Identification of short open reading frames in plant genomes.

The roles of short/small open reading frames (sORFs) have been increasingly recognized in recent years due to the rapidly growing number of sORFs identified in various organisms due to the development and application of the Ribo-Seq technique, which sequences the ribosome-protected footprints (RPFs) of the translating mRNAs. However, special attention should be paid to RPFs used to identify sORFs in plants due to their small size (~30 nt) and the high complexity and repetitiveness of the plant genome, particularly for polyploidy species. In this work, we compare different approaches to the identification of plant sORFs, discuss the advantages and disadvantages of each method, and provide a guide for choosing different methods in plant sORF studies.

Long non-coding RNA PRNCR1 promotes ovarian cancer cell proliferation, migration and invasion by targeting the miR-653-5p/ELF2 axis.

Recent studies have shown that prostate cancer-associated long non-coding RNA, PRNCR1, plays crucial roles in the development of multiple human cancers. However, its role in ovarian cancer is barely known. This study was carried out to investigate the role of PRNCR1 and the underlying mechanisms in OC. The expression of PRNCR1 and miR-653-5p in OC cell lines and tissues were detected by qRT-PCR. The expression of ELF2 protein was evaluated by Western blot analysis. Cell proliferation was measured by colony formation and MTT assay. Cell invasion and migration were evaluated by Transwell and wound healing assay. Luciferase reporter assay and RNA-binding protein immunoprecipitation assay were performed to determine the interaction between miR-653-5p and PRNCR1, as well as between miR-653-5p and ELF2. In vivo tumor xenograft model was established to evaluate the role of PRNCR1 in tumor growth. Our results demonstrated that PRNCR1 was significantly upregulated in both OC cell lines and tissues, and high expression of PRNCR1 was correlated with poor survival of OC patients. Overexpression of PRNCR1 accelerated OC cell invasion, migration and proliferation. Besides, the expression of PRNCR1 was negatively correlated with the expression of miR-653-5p, while positively correlated with the expression of E74-like factor 2 in OC tissues. Importantly, ELF2 could target miR-653-5p, and PRNCR1 increased the expression levels of ELF2 by sponging miR-653-5p in OC cells. Furthermore, the miR-145-5p/ELF2 axis was involved in the regulation of PRNCR1 in OC progression in vivo. PRNCR1 promotes OC tumor progress via the miR-653-5p/ELF2 axis and might be a potential therapeutic target for OC.

The novel circ_0084904/miR-802/MAL2 axis promotes the development of cervical cancer.

Circular RNAs (circRNAs) have been identified as critical regulators in human cancers, including cervical cancer (CC). However, the precise action of circ_0084904 in cervical carcinogenesis remains to be elucidated. The levels of circ_0084904, microRNA (miR)-802, and Mal, T cell differentiation protein 2 (MAL2) were checked by quantitative real-time PCR (qRT-PCR) or western blot. Ribonuclease R (RNase R) and subcellular localization assays were used to detect the stability and localization of circ_0084904, respectively. Cell colony formation ability was assessed by colony formation assay. Cell cycle and apoptosis were detected by flow cytometry. Cell migration and invasion abilities were gauged by transwell assay. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were applied to determine the direct relationship between miR-802 and circ_0084904 or MAL2. The xenograft experiments were performed to evaluate the role of circ_0084904 in tumor growth in vivo. Circ_0084904 was markedly up-regulated in CC tissues and cell lines. Silencing endogenous circ_0084904 impeded cell colony formation, cell cycle progression, migration, invasion, epithelial-mesenchymal transition (EMT), and promoted apoptosis in vitro, as well as diminished tumor growth in vivo. Mechanistically, circ_0084904 targeted miR-802, and the effects of circ_0084904 silencing were mediated by miR-802. MAL2 was directly targeted and inhibited by miR-802, and MAL2 was a functional target of miR-802. Moreover, circ_0084904 modulated MAL2 expression via miR-802. Our study identified circ_0084904 as a novel oncogenic driver in CC depending on the modulation of the miR-802/MAL2 axis, establishing the notion that silencing of circ_0084904 might represent a promising targeted therapy for CC.

The genome of cultivated peanut provides insight into legume karyotypes, polyploid evolution and crop domestication.

High oil and protein content make tetraploid peanut a leading oil and food legume. Here we report a high-quality peanut genome sequence, comprising 2.54 Gb with 20 pseudomolecules and 83,709 protein-coding gene models. We characterize gene functional groups implicated in seed size evolution, seed oil content, disease resistance and symbiotic nitrogen fixation. The peanut B subgenome has more genes and general expression dominance, temporally associated with long-terminal-repeat expansion in the A subgenome that also raises questions about the A-genome progenitor. The polyploid genome provided insights into the evolution of Arachis hypogaea and other legume chromosomes. Resequencing of 52 accessions suggests that independent domestications formed peanut ecotypes. Whereas 0.42-0.47 million years ago (Ma) polyploidy constrained genetic variation, the peanut genome sequence aids mapping and candidate-gene discovery for traits such as seed size and color, foliar disease resistance and others, also providing a cornerstone for functional genomics and peanut improvement.

Efficient Production of a Bioactive Bevacizumab Monoclonal Antibody Using the 2A Self-cleavage Peptide in Transgenic Rice Callus.

Bevacizumab, a humanized monoclonal antibody (mAb) targeting to the vascular endothelial growth factor (VEGF), has been widely used in clinical practice for the treatment of multiple cancers. Bevacizumab was mostly produced by the mammalian cell expression system. We here reported the first plant-derived Bevacizumab by using transgenic rice callus as an alternative gene expression system. Codon-optimized Bevacizumab light chain (BLC) and Bevacizumab heavy chain (BHC) genes were designed, synthesized as a polyprotein with a 2A self-cleavage linker peptide from the Foot-and-mouth disease virus, cloned into a plant binary vector under a constitutive maize ubiquitin promoter, and transformed into rice nuclear genome through Agrobacterium-mediated transformation. Southern blot and western blot analyses confirmed the integration and expression of BLC and BHC genes in transgenic rice callus. Enzyme-linked immunosorbent assay (ELISA) analysis indicated that the rice-derived Bevacizumab mAb was biologically active and the recombinant mAb was expressed at high levels (160.7-242.8 mg/Kg) in transgenic rice callus. The mAb was purified by using protein A affinity chromatography and the purified antibody was tested for its binding affinity with its target human VEGF (hVEGF) antigen by ELISA. Rice callus produced Bevacizumab and a commercial Bevacizumab (Avastin) were shown to have similar binding affinity to hVEGF. These results indicated that rice callus produced Bevacizumab could have similar biological activity and might potentially be used as a cost-effective biosimilar molecule in future cancer treatment.

Rapid proliferation and nucleolar organizer targeting centromeric retrotransposons in cotton.

Centromeric chromatin in most eukaryotes is composed of highly repetitive centromeric retrotransposons and satellite repeats that are highly variable even among closely related species. The evolutionary mechanisms that underlie the rapid evolution of centromeric repeats remain unknown. To obtain insight into the evolution of centromeric repeats following polyploidy, we studied a model diploid progenitor (Gossypium raimondii, D-genome) of the allopolyploid (AD-genome) cottons, G. hirsutum and G. barbadense. Sequence analysis of chromatin-immunoprecipitated DNA showed that the G. raimondii centromeric repeats originated from retrotransposon-related sequences. Comparative analysis showed that nine of the 10 analyzed centromeric repeats were absent from the centromeres in the A-genome and related diploid species (B-, F- and G-genomes), indicating that they colonized the centromeres of D-genome lineage after the divergence of the A- and D- ancestral species or that they were ancestrally retained prior to the origin of Gossypium. Notably, six of the nine repeats were present in both the A- and D-subgenomes in tetraploid G. hirsutum, and increased in abundance in both subgenomes. This finding suggests that centromeric repeats may spread and proliferate between genomes subsequent to polyploidization. Two repeats, Gr334 and Gr359 occurred in both the centromeres and nucleolar organizer regions (NORs) in D- and AD-genome species, yet localized to just the NORs in A-, B-, F-, and G-genome species. Contained within is a story of an established centromeric repeat that is eliminated and allopolyploidization provides an opportunity for reinvasion and reestablishment, which broadens our evolutionary understanding behind the cycles of centromeric repeat establishment and targeting.

The cauliflower mosaic virus protein P6 forms motile inclusions that traffic along actin microfilaments and stabilize microtubules.

The gene VI product (P6) of Cauliflower mosaic virus (CaMV) is a multifunctional protein known to be a major component of cytoplasmic inclusion bodies formed during CaMV infection. Although these inclusions are known to contain virions and are thought to be sites of translation from the CaMV 35S polycistronic RNA intermediate, the precise role of these bodies in the CaMV infection cycle remains unclear. Here, we examine the functionality and intracellular location of a fusion between P6 and GFP (P6-GFP). We initially show that the ability of P6-GFP to transactivate translation is comparable to unmodified P6. Consequently, our work has direct application for the large body of literature in which P6 has been expressed ectopically and its functions characterized. We subsequently found that P6-GFP forms highly motile cytoplasmic inclusion bodies and revealed through fluorescence colocalization studies that these P6-GFP bodies associate with the actin/endoplasmic reticulum network as well as microtubules. We demonstrate that while P6-GFP inclusions traffic along microfilaments, those associated with microtubules appear stationary. Additionally, inhibitor studies reveal that the intracellular movement of P6-GFP inclusions is sensitive to the actin inhibitor, latrunculin B, which also inhibits the formation of local lesions by CaMV in Nicotiana edwardsonii leaves. The motility of P6 along microfilaments represents an entirely new property for this protein, and these results imply a role for P6 in intracellular and cell-to-cell movement of CaMV.

[Effect of connexin 43 knockout on acupuncture-induced down-regulation of c-fos expression in spinal dorsal horn in visceral pain mice].

OBJECTIVE: To explore the effect of connexin 43 gene knockout on acupuncture analgesia. METHODS: Seventy-two wide type (WT) and connexin 43 gene knockout mice were separately and randomly divided into: WT control group, WT model group, WT acupuncture group, heterozygous (HT) control group, HT model group and HT acupuncture group, with 12 cases in each. Visceral pain model was established by intraperitoneal administration of acetic acid. "Zhongwan" (CV 12) and bilateral "Zusanli" (ST 36) were punctured with a filiform needle for 30 min and stimulated by manipulating the needle 30 s every 5 min. The expression of c-fos in the spinal dorsal horn was assayed by using RT-PCR and western blot techniques. RESULTS: There was no significant difference between HT and WT control mice in relative grey value of spinal c-fos mRNA expression (P>0.05), in which few c-fos mRNA and protein expressed. The expression of c-fos mRNA and protein was increased significantly following intraperitoneal acetic acid injection compared with control groups in both HT and WT mice (P<0.01). And no significant difference was found between HT and WT model groups in c-fos mRNA expression (P>0.05). Compared with WT model group, the expression of both c-fos mRNA and c-fos protein in WT acupuncture group was down-regulated significantly (P<0.01). In comparison with HT model group, the expression of both c-fos mRNA and protein in HT acupuncture group was down-regulated but without statistically significant difference (P>0.05). And the expression of c-fos mRNA and protein in HT acupuncture group was significantly higher than that in WT acupuncture group (P<0.05, 0.01). CONCLUSION: Acupuncture has a marked antinociceptive effect in visceral pain mice, and simultaneously suppresses the expression of c-fos mRNA and protein evoked by noxious stimulation in the spinal dorsal horn. Connexin 43 gene knockout may weaken acupuncture analgesia and reduce EA-induced down-regulation of c-fos expression, suggesting an involvement of connexin 43 in the analgesic effect of acupuncture.

Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors.

High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant L-cysteine alone or L-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.

Construction and behavior of engineered minichromosomes in maize.

Engineered minichromosomes were constructed in maize by modifying natural A and supernumerary B chromosomes. By using telomere-mediated chromosomal truncation, it was demonstrated that such an approach is feasible for the generation of minichromosomes of normal A chromosomes by selection of spontaneous polyploid events that compensate for the deficiencies produced. B chromosomes are readily fractionated by biolistic transformation of truncating plasmids. Foreign genes were faithfully expressed from integrations into normal B chromosomes and from truncated miniB chromosomes. Site-specific recombination between the terminal transgene on a miniA chromosome and a terminal site on a normal chromosome was demonstrated. It was also found that the miniA chromosome did not pair with its progenitor chromosomes during meiosis, indicating a useful property for such constructs. The miniB chromosomes are faithfully transmitted from one generation to the next but can be changed in dosage in the presence of normal B chromosomes. This approach for construction of engineered chromosomes can be easily extended to other plant species because it does not rely on cloned centromere sequences, which are species-specific. These platforms will provide avenues for studies on plant chromosome structure and function and for future developments in biotechnology and agriculture.