YTH Domain: A Family of N6-methyladenosine (m6A) Readers

Like protein and DNA, different types of RNA molecules undergo various modifications. Accumulating evidence suggests that these RNA modifications serve as sophisticated codes to mediate RNA behaviors and many important biological functions. N⁶-methyladenosine (m⁶A) is the most abundant internal RNA modification found in a variety of eukaryotic RNAs, including but not limited to mRNAs, tRNAs, rRNAs, and long non-coding RNAs (lncRNAs). In mammalian cells, m⁶A can be incorporated by a methyltransferase complex and removed by demethylases, which ensures that the m⁶A modification is reversible and dynamic. Moreover, m⁶A is recognized by the YT521-B homology (YTH) domain-containing proteins, which subsequently direct different complexes to regulate RNA signaling pathways, such as RNA metabolism, RNA splicing, RNA folding, and protein translation. Herein, we summarize the recent progresses made in understanding the molecular mechanisms underlying the m⁶A recognition by YTH domain-containing proteins, which would shed new light on m⁶A-specific recognition and provide clues to the future identification of reader proteins of many other RNA modifications.     

Two novel RNA-binding proteins identification through computational prediction and experimental validation

Since RBPs play important roles in the cell, it's particularly important to find new RBPs. We performed iRIP-seq and CLIP-seq to verify two proteins, CLIP1 and DMD, predicted by RBPPred whether are RBPs or not. The experimental results confirm that these two proteins have RNA-binding activity. We identified significantly enriched binding motifs UGGGGAGG, CUUCCG and CCCGU for CLIP1 (iRIP-seq), DMD (iRIP-seq) and DMD (CLIP-seq), respectively. The computational KEGG and GO analysis show that the CLIP1 and DMD share some biological processes and functions. Besides, we found that the SNPs between DMD and its RNA partners may be associated with Becker muscular dystrophy, Duchenne muscular dystrophy, Dilated cardiomyopathy 3B and Cardiovascular phenotype. Among the thirteen cancers data, CLIP1 and another 300 oncogenes always co-occur, and 123 of these 300 genes interact with CLIP1. These cancers may be associated with the mutations occurred in both CLIP1 and the genes it interacts with.     

A comparison of Xenopus laevis oocyte and embryo mRNA

Total RNA, extracted from mature oocytes and tadpoles of Xenopus laevis, was used as a template for in vitro protein synthesis. The oocyte RNA is markedly deficient in abundant mRNA species by comparison to tadpole RNA or other somatic RNAs, in agreement with previous experiments using RNA-cDNA hybridization analysis (S. Perlman and M. Rosbash, 1978, Develop. Biol.63, 197–212). Oocyte pA⁺ RNA is also larger than tadpole pA⁺ RNA or other somatic pA⁺ populations. The larger oocyte pA⁺ RNA and smaller oocyte pA⁺ RNA are equally good templates for in vitro protein synthesis, which implies that much, and perhaps all, of the large oocyte pA⁺ RNA is bona fide mRNA. We suggest that the relatively large size of the oocyte pA⁺ RNA population is due, at least in part, to the relative lack of abundant mRNA species in the population. This suggestion follows from the observation of 0. Meyuhas and R. P. Perry (1979, Cell16, 139–148) that L-cell-abundant mRNAs are preferentially small and rare mRNAs preferentially large. Most of the oocyte pA⁺ sequences are also present in tadpoles and are still adenylated at this stage. Oocyte proteins synthesized in vivo do not appear deficient in abundant proteins, suggesting that a translational control mechanism operates to select certain pA⁺ RNAs at higher frequencies than others.     

Defining Proximity Proteome of Histone Modifications by Antibody-mediated Protein A-APEX2 Labeling

Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein–protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2 (pA-APEX2) labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a pA-APEX2 fusion protein. Activation of APEX2 labels the nearby proteins with biotin; the biotinylated proteins are then purified using streptavidin beads and identified by mass spectrometry. We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac, and H4K12ac, as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient method to identify proteins that are proximal to modified histones.     

Rapid extraction method for detection of outer membrane protein F of Pseudomonas aeruginosa

A filtration technique has been developed to trap the peptidoglycan sacculus for rapid extraction of the outer membrane protein F of Pseudomonas aeruginosa. The method consists of the following three steps: (i) removal of cell components except peptidoglycan associated with some proteins, (ii) trapping of peptidoglycan with a glass filter and (iii) extraction with a hot SDS solution of proteins, mainly F protein, associated with peptidoglycan. The method is simple and rapid, providing for efficient screening for protein F-deficient mutants of P. aeruginosa. Using this method of screening, three mutants were isolated among 500 mutagenized clones.     

The development of new molecular tools containing a chemically synthesized carbohydrate ligand for the elucidation of carbohydrate roles via photoaffinity labeling: Carbohydrate–protein interactions are affected by the structures of the glycosidic bonds and the reducing-end sugar

Photoaffinity labeling technology is a highly efficient method for cloning carbohydrate-binding proteins. When the carbohydrate probes are synthesized according to conventional methods, however, the reducing terminus of the sugar is opened to provide an acyclic structure. Our continued efforts to solve this problem led to the development of new molecular tools with an oligosaccharide structure that contains a phenyldiazirine group for the elucidation of carbohydrate–protein interactions. We investigated whether carbohydrate–lectin interactions are affected by differences in the glycosidic formation and synthesized three types of molecular tools containing Galp–GlcpNAc disaccharide ligands and a photoreactive group (1, 2, 3). Photoaffinity labeling validated the recognition of the new ligand by different glycosidic bonds. Photoaffinity labeling also demonstrated that both the reducing end sugar and non-reducing end sugar recognized the Erythrina cristagalli agglutinin.     

N-Benzyloxycarbonyl-S-(2,4-dinitrophenyl)glutathione dibutyl diester is inhibitory to melarsoprol resistant cell lines overexpressing the T. bruceiMRPA transporter

A series of glutathione derivatives 1–4, modified at the N,S and/or COOH sites, with in vitro antitrypanosomal activity were tested against bloodstream form Trypanosoma brucei 247 wild type and a T. b. brucei 247 strain over-expressing the multiple drug resistance protein (MRPA) by 50–100x to assess the susceptibility of these compounds to resistance by the TbMRP protein. Of the compounds tested, only compound 1 inhibited both bloodstream form T. brucei and T. bruceiMRPA, with a resistance factor of 1.4, indicating it to be an inhibitor of this protein and proteins acting in synergy with the transporter, whilst 2 & 3 and its derivatives showed reduced inhibitory activity against T. bruceiMRPA, indicating them to be substrates and susceptible to resistance.     

Structural and SAXS analysis of the budding yeast SHU-complex proteins

In Saccharomyces cerevisiae, four proteins, Shu1, Shu2, Psy3 and Csm2, form a stable SHU-complex both in vivo and in vitro. These proteins are involved in the early stages of the homologous recombination DNA damage repair process. In this paper, the crystal structure of the Psy3–Csm2 sub-complex is presented at 1.8 Å resolution and successfully fitted into our small angle X-ray scattering (SAXS) data of the SHU-complex. Taken together with our electrophoretic mobility shift assay (EMSA) results, a model is proposed for the SHU–protein complex coupled with DNA.Structured summary of protein interactions:PSY3 and CSM2 bind by X-ray crystallography (View interaction) PSY3, CSM2, Shu 1 and Shu 2 physically interact by x ray scattering (View interaction)     

Peptide Linkage to the α-Subunit of MHCII Creates a Stably Inverted Antigen Presentation Complex

Class II proteins of the major histocompatibility complex (MHCII) typically present exogenous antigenic peptides to cognate T cell receptors of CD4-T lymphocytes. The exact conformation of peptide–MHCII complexes (pMHCII) can vary depending on the length, register and orientation of the bound peptide. We have recently found the self-peptide CLIP (class‐II-associated invariant chain‐derived peptide) to adopt a dynamic bidirectional binding mode with regard to the human MHCII HLA-DR1 (HLA, human leukocyte antigen). We suggested that inversely bound peptides could activate specific T cell clones in the context of autoimmunity. As a first step to prove this hypothesis, pMHC complexes restricted to either the canonical or the inverted peptide orientation have to be constructed. Here, we show that genetically encoded linkage of CLIP and two other antigenic peptides to the HLA-DR1 α-chain results in stable complexes with inversely bound ligands. Two‐dimensional NMR and biophysical analyses indicate that the CLIP-bound pMHC^inv complex (pMHC^inv, inverted MHCII–peptide complex) displays high thermodynamic stability but still allows for the exchange against higher‐affinity viral antigen. Complemented by comparable data on a corresponding β-chain-fused canonical HLA-DR1/CLIP complex, we further show that linkage of CLIP leads to a binding mode exactly the same as that of the corresponding unlinked constructs. We suggest that our approach constitutes a general strategy to create pMHC^inv complexes. Such engineering is needed to create orientation-specific antibodies and raise T cells to study phenomena of autoimmunity caused by isomeric pMHCs.     

Comparative Secretome Analysis of Magnaporthe oryzae Identified Proteins Involved in Virulence and Cell Wall Integrity

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.     

Determination of membrane-bound chloroplast RNA by the ethidium bromide fluorometric method

The method of El-Hamalawi et al. [(1975) Anal. Biochem.67, 384–391] for the fluorometric determination of nucleic acids with ethidium bromide has been adapted for the assay of membrane-associated chloroplast RNA. Membranes are stripped of RNA by incubation in a high-salt buffer lacking Mg²⁺, and the RNA is collected by magnesium phosphate-ethanol coprecipitation. RNA levels are determined by measuring the degree of enhancement of ethidium bromide fluorescence.     

The human MAPT locus generates circular RNAs

The microtubule-associated protein Tau, generated by the MAPT gene is involved in dozens of neurodegenerative conditions (“tauopathies”), including Alzheimer's disease (AD) and frontotemporal lobar degeneration/frontotemporal dementia (FTLD/FTD). The pre-mRNA of MAPT is well studied and its aberrant pre-mRNA splicing is associated with frontotemporal dementia. Using a PCR screen of RNA from human brain tissues, we found that the MAPT locus generates circular RNAs through a backsplicing mechanism from exon 12 to either exon 10 or 7. MAPT circular RNAs are localized in the cytosol and contain open reading frames encoding Tau protein fragments. The MAPT exon 10 is alternatively spliced and proteins involved in its regulation, such as CLK2, SRSF7/9G8, PP1 (protein phosphatase 1) and NIPP1 (nuclear inhibitor of PP1) reduce the abundance of the circular MAPT exon 12?→?10 backsplice RNA after being transfected into cultured HEK293 cells. In summary, we report the identification of new bona fide human brain RNAs produced from the MAPT locus. These may be a component of normal human brain Tau regulation and, since the circular RNAs could generate high molecular weight proteins with multiple microtubule binding sites, they could contribute to taupathies.     

The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein

Background: Colicin E7 (ColE7) is one of the bacterial toxins classified as a DNase-type E-group colicin. The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein. This biological event is a good model system for the investigation of protein recognition.Results: The crystal structure of a one-to-one complex between the DNase domain of colicin E7 and its cognate immunity protein Im7 has been determined at 2.3 Å resolution. Im7 in the complex is a varied four-helix bundle that is identical to the structure previously determined for uncomplexed Im7. The structure of the DNase domain of ColE7 displays a novel α/β fold and contains a Zn²⁺ ion bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. Im7 has a V-shaped structure, extending two arms to clamp the DNase domain of ColE7. One arm (α1¹–loop12–α2¹; where ¹ represents helices in Im7) is located in the region that displays the greatest sequence variation among members of the immunity proteins in the same subfamily. This arm mainly uses acidic sidechains to interact with the basic sidechains in the DNase domain of ColE7. The other arm (loop 23–α3¹–loop 34) is more conserved and it interacts not only with the sidechain but also with the mainchain atoms of the DNase domain of ColE7.Conclusions: The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site.