The RNA binding protein EHD6 recruits the m6A reader YTH07 and sequesters OsCOL4 mRNA into phase-separated ribonucleoprotein condensates to promote rice flowering.

N6-Methyladenosine (m6A) is one of the most abundant modifications of eukaryotic mRNA, but its comprehensive biological functionality remains further exploration. In this study, we identified and characterized a new flowering-promoting gene, EARLY HEADING DATE6 (EHD6), in rice. EHD6 encodes an RNA recognition motif (RRM)-containing RNA binding protein that is localized in the non-membranous cytoplasm ribonucleoprotein (RNP) granules and can bind both m6A-modified RNA and unmodified RNA indiscriminately. We found that EHD6 can physically interact with YTH07, a YTH (YT521-B homology) domain-containing m6A reader. We showed that their interaction enhances the binding of an m6A-modified RNA and triggers relocation of a portion of YTH07 from the cytoplasm into RNP granules through phase-separated condensation. Within these condensates, the mRNA of a rice flowering repressor, CONSTANS-like 4 (OsCOL4), becomes sequestered, leading to a reduction in its protein abundance and thus accelerated flowering through the Early heading date 1 pathway. Taken together, these results not only shed new light on the molecular mechanism of efficient m6A recognition by the collaboration between an RNA binding protein and YTH family m6A reader, but also uncover the potential for m6A-mediated translation regulation through phase-separated ribonucleoprotein condensation in rice.

A novel multimeric sCD19-streptavidin fusion protein for functional detection and selective expansion of CD19-targeted CAR-T cells.

BACKGROUND: CARs are engineered receptors comprising an immunoglobulin single-chain variable fragment (scFv) that identifies and binds to the target antigen, a transmembrane domain, and an intracellular T-cell signaling domain. CD19 is a B lineage-specific transmembrane glycoprotein and is expressed in more than 95% of B-cell malignancies. Streptavidin (SA) is a homo-tetrameric protein derived from Streptomyces avidinii, which can bind four biotin molecules with an extremely high affinity at a Kd value of 10-15 M. AIMS: The aim of the study is to generate a novel soluble multimeric fusion protein, sCD19-streptavidin (sCD19-SA) for functional detection and selective expansion of CD19-targeted CAR-T cells. METHODS: The fusion proteins CD19-SA was expressed in CHO cells and purified by use of Ni-nitrilotriacetic acid agarose beads. RESULTS: A novel fusion protein (sCD19-SA) was generated, consisting of the extracellular domain of human CD19 and the core region of SA, and could be used to functionally detect CD19-targeted CAR-T cells. Furthermore, this protein was demonstrated to form multimers to activate CAR-T cells to induce their selective expansion. Importantly, sCD19-SA-stimulated CD19-targeted CAR-T cells could improve antitumor effects in vivo. CONCLUSIONS: Our study has highlighted the potential of utilizing antigen-SA fusion proteins such as sCD19-SA for CAR-T therapy for the functional detection of CAR expression and selective expansion of CAR-T cells.

The pilot-scale preparation of the SA-hGM-CSF bi-functional fusion protein.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) can be used to induce a powerful immune response. Based on the specific binding of biotin and streptavidin, SA-hGM-CSF was anchored on the surface of biotinylated tumor cells, which could enhance the anti-tumor effect of tumor cell vaccines in our previous reports, suggesting it would have potential clinical value. Preparation of the biologically active proteins in large-scale production is the basis of clinical application, however, only a small amount of biologically active protein was obtained according to previous studies. In this study, we researched the effects of various factors on the purification and simultaneous renaturation of SA-hGM-CSF fusion protein by single factor experiment and orthogonal experiment. Here, we developed a viable pilot-scale trial in the fermentation, purification, refolding and freeze-drying of SA-hGM-CSF proteins in order to efficiently obtain more biologically active proteins with high purity, which will lay the foundation for industrial production.

[Preparation of streptavidin-C3d fusion protein and detection of its function in vitro].

Objective To detect the prokaryotic expression of streptavidin-complement 3d (SA-C3d) fusion protein and verify its function in vitro. Methods The C3d DNA was amplified using C3 cDNA as a template, and the C3d fragment was ligated with the vector plasmid pET-24a-6His-SA-IL15 after the digestion with a one-step cloning method to obtain the SA-C3d prokaryotic expression plasmid. The correctly sequenced plasmid was transformed into expression competent Rosetta to induce protein expression. The target protein was obtained by nickel column affinity chromatography and urea dialyzed refolding. The function of SA was demonstrated by anchoring the biotinylated MB49 cell experiment, and the function of C3d was detected by an experiment that promoted the growth of Raji cells. Results The prokaryotic expression vector of SA-C3d was successfully constructed. The purified target protein was obtained by nickel column purification and dialysis refolding. The protein was specifically bound to biotinylated MB49 cells, which promoted the proliferation of Raji cells in a dose-dependent manner, indicating that the protein SA-C3d had a bifunctional activity. Conclusion The successfully prepared SA-C3d fusion protein can be bound to biotinylated MB49 cells in vitro and promote Raji cell proliferation.

The ethylene response factor OsERF109 negatively affects ethylene biosynthesis and drought tolerance in rice.

Drought is an important factor limiting plant development and crop production. Dissecting the factors involved in this process is the key for enhancement of plant tolerance to drought stress by genetic approach. Here, we evaluated the regulatory function of a novel rice ethylene response factor (ERF) OsERF109 in drought stress. Expression of OsERF109 was rapidly induced by stress and phytohormones. Subcellular localization and transactivation assay demonstrated that OsERF109 was localized in nucleus and possessed transactivation activity. Transgenic plants overexpressing (OE) and knockdown with RNA interfering (RI) OsERF109 exhibited significantly reduced and improved drought resistance, respectively, indicating that OsERF109 negatively regulates drought resistance in rice. Furthermore, measurement by gas chromatography showed that ethylene contents were less in OE while more in RI lines than these in wild types, supporting the data of drought tolerance and water loss in transgenic lines. Quantitative real-time PCR analysis also proved the regulation of OsERF109 in the expression of OSACS6, OSACO2, and OsERF3, which have been identified to play important roles in ethylene biosynthesis. Based on these results, our data evidence that OsERF109 regulates drought resistance by affecting the ethylene biosynthesis in rice. Overall, our study reveals the negative role of OsERF109 in ethylene biosynthesis and drought tolerance in rice.

An ethylene response factor OsWR1 responsive to drought stress transcriptionally activates wax synthesis related genes and increases wax production in rice.

Increasing evidence has revealed the major enzymes-involved in Arabidopsis and maize wax/cutin synthesis; however, there is limited information about the genes-associated with wax/cutin synthesis in rice. Here we report the characterization of an ethylene response factor gene in rice. This rice wax synthesis regulatory gene 1 (OsWR1) is a homolog of Arabidopsis wax/cutin synthesis regulatory gene WIN1/SHN1. Transcript analysis showed that OsWR1 is induced by drought, abscisic acid and salt, and is predominantly expressed in leaves. Functional analyses indicated that overexpressing OsWR1 (Ox-WR1) improved while RNA interference OsWR1 rice (RI-WR1) decreased drought tolerance, consistent with water loss and cuticular permeability, suggesting that OsWR1-triggered drought response might be associated with cuticular characteristics. In addition, OsWR1 activated the expression of the genes-related to oxidative stress response and membrane stability. Gas chromatograph-mass spectrometry analysis further showed that OsWR1 modulated the wax synthesis through alteration of long chain fatty acids and alkanes, evidencing the regulation of OsWR1 in wax synthesis. Detection with real-time PCR amplification indicated that Ox-WR1 enhanced while RI-WR1 decreased the expression of wax/cutin synthesis related genes. Furthermore, OsWR1 physically interacted with the DRE and GCC box in the promoters of wax related genes OsLACS2 and OsFAE1'-L, indicating that OsWR1 at least directly modulates the expression of these genes. Thus our results indicate that OsWR1 is a positive regulator of wax synthesis related genes in rice, and this regulation, distinct from its homology regulator of WIN1/SHN1 in cutin synthesis, subsequently contributes to reduced water loss and enhanced drought tolerance.

Transcriptional activation of OsDERF1 in OsERF3 and OsAP2-39 negatively modulates ethylene synthesis and drought tolerance in rice.

The phytohormone ethylene is a key signaling molecule that regulates a variety of developmental processes and stress responses in plants. Transcriptional modulation is a pivotal process controlling ethylene synthesis, which further triggers the expression of stress-related genes and plant adaptation to stresses; however, it is unclear how this process is transcriptionally modulated in rice. In the present research, we report the transcriptional regulation of a novel rice ethylene response factor (ERF) in ethylene synthesis and drought tolerance. Through analysis of transcriptional data, one of the drought-responsive ERF genes, OsDERF1, was identified for its activation in response to drought, ethylene and abscisic acid. Transgenic plants overexpressing OsDERF1 (OE) led to reduced tolerance to drought stress in rice at seedling stage, while knockdown of OsDERF1 (RI) expression conferred enhanced tolerance at seedling and tillering stages. This regulation was supported by negative modulation in osmotic adjustment response. To elucidate the molecular basis of drought tolerance, we identified the target genes of OsDERF1 using the Affymetrix GeneChip, including the activation of cluster stress-related negative regulators such as ERF repressors. Biochemical and molecular approaches showed that OsDERF1 at least directly interacted with the GCC box in the promoters of ERF repressors OsERF3 and OsAP2-39. Further investigations showed that OE seedlings had reduced expression (while RI lines showed enhanced expression) of ethylene synthesis genes, thereby resulting in changes in ethylene production. Moreover, overexpression of OsERF3/OsAP2-39 suppressed ethylene synthesis. In addition, application of ACC recovered the drought-sensitive phenotype in the lines overexpressing OsERF3, showing that ethylene production contributed to drought response in rice. Thus our data reveal that a novel ERF transcriptional cascade modulates drought response through controlling the ethylene synthesis, deepening our understanding of the regulation of ERF proteins in ethylene related drought response.