Characterization and expression patterns of small RNAs in synthesized Brassica hexaploids

Polyploidy has played an important role in promoting plant evolution through genomic merging and doubling. We used high-throughput sequencing to compare miRNA expression profiles between Brassica hexaploid and its parents. A total of 613, 784 and 742 known miRNAs were identified in Brassica rapa, Brassica carinata, and Brassica hexaploid, respectively. We detected 618 miRNAs were differentially expressed (log₂Ratio ≥ 1, P ≤ 0.05) between Brassica hexaploid and its parents, and 425 miRNAs were non-additively expressed in Brassica hexaploid, which suggest a trend of non-additive miRNA regulation following hybridization and polyploidization. Remarkably, majority of the non-additively expressed miRNAs in the Brassica hexaploid are repressed, and there was a bias toward repression of B. rapa miRNAs, which is consistent with the progenitor-biased gene repression in the synthetic allopolyploids. In addition, we identified 653 novel mature miRNAs in Brassica hexaploid and its parents. Finally, we found that almost all the non-additive accumulation of siRNA clusters exhibited a low-parent pattern in Brassica hexaploid. Non-additive small RNA regulation is involved in a range of biological pathways, probably providing a driving force for variation and adaptation in allopolyploids.     

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The importance of long non‐coding RNAs (lncRNAs) in plant development has been established, but a systematic analysis of lncRNAs expressed during pollen development and fertilization has been elusive. We performed a time series of RNA‐seq experiments at five developmental stages during pollen development and three different time points after pollination in Brassica rapa and identified 12 051 putative lncRNAs. A comprehensive view of dynamic lncRNA expression networks underpinning pollen development and fertilization was provided. B. rapa lncRNAs share many common characteristics of lncRNAs: relatively short length, low expression but specific in narrow time windows, and low evolutionary conservation. Gene modules and key lncRNAs regulating reproductive development such as exine formation were uncovered. Forty‐seven cis‐acting lncRNAs and 451 trans‐acting lncRNAs were revealed to be highly coexpressed with their target protein‐coding genes. Of particular importance are the discoveries of 14 lncRNAs that were highly coexpressed with 10 function‐known pollen‐associated coding genes. Fifteen lncRNAs were predicted as endogenous target mimics for 13 miRNAs, and two lncRNAs were proved to be functional target mimics for miR160 after experimental verification and shown to function in pollen development. Our study provides the systematic identification of lncRNAs during pollen development and fertilization in B. rapa and forms the foundation for future genetic, genomic, and evolutionary studies.     

Vernalization can regulate flowering time through microRNA mechanism in Brassica rapa

Vernalization is an important process that regulates the floral transition in plants. MicroRNAs (miRNAs) are endogenous non‐coding small RNA (sRNA) molecules that function in plant growth and development. Despite that miRNAs related to flowering have previously been characterized, their roles in response to vernalization in pak‐choi (Brassica rapa ssp. chinensis) has never been studied. Here, two sRNA libraries from B. rapa leaves (vernalized and non‐vernalized plants) were constructed and sequenced. Two hundred eight known and 535 novel miRNAs were obtained, of which 20 known and 66 new miRNAs were significantly differentially expressed and considered as vernalization‐related miRNAs. The corresponding targets were predicted on the basic of sequence homology search. In addition, 11 miRNAs and eight targets were selected for real‐time quantitative PCR to confirm their expression profiles. Functional annotation of targets using gene ontology and Kyoto encyclopedia of genes and genomes results suggested that most targets were significantly enriched in the hormone signaling pathway. Moreover, a decreased indole‐3‐acetic acid (IAA) and an increased GA₃ hormone were detected after vernalization, indicating that the IAA and GA₃ might response to vernalization. These results indicated that vernalization regulates flowering through microRNA mechanism by affecting endogenous hormone level in B. rapa. This study provides useful insights of promising miRNAs candidates involved in vernalization in B. rapa, and facilitates further investigation of the miRNA‐mediated molecular mechanisms of vernalization in B. rapa.     

Fraction I protein analysis of parasexual hybrid plants Arabidopsis thaliana + Brassica campestris

The polypeptide composition of Fraction I protein (ribulose-1,5-bisphosphate carboxylase) prepared from leaves of two clones of the parasexual hybrid plant Arabidopsis thaliana + Brassica campestris as well as their parents was analyzed by isoelectric focusing. The protein in hybrid plants contained a heterogenous population of small subunits resulting from the expression of both Arbabidopsis and Brassica nuclear genes, whereas the large subunit polypeptides, and hence the functional chloroplast DNA, were from the Brassica parent.     

Non-coding RNAs: Functional roles in the regulation of stress response in Brassica crops

Brassica crops face a combination of different abiotic and biotic stresses in the field that can reduce plant growth and development by affecting biochemical and morpho-physiological processes. Emerging evidence suggests that non-coding RNAs (ncRNAs), especially microRNAs (miRNAs) and long ncRNAs (lncRNAs), play a significant role in the modulation of gene expression in response to plant stresses. Recent advances in computational and experimental approaches are of great interest for identifying and functionally characterizing ncRNAs. While progress in this field is limited, numerous ncRNAs involved in the regulation of gene expression in response to stress have been reported in Brassica. In this review, we summarize the modes of action and functions of stress-related miRNAs and lncRNAs in Brassica as well as the approaches used to identify ncRNAs.     

Deregulated lncRNA expression profile in the mouse lung adenocarcinomas with KRAS‐G12D mutation and P53 knockout

Recent studies have demonstrated that aberrant long non‐coding RNAs (lncRNAs) expression are suggested to be closely associated with multiple human diseases, lung cancer included. However, the roles of lncRNAs in lung cancer are not well understood. In this study, we used microarrays to investigate the aberrantly expressed lncRNAs in the mouse lung adenocarcinoma with P53 knockout and the Kras^G12D mutation. Results revealed that 6424 lncRNAs were differentially expressed (≥ 2‐fold change, P < .05). Two hundred and ten lncRNAs showed more than 8‐fold change and conserved across human and were further analysed in the primary mouse lung adenocarcinoma KP cells, which were isolated from the p53 knockout and the Kras^G12D mutation mice. Among all the 210 lncRNAs, 11 lncRNAs' expression was regulated by P53, 33 lncRNAs by KRAS and 13 lncRNAs by hypoxia in the primary KP cells, respectively. NONMMUT015812, which was remarkably up‐regulated in the mouse lung adenocarcinoma and negatively regulated by the P53 re‐expression, was detected to analyse its cellular function. Results showed that knockdown of NONMMUT015812 by shRNAs decreased proliferation and migration abilities of KP cells. Among those aberrantly expressed lncRNAs in the mouse lung adenocarcinoma, NONMMUT015812 was a potential oncogene.     

Three vital RNA functions and interactions in the process of silk gland apoptosis in silkworm Bombyx mori

The Silkworm Bombyx mori is an important insect in terms of economics and a model organism with a complete metamorphosis. The economic importance of silkworms is dependent on the functions of the silkgland, a specialized organ that synthesizes silk proteins. The silk gland undergoes massive degeneration during the larval to pupal stage, which involves in cell apoptosis. In this paper, high throughput sequencing was used to detect the expression of messenger RNA (mRNA), long noncoding RNA (lncRNA), and microRNA (miRNA) from silk glands of Day 3 in the fifth instar larvae (L5D3) and the spinning 36h (sp36h). We analyzed the Gene Ontology (GO) functions of target genes of the differentially expressed lncRNAs and miRNAs. We investigated the regulations of mRNA, lncRNA, and miRNA on silk gland apoptosis in L5D3 and sp36h. In total, 10,947 lncRNAs were detected in the silk gland and the index number TCONS‐00021360 lncRNA may be involved in the process of apoptosis. In addition, 344 miRNAs targeted 285 mRNAs were related to the death process under GO entry. The results indicated that miRNAs play an important role in the molecular regulation of the silk gland apoptosis compared with that of lncRNAs. Finally, we screened 746 lncRNAs and 20 miRNAs that might interact with BmDredd, and drew an interaction network among them.     

Characterization of miRNAs involved in response to poly(I:C) in porcine airway epithelial cells

MicroRNAs (miRNA) have been implicated in a variety of pathological conditions including infectious diseases. Knowledge of the miRNAs affected by poly(I:C), a synthetic analog of viral double‐stranded RNA, in porcine airway epithelial cells (PAECs) contributes to understanding the mechanisms of swine viral respiratory diseases, which bring enormous economic loss worldwide every year. In this study, we used high throughput sequencing to profile miRNA expression in PAECs treated with poly(I:C) as compared to the untreated control. This approach revealed 23 differentially expressed miRNAs (DEMs), five of which have not been implicated in viral infection before. Nineteen of the 23 miRNAs were down‐regulated including members of the miR‐17‐92 cluster, a well‐known polycistronic oncomir and extensively involved in viral infection in humans. Target genes of DEMs, predicted using bioinformatic methods and validated by luciferase reporter analysis on two representative DEMs, were significantly enriched in several pathways including transforming growth factor‐β signaling. A large quantity of sequence variations (isomiRs) were found including a substitution at position 5, which was verified to redirect miRNAs to a new spectrum of targets by luciferase reporter assay together with bioinformatics analysis. Twelve novel porcine miRNAs conserved in other species were identified by homology analysis together with cloning verification. Furthermore, the expression analysis revealed the potential importance of three novel miRNAs in porcine immune response to viruses. Overall, our data contribute to clarifying the mechanisms underlying the host immune response against respiratory viruses in pigs, and enriches the repertoire of porcine miRNAs.