNA (phasiRNA) biogenesis and function. However, the specific phasiRNAs and their targets underlying the temperature-sensitive male sterility in the ago1d mutant remain unknown.

Temperature-sensitive male sterility in rice determined by the roles of AGO1d in reproductive phasiRNA biogenesis and function.

Phased secondary siRNAs (phasiRNAs) are broadly present in the reproductive tissues of flowering plants, with spatial-temporal specificity. However, the ARGONAUTE (AGO) proteins associated with phasiRNAs and their miRNA triggers remain elusive. Here, through histological and high-throughput sequencing analyses, we show that rice AGO1d, which is specifically expressed in anther wall cells before and during meiosis, associates with both miR2118 and miR2275 to mediate phasiRNA biogenesis.

Chloroplast genomes in Populus (Salicaceae): comparisons from an intensively sampled genus reveal dynamic patterns of evolution.

The chloroplast is one of two organelles containing a separate genome that codes for essential and distinct cellular functions such as photosynthesis. Given the importance of chloroplasts in plant metabolism, the genomic architecture and gene content have been strongly conserved through long periods of time and as such are useful molecular tools for evolutionary inferences. At present, complete chloroplast genomes from over 4000 species have been deposited into publicly accessible databases.

Stabilization of ERK-Phosphorylated METTL3 by USP5 Increases mA Methylation.

N-methyladenosine (mA) is the most abundant mRNA modification and is installed by the METTL3-METTL14-WTAP methyltransferase complex. Although the importance of mA methylation in mRNA metabolism has been well documented recently, regulation of the mA machinery remains obscure. Through a genome-wide CRISPR screen, we identify the ERK pathway and USP5 as positive regulators of the mA deposition.

YTHDF2 promotes mitotic entry and is regulated by cell cycle mediators.

The N6-methyladenosine (m6A) modification regulates mRNA stability and translation. Here, we show that transcriptomic m6A modification can be dynamic and the m6A reader protein YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) promotes mRNA decay during cell cycle. Depletion of YTHDF2 in HeLa cells leads to the delay of mitotic entry due to overaccumulation of negative regulators of cell cycle such as Wee1-like protein kinase (WEE1).

mA mRNA Methylation Is Essential for Oligodendrocyte Maturation and CNS Myelination.

The molecular mechanisms that govern the maturation of oligodendrocyte lineage cells remain unclear. Emerging studies have shown that N-methyladenosine (mA), the most common internal RNA modification of mammalian mRNA, plays a critical role in various developmental processes. Here, we demonstrate that oligodendrocyte lineage progression is accompanied by dynamic changes in mA modification on numerous transcripts.

Disruption of miRNA sequences by TALENs and CRISPR/Cas9 induces varied lengths of miRNA production.

MicroRNAs (miRNAs) are 20-24 nucleotides (nt) small RNAs functioning in eukaryotes. The length and sequence of miRNAs are not only related to the biogenesis of miRNAs but are also important for downstream physiological processes like ta-siRNA production. To investigate these roles, it is informative to create small mutations within mature miRNA sequences.

Single base resolution mapping of 2'-O-methylation sites in human mRNA and in 3' terminal ends of small RNAs.

The post-transcriptional modification 2'-O-Methyl (2'OMe) could be present on the ribose of all four ribonucleosides, and is highly prevalent in a wide variety of RNA species, including the 5' RNA cap of viruses and higher eukaryotes, as well as internally in transfer RNA and ribosomal RNA. Recent studies have suggested that 2'OMe is also located internally in low-abundance RNA species such as viral RNA and mRNA. To profile 2'OMe on different RNA species, we have developed Nm-seq, which could identify 2'OMe sites at single base resolution.

Differential mA, mA, and mA Demethylation Mediated by FTO in the Cell Nucleus and Cytoplasm.

FTO, the first RNA demethylase discovered, mediates the demethylation of internal N-methyladenosine (mA) and N, 2-O-dimethyladenosine (mA) at the +1 position from the 5' cap in mRNA. Here we demonstrate that the cellular distribution of FTO is distinct among different cell lines, affecting the access of FTO to different RNA substrates. We find that FTO binds multiple RNA species, including mRNA, snRNA, and tRNA, and can demethylate internal mA and cap mA in mRNA, internal mA in U6 RNA, internal and cap mA in snRNAs, and N-methyladenosine (mA) in tRNA.

Biogenesis of a 22-nt microRNA in Phaseoleae species by precursor-programmed uridylation.

Phased, secondary siRNAs (phasiRNAs) represent a class of small RNAs in plants generated via distinct biogenesis pathways, predominantly dependent on the activity of 22-nt miRNAs. Most 22-nt miRNAs are processed by DCL1 from miRNA precursors containing an asymmetric bulge, yielding a 22/21-nt miRNA/miRNA* duplex. Here we show that miR1510, a soybean miRNA capable of triggering phasiRNA production from numerous (s), previously described as 21 nt in its mature form, primarily accumulates as a 22-nt isoform via monouridylation.

CRISPR/Cas9-mediated resistance to cauliflower mosaic virus.

Viral diseases are a leading cause of worldwide yield losses in crop production. Breeding of resistance genes ( gene) into elite crop cultivars has been the standard and most cost-effective practice. However, gene-mediated resistance is limited by the available genes within genetic resources and in many cases, by strain specificity.

N-Allyladenosine: A New Small Molecule for RNA Labeling Identified by Mutation Assay.

RNA labeling is crucial for the study of RNA structure and metabolism. Herein we report N-allyladenosine (aA) as a new small molecule for RNA labeling through both metabolic and enzyme-assisted manners. aA behaves like A and can be metabolically incorporated into newly synthesized RNAs inside mammalian cells.

Dynamic changes of small RNAs in rice spikelet development reveal specialized reproductive phasiRNA pathways.

Dissection of the genetic pathways and mechanisms by which anther development occurs in grasses is crucial for both a basic understanding of plant development and for examining traits of agronomic importance such as male sterility. In rice, MULTIPLE SPOROCYTES1 (MSP1), a leucine-rich-repeat receptor kinase, plays an important role in anther development by limiting the number of sporocytes. OsTDL1a (a TPD1-like gene in rice) encodes a small protein that acts as a cofactor of MSP1 in the same regulatory pathway.

Regulatory Role of a Receptor-Like Kinase in Specifying Anther Cell Identity.

In flowering plants, sequential formation of anther cell types is a highly ordered process that is essential for successful meiosis and sexual reproduction. Differentiation of meristematic cells and cell-cell communication are proposed to coordinate anther development. Among the proposed mechanisms of cell fate specification are cell surface-localized Leu-rich repeat receptor-like kinases (LRR-RLKs) and their putative ligands.

Small RNAs Add Zing to the Zig-Zag-Zig Model of Plant Defenses.

Plant small RNAs play important roles in transcriptional and posttranscriptional regulation, with ongoing work demonstrating their functions in diverse pathways. Their roles in defense responses are a topic of active investigation, particularly the rich set of micro (mi)RNAs that target disease resistance genes such as nucleotide binding/leucine-rich repeat (NB-LRR) genes. The miRNA-NB-LRR interactions result in the production of phased, secondary small interfering (phasi)RNAs, and phasiRNAs function in both cis and trans to propagate negative regulatory effects across additional members of the target gene family.

Secondary siRNAs from Medicago NB-LRRs modulated via miRNA-target interactions and their abundances.

Small RNAs are a class of non-coding RNAs that are of great importance in gene expression regulatory networks. Different families of small RNAs are generated via distinct biogenesis pathways. One such family specific to plants is that of phased, secondary siRNAs (phasiRNAs); these require RDR6, DCL4, and (typically) a microRNA (miRNA) trigger for their biogenesis.

Phased, secondary, small interfering RNAs in posttranscriptional regulatory networks.

Plant genomes are the source of large numbers of small RNAs, generated via a variety of genetically separable pathways. Several of these pathways converge in the production of phased, secondary, small interfering RNAs (phasiRNAs), originally designated as trans-acting small interfering RNAs or tasiRNAs. PhasiRNA biogenesis requires the involvement of microRNAs as well as the cellular machinery for the production of siRNAs.

[Evaluation of reconstruction of extensor pollicis function by transfer of extensor indicis].

Objective: To assess the long-time results of reconstruction of the extensor pollicis longus (EPL) function by transfer of the extensor indicis (EI).

Methods: From August 1978 to March 2003, 46 cases of loss of the EPL function were treated by transfer of the extensor indicis. Of 46 cases, there were 32 males and 14 females, aged 16-51 years with an average of 36 years; there were 24 cases of old traumatic rupture and 22 cases of secondary rupture.

[Treatment of old scaphoid fracture with Russe technique].

Objective: To report the effect of Russe technique in treating old scaphoid fracture.

Methods: From January 1987 to February 1999, 11 cases of old scaphoid fractures were treated with Russe technique. The follow-up period averaged 5 years and 7 months.