Yu-Chan Zhang

Deep Sequencing of Human Nuclear and Cytoplasmic Small RNAs Reveals an Unexpectedly Complex Subcellular Distribution of miRNAs and tRNA 3′ Trailers

Background MicroRNAs (miRNAs) are ∼22-nt small non-coding regulatory RNAs that have generally been considered to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in the nucleus. Methodology/Principal Findings To determine the number of miRNAs localized to the nucleus, we systematically investigated the subcellular distribution of small RNAs (sRNAs) by independent deep sequencing sequenced of the nuclear and cytoplasmic pools of 18- to 30-nucleotide sRNAs from human cells. We identified 339 nuclear and 324 cytoplasmic known miRNAs, 300 of which overlap, suggesting that the majority of miRNAs are imported into the nucleus. With the exception of a few miRNAs evidently enriched in the nuclear pool, such as the mir-29b, the ratio of miRNA abundances in the nuclear fraction versus in the cytoplasmic fraction vary to some extent. Moreover, our results revealed that a large number of tRNA 3′trailers are exported from the nucleus and accumulate in the cytoplasm. These tRNA 3′ trailers accumulate in a variety of cell types, implying that the biogenesis of tRNA 3′ trailers is conserved and that they have a potential functional role in vertebrate cells. Conclusion/Significance Our results provide the first comprehensive view of the subcellular distribution of diverse sRNAs and new insights into the roles of miRNAs and tRNA 3′ trailers in the cell.

Expression analysis of phytohormone‐regulated microRNAs in rice, implying their regulation roles in plant hormone signaling

Twenty‐two conserved miRNAs were chosen to investigate the expression pattern in response to phytohormone treatments, in which the effects of five classic plant hormone stresses were surveyed in Oryza sativa. The results showed that 11 miRNAs were found to be dysregulated by one or more phytohormone treatments. The target genes of these miRNAs were validated in vivo and their expression profiling were revealed. We also analyzed the promoter regions of the 22 conserved miRNAs for phytohormone‐responsive elements and the existence of the elements provided further evidences supporting our results. These findings enable us to further investigate the role of miRNAs in phytohormone signaling.

Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching

Increasing grain yields is a major focus of crop breeders around the world. Here we report that overexpression of the rice microRNA (miRNA) OsmiR397, which is naturally highly expressed in young panicles and grains, enlarges grain size and promotes panicle branching, leading to an increase in overall grain yield of up to 25% in a field trial. To our knowledge, no previous report has shown a positive regulatory role of miRNA in the control of plant seed size and grain yield. We determined that OsmiR397 increases grain yield by downregulating its target, OsLAC, whose product is a laccase-like protein that we found to be involved in the sensitivity of plants to brassinosteroids. As miR397 is highly conserved across different species, our results suggest that manipulating miR397 may be useful for increasing grain yield not only in rice but also in other cereal crops.

Both endo-siRNAs and tRNA-derived small RNAs are involved in the differentiation of primitive eukaryote Giardia lamblia

Significance Small RNAs (sRNAs) are most important regulators in eukaryotes. Although different kinds of sRNAs have been extensively studied in higher eukaryotes, their role remains largely unknown in protozoa. We have systematically investigated in the full genome the sRNAs of Giardia lamblia, the most primitive eukaryote known. Surprisingly, we have found that two major types of sRNAs (i.e., endogenous siRNAs and tRNA-derived sRNAs) are largely encoded in the genome of G. lamblia, whereas canonical microRNAs could not be identified in this parasite. Additional studies showed that both sRNAs might be involved in the differentiation regulation of G. lamblia. This study indicates that these two kinds of eukaryotic sRNAs emerged in the early evolution of eukaryotes. Small RNAs (sRNAs), including microRNAs and endogenous siRNAs (endo-siRNAs), regulate most important biologic processes in eukaryotes, such as cell division and differentiation. Although sRNAs have been extensively studied in various eukaryotes, the role of sRNAs in the early emergence of eukaryotes is unclear. To address these questions, we deep sequenced the sRNA transcriptome of four different stages in the differentiation of Giardia lamblia, one of the most primitive eukaryotes. We identified a large number of endo-siRNAs in this fascinating parasitic protozoan and found that they were produced from live telomeric retrotransposons and three genomic regions (i.e., endo-siRNA generating regions [eSGRs]). eSGR-derived endo-siRNAs were proven to target mRNAs in trans. Gradual up-regulation of endo-siRNAs in the differentiation of Giardia suggested that they might be involved in the regulation of this process. This hypothesis was supported by the impairment of the differentiation ability of Giardia when GLDICER, essential for the biogenesis of endo-siRNAs, was knocked down. Endo-siRNAs are not the only sRNA regulators in Giardia differentiation, because a great number of tRNAs-derived sRNAs showed more dramatic expression changes than endo-siRNAs in this process. We totally identified five novel kinds of tRNAs-derived sRNAs and found that the biogenesis in four of them might be correlated with that of stress-induced tRNA-derived RNA (sitRNA), which was discovered in our previous studies. Our studies reveal an unexpected complex panorama of sRNA in G. lamblia and shed light on the origin and functional evolution of eukaryotic sRNAs.

Large-scale rewiring of innate immunity circuitry and microRNA regulation during initial rice blast infection

Rice blast is a recurrent fungal disease, and resistance to fungal infection is a complex trait. Therefore, a comprehensive examination of rice transcriptome and its variation during fungal infection is necessary to understand the complex gene regulatory networks. In this study, adopting Next-Generation Sequencing we profiled the transcriptomes and microRNAomes of rice varieties, one susceptible and the other resistant to M. oryzae, at multiple time points during the fungal infection. Our results revealed a substantial variation in the plant transcriptome and microRNAome as well as change to rice innate immunity during fungal infection. A number of putative R gene candidates were identified from a perturbed rice transcriptome analysis. The expression of genes and non-coding RNA molecules changed in both fungal resistant and susceptible plants during M. oryzae invasion discovered distinct pathways triggered in the susceptible and resistant plants. In addition, a number of fungus genes in the susceptible and resistant plants were constantly expressed at different time points, suggesting that they were likely to be the potential AVR genes. Our results revealed large-scale rewiring of innate immunity circuitry and microRNA regulation during initial rice blast infection, which would help to develop more robust blast-resistant rice plants.

Circular RNAs roll into the regulatory network of plants

As a novel class of endogenous non-coding RNAs, circular RNAs (circRNAs) have become a new research hotspot in recent years. The wide distribution of circRNAs in different plant species has been proven. Furthermore, circRNAs show significant tissue-specific expression patterns in plant development and are responsive to a variety of biotic and abiotic stresses, indicating that circRNAs might have important biological functions in plant development. Here, we summarize the current knowledge of plant circRNAs in recent years and discuss views and perspectives on the possible regulatory roles of plant circRNAs, including the function of miRNA sponges, regulating the expression of their parental genes or linear mRNAs, translating into peptides or proteins and responses to different stresses. These advances have sculpted a framework of plant circRNAs and provide new insights for functional RNA regulation research in the future.

miRNA MiR408 regulates grain yield and photosynthesis via a phytocyanin protein

MiR408 positively regulates rice grain yield by targeting the uclacyanin gene OsUCL8 and, in turn, affects photosynthesis. Increasing grain yield is the most important object of crop breeding. Here, we report that the elevated expression of a conserved microRNA, OsmiR408, could positively regulate grain yield in rice (Oryza sativa) by increasing panicle branches and grain number. We further showed that OsmiR408 regulates grain yield by down-regulating its downstream target, OsUCL8, which is an uclacyanin (UCL) gene of the phytocyanin family. The knock down or knock out of OsUCL8 also increases grain yield, while the overexpression of OsUCL8 results in an opposite phenotype. Spatial and temporal expression analyses showed that OsUCL8 was highly expressed in pistils, young panicles, developing seeds, and inflorescence meristem and was nearly complementary to that of OsmiR408. Interestingly, the OsUCL8 protein was localized to the cytoplasm, distinct from a majority of phytocyanins, which localize to the plasma membrane. Further studies revealed that the cleavage of OsUCL8 by miR408 affects copper homeostasis in the plant cell, which, in turn, affects the abundance of plastocyanin proteins and photosynthesis in rice. To our knowledge, this is the first report of the effects of miR408-OsUCL8 in regulating rice photosynthesis and grain yield. Our study further broadens the perspective of microRNAs and UCLs and provides important information for breeding high-yielding crops through genetic engineering.

Grass phasiRNAs and male fertility

Recent studies have indicated that a special type of small noncoding RNAs, phased small-interfering RNAs (phasiRNAs) play crucial roles in many cellular processes of plant development. PhasiRNAs are generated from long RNA precursors at intervals of 21 or 24 nt in plants, and they are produced from both protein-coding gene and long noncoding RNA genes. Different from those in eudicots, grass phasiRNAs include a special class of small RNAs that are specifically expressed in reproductive organs. These grass phasiRNAs are associated with gametogenesis, especially with anther development and male fertility. In this review, we summarized current knowledge on these small noncoding RNAs in male germ cells and their possible biological functions and mechanisms in grass species.

Laccase-13 Regulates Seed Setting Rate by Affecting Hydrogen Peroxide Dynamics and Mitochondrial Integrity in Rice

Seed setting rate is one of the most important components of rice grain yield. To date, only several genes regulating setting rate have been identified in plant. In this study, we showed that laccase-13 (OsLAC13), a member of laccase family genes which are known for their roles in modulating phenylpropanoid pathway and secondary lignification in cell wall, exerts a regulatory function in rice seed setting rate. OsLAC13 expressed in anthers and promotes hydrogen peroxide production both in vitro and in the filaments and anther connectives. Knock-out of OsLAC13 showed significantly increased seed setting rate, while overexpression of this gene exhibited induced mitochondrial damage and suppressed sugar transportation in anthers, which in turn affected seed setting rate. OsLAC13 also induced H2O2 production and mitochondrial damage in the root tip cells which caused the lethal phenotype. We also showed that high abundant of OsmiR397, the suppressor of OsLAC13 mRNA, increased the seed setting rate of rice plants, and restrains H2O2 accumulation in roots during oxidative stress. Our results suggested a novel regulatory role of OsLAC13 gene in regulating seed setting rate by affecting H2O2 dynamics and mitochondrial integrity in rice.