N6-methyladenosine in 7SK small nuclear RNA underlies RNA polymerase II transcription regulation.

N6-methyladenosine (m6A) modifications play crucial roles in RNA metabolism. How m6A regulates RNA polymerase II (RNA Pol II) transcription remains unclear. We find that 7SK small nuclear RNA (snRNA), a regulator of RNA Pol II promoter-proximal pausing, is highly m6A-modified in non-small cell lung cancer (NSCLC) cells. In A549 cells, we identified eight m6A sites on 7SK and discovered methyltransferase-like 3 (METTL3) and alkB homolog 5 (ALKBH5) as the responsible writer and eraser. When the m6A-7SK is specifically erased by a dCasRx-ALKBH5 fusion protein, A549 cell growth is attenuated due to reduction of RNA Pol II transcription. Mechanistically, removal of m6A leads to 7SK structural rearrangements that facilitate sequestration of the positive transcription elongation factor b (P-TEFb) complex, which results in reduction of serine 2 phosphorylation (Ser2P) in the RNA Pol II C-terminal domain and accumulation of RNA Pol II in the promoter-proximal region. Taken together, we uncover that m6A modifications of a non-coding RNA regulate RNA Pol II transcription and NSCLC tumorigenesis.

Functional Interrogation of Ca2+ Signals in Human Cancer Cells In Vitro and Ex Vivo by Fluorescent Microscopy and Molecular Tools.

Genetically encoded calcium indicators (GECIs) and high-resolution confocal microscopy enable dynamic visualization of calcium signals in cells and tissues. Two-dimensional and 3D biocompatible materials mimic the mechanical microenvironments of tumor and healthy tissues in a programmable manner. Cancer xenograft models and ex vivo functional imaging of tumor slices reveal physiologically relevant functions of calcium dynamics in tumors at different progression stages. Integration of these powerful techniques allows us to quantify, diagnose, model, and understand cancer pathobiology. Here, we describe detailed materials and methods used to establish this integrated interrogation platform, from generating transduced cancer cell lines that stably express CaViar (GCaMP5G + QuasAr2) to in vitro and ex vivo calcium imaging of the cells in 2D/3D hydrogels and tumor tissues. These tools open the possibility for detailed explorations of mechano-electro-chemical network dynamics in living systems.

Screening of Drosophila microRNA-degradation sequences reveals Argonaute1 mRNA's role in regulating miR-999.

MicroRNAs (miRNA) load onto AGO proteins to target mRNAs for translational repression or degradation. However, miRNA degradation can be triggered when extensively base-paired with target RNAs, which induces confirmational change of AGO and recruitment of ZSWIM8 ubiquitin ligase to mark AGO for proteasomal degradation. This target RNA-directed miRNA degradation (TDMD) mechanism appears to be evolutionarily conserved, but recent studies have focused on mammalian systems. Here, we performed AGO1-CLASH in Drosophila S2 cells, with Dora (ortholog of vertebrate ZSWIM8) knockout mediated by CRISPR-Cas9 to identify five TDMD triggers (sequences that can induce miRNA degradation). Interestingly, one trigger in the 3' UTR of AGO1 mRNA induces miR-999 degradation. CRISPR-Cas9 knockout of the AGO1 trigger in S2 cells and in Drosophila specifically elevates miR-999, with concurrent repression of the miR-999 targets. AGO1 trigger knockout flies respond poorly to hydrogen peroxide-induced stress, demonstrating the physiological importance of this TDMD event.

A regulatory loop establishes the link between the circadian clock and abscisic acid signaling in rice.

There is a close regulatory relationship between the circadian clock and the abscisic acid (ABA) signaling pathway in regulating many developmental processes and stress responses. However, the exact feedback regulation mechanism between them is still poorly understood. Here, we identified the rice (Oryza sativa) clock component PSEUDO-RESPONSE REGULATOR 95 (OsPRR95) as a transcriptional regulator that accelerates seed germination and seedling growth by inhibiting ABA signaling. We also found that OsPRR95 binds to the ABA receptor gene REGULATORY COMPONENTS OF ABA RECEPTORS10 (OsRCAR10) DNA and inhibits its expression. Genetic analysis showed OsRCAR10 acts downstream of OsPRR95 in mediating ABA responses. In addition, the induction of OsPRR95 by ABA partly required a functional OsRCAR10, and the ABA-responsive element-binding factor ABSCISIC ACID INSENSITIVE5 (OsABI5) bound directly to the promoter of OsPRR95 and activated its expression, thus establishing a regulatory feedback loop between OsPRR95, OsRCAR10, and OsABI5. Taken together, our results demonstrated that the OsRCAR10-OsABI5-OsPRR95 feedback loop modulates ABA signaling to fine-tune seed germination and seedling growth, thus establishing the molecular link between ABA signaling and the circadian clock.

Sequencing of Argonaute-bound microRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a.

MicroRNAs (miRNA) are short non-coding RNAs widely implicated in gene regulation. Most metazoan miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis does not require Drosha. The functional importance of the TSS-miRNA biogenesis is uncertain. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (AGO-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without DROSHA knockout. We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by AGO-qCLASH. Targets for miR-320a are enriched for the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and antagomir- transfected cells, differentially expressed gene products are associated with eIF2 signaling. Within the AGO-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone calnexin as a direct miR-320a down-regulated target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. In summary, our study investigates the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.

Widespread microRNA degradation elements in target mRNAs can assist the encoded proteins.

Binding of microRNAs (miRNAs) to mRNAs normally results in post-transcriptional repression of gene expression. However, extensive base-pairing between miRNAs and target RNAs can trigger miRNA degradation, a phenomenon called target RNA-directed miRNA degradation (TDMD). Here, we systematically analyzed Argonaute-CLASH (cross-linking, ligation, and sequencing of miRNA-target RNA hybrids) data and identified numerous candidate TDMD triggers, focusing on their ability to induce nontemplated nucleotide addition at the miRNA 3' end. When exogenously expressed in various cell lines, eight triggers induce degradation of corresponding miRNAs. Both the TDMD base-pairing and surrounding sequences are essential for TDMD. CRISPR knockout of endogenous trigger or ZSWIM8, a ubiquitin ligase essential for TDMD, reduced miRNA degradation. Furthermore, degradation of miR-221 and miR-222 by a trigger in BCL2L11, which encodes a proapoptotic protein, enhances apoptosis. Therefore, we uncovered widespread TDMD triggers in target RNAs and demonstrated an example that could functionally cooperate with the encoded protein.

OsRE1 interacts with OsRIP1 to regulate rice heading date by finely modulating Ehd1 expression.

Heading date is a key agronomic trait affecting crop yield. In rice, Early heading date 1 (Ehd1) is an important B-type response regulator in determination of heading date. Although many regulatory factors of Ehd1 expression have been functionally characterized, the direct regulators of Ehd1 largely remain to be identified. Here, we identified a new regulator of Ehd1, OsRE1, that directly binds to the A-box motif in the Ehd1 promoter. Osre1 confers an early heading phenotype due to elevated expression levels of Ehd1. OsRE1 is a nucleus-localized bZIP transcription factor with a diurnal rhythmic expression pattern. Furthermore, we identified an OsRE1-interacting protein, OsRIP1, and demonstrated that OsRIP1 can repress the transcript expression of Ehd1 in an OsRE1-dependent manner. Our genetic data showed that OsRE1 and OsRIP1 may function upstream of Ehd1 in regulating heading date. Together, our results suggest that OsRE1 functions cooperatively with OsRIP1 to regulate heading date through finely modulating the expression of Ehd1. In addition, OsRE1 and OsRIP1 are two minor heading date regulators, which are more desirable for fine-tuning heading date to improve rice regional adaptability.

Short Hairpin RNAs for Strand-Specific Small Interfering RNA Production.

RNA interference (RNAi) is an effective mechanism for inhibiting gene expression at the post-transcriptional level. Expression of a messenger RNA (mRNA) can be inhibited by a ∼22-nucleotide (nt) small interfering (si)RNA with the corresponding reverse complementary sequence. Typically, a duplex of siRNA, composed of the desired siRNA and a passenger strand, is processed from a short hairpin RNA (shRNA) precursor by Dicer. Subsequently, one strand of the siRNA duplex is associated with Argonaute (Ago) protein for RNAi. Although RNAi is widely used, the off-target effect induced by the passenger strand remains a potential problem. Here, based on current understanding of endogenous precursor microRNA (pre-miRNA) hairpins, called Ago-shRNA and m7G-capped pre-miRNA, we discuss the principles of shRNA designs that produce a single siRNA from one strand of the hairpin.

Catalytically inactive Dnmt3b rescues mouse embryonic development by accessory and repressive functions.

DNA methylation regulates gene expression in a variety of processes, including mouse embryonic development. Four catalytically active enzymes function in mice as DNA methyltransferases (Dnmts) and as transcriptional regulators. Inactivation of Dnmt3b results in mouse embryonic lethality, but which activities are involved is unclear. Here we show that catalytically inactive Dnmt3b restores a majority of methylation and expression changes deregulated in the absence of Dnmt3b, and as a result, mice survive embryonic development. Thus, Dnmt3b functions as an accessory cofactor supporting catalytic activities performed by other Dnmts. We further demonstrate that Dnmt3b is linked to a control of major developmental pathways, including Wnt and hedgehog signaling. Dnmt3b directly represses Wnt9b whose aberrant up-regulation contributes to embryonic lethality of Dnmt3b knockout embryos. Our results highlight that Dnmt3b is a multifaceted protein that serves as an enzyme, an accessory factor for other methyltransferases, and as a transcriptional repressor in mouse embryogenesis.

Northern Blot with IR Fluorescent Probes: Strategies for Probe Preparation.

Northern blot is a molecular biology technique that can detect, quantify, and determine the molecular weight of RNA. Recently, we published a protocol utilizing near-infrared (IR) fluorescent probes in Northern blot (irNorthern). Our method is as sensitive as other non-radioactive methods but is more straightforward and versatile. Additionally, we found that IR-labeled probes can be used to multiplex or detect different species of RNA at the same time. Here we describe three methods for generating an IR-labeled probe as well as how to perform irNorthern blot. In conclusion, our irNorthern protocol offers a convenient method for RNA detection.

DELAYED HEADING DATE1 interacts with OsHAP5C/D, delays flowering time and enhances yield in rice.

Heading date is an important agronomic trait affecting crop yield. The GRAS protein family is a plant-specific super family extensively involved in plant growth and signal transduction. However, GRAS proteins are rarely reported have a role in regulating rice heading date. Here, we report a GRAS protein DHD1 (Delayed Heading Date1) delays heading and enhances yield in rice. Biochemical assays showed DHD1 physically interacts with OsHAP5C/D both in vitro and in vivo. DHD1 and OsHAP5C/D located in the nucleus and showed that rhythmic expression. Both DHD1 and OsHAP5C/D affect heading date by regulating expression of Ehd1. We propose that DHD1 interacts with OsHAP5C/D to delay heading date by inhibiting expression of Ehd1.

Near-infrared fluorescent northern blot.

Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive 32P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than 32P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.

Dicer cleaves 5'-extended microRNA precursors originating from RNA polymerase II transcription start sites.

MicroRNAs (miRNAs) are approximately 22 nucleotide (nt) long and play important roles in post-transcriptional regulation in both plants and animals. In animals, precursor (pre-) miRNAs are ∼70 nt hairpins produced by Drosha cleavage of long primary (pri-) miRNAs in the nucleus. Exportin-5 (XPO5) transports pre-miRNAs into the cytoplasm for Dicer processing. Alternatively, pre-miRNAs containing a 5' 7-methylguanine (m7G-) cap can be generated independently of Drosha and XPO5. Here we identify a class of m7G-capped pre-miRNAs with 5' extensions up to 39 nt long. The 5'-extended pre-miRNAs are transported by Exportin-1 (XPO1). Unexpectedly, a long 5' extension does not block Dicer processing. Rather, Dicer directly cleaves 5'-extended pre-miRNAs by recognizing its 3' end to produce mature 3p miRNA and extended 5p miRNA both in vivo and in vitro. The recognition of 5'-extended pre-miRNAs by the Dicer Platform-PAZ-Connector (PPC) domain can be traced back to ancestral animal Dicers, suggesting that this previously unrecognized Dicer reaction mode is evolutionarily conserved. Our work reveals additional genetic sources for small regulatory RNAs and substantiates Dicer's essential role in RNAi-based gene regulation.

The OsHAPL1-DTH8-Hd1 complex functions as the transcription regulator to repress heading date in rice.

Heading date is an important agronomic trait related to crop yield. Many genes related to heading date have already been identified in rice (Oryza sativa), and a complicated, preliminary regulatory genetic network has also already been established, but the protein regulatory network is poorly understood. We have identified a novel heading date regulator, Heme Activator Protein like 1 (OsHAPL1), which inhibits flowering under long-day conditions. OsHAPL1 is a nuclear-localized protein that is highly expressed in leaves in a rhythmic manner. OsHAPL1 can physically interact with Days To Heading on chromosome 8 (DTH8), which physically interacts with Heading date 1 (Hd1) both in vitro and in vivo. OsHAPL1 forms a complex with DTH8 and Hd1 in Escherichia coli. OsHAPL1, DTH8, and Hd1 physically interact with the HAP complex, and also with general transcription factors in yeast (Saccharomyces cerevisiae). Further studies showed that OsHAPL1 represses the expression of the florigen genes and FLOWERING LOCUS T 1 (RFT1) and Hd3a through Early heading date 1 (Ehd1). We propose that OsHAPL1 functions as a transcriptional regulator and, together with DTH8, Hd1, the HAP complex, and general transcription factors, regulates the expression of target genes and then affects heading date by influencing the expression of Hd3a and RFT1 through Ehd1.

The LBD12-1 Transcription Factor Suppresses Apical Meristem Size by Repressing Argonaute 10 Expression.

The shoot apical meristem (SAM) consists of a population of multipotent cells that generates all aerial structures and regenerates itself. SAM maintenance and lateral organ development are regulated by several complex signaling pathways, in which the Argonaute gene-mediated pathway plays a key role. One Argonaute gene, AGO10, functions as a microRNA locker that attenuates miR165/166 activity and positively regulates shoot apical meristem development, but little is known about when and how AGO10 is regulated at the transcriptional level. In this work, we showed that transgenic rice plants overexpressing LBD12-1, an LBD family transcription factor, exhibited stunted growth, twisted leaves, abnormal anthers, and reduced SAM size. Further research revealed that LBD12-1 directly binds to the promoter region and represses the expression of AGO10. Overexpression of AGO10 in an LBD12-1 overexpression background rescued the growth defect phenotype of LBD12-1-overexpressing plants. The expression of LBD12-1 and its binding ability to the AGO10 promoter is induced by stress. lbd12-1 loss-of-function mutants showed similar phenotypes and SAM size to the wild type under normal conditions, but lbd12-1 had a larger SAM under salt stress. Our findings provide novel insights into the regulatory mechanism of AGO10 by which SAM size is controlled under stress conditions.

Overexpression of OsMYB1R1-VP64 fusion protein increases grain yield in rice by delaying flowering time.

VP64 is widely used as a transcriptional activator to investigate the biological function of genes, but its potential for application in genetic improvement of crops has not been fully investigated. Here, we characterized an OsMYB1R1-VP64 fusion protein that enhanced the grain yield of rice cultivar 'Kita-ake' by 35%. OsMYB1R1-VP64 regulated grain yield of transgenic plants mainly by extending the vegetative growth stage. Further analysis indicated that OsMYB1R1-VP64 delayed flowering by down-regulating expression of Heading date 3a (Hd3a) and RICE FLOWERING LOCUST 1 (RFT1) through repression of Early heading date 1 (Ehd1). The expression pattern of OsMYB1R1 was constitutive and rhythmically controlled. OsMYB1R1 protein had transcriptional activation activity and was localized to the nucleus. Our findings suggest that the OsMYB1R1-VP64 construct can be used to increase grain yield in rice.

A CONSTANS-like transcriptional activator, OsCOL13, functions as a negative regulator of flowering downstream of OsphyB and upstream of Ehd1 in rice.

Flowering time determines the adaptability of crop plants to different local environments, thus being one of the most important agronomic traits targeted in breeding programs. Photoperiod is one of the key factors that control flowering in plant. A number of genes that participate in the photoperiod pathway have been characterized in long-day plants such as Arabidopsis, as well as in short-day plants such as Oryza sativa. Of those, CONSTANS (CO) as a floral integrator promotes flowering in Arabidopsis under long day conditions. In rice, Heading date1 (Hd1), a homologue of CO, functions in an opposite way, which inhibits flowering under long day conditions and induces flowering under short day conditions. Here, we show that another CONSTANS-like (COL) gene, OsCOL13, negatively regulates flowering in rice under both long and short day conditions. Overexpression of OsCOL13 delays flowering regardless of day length. We also demonstrated that OsCOL13 has a constitutive and rhythmic expression pattern, and that OsCOL13 is localized to the nucleus. OsCOL13 displays transcriptional activation activity in the yeast assays and likely forms homodimers in vivo. OsCOL13 suppresses the florigen genes Hd3a and RFT1 by repressing Ehd1, but has no relationship with other known Ehd1 regulators as determined by using mutants or near isogenic lines. In addition, the transcriptional level of OsCOL13 significantly decreased in the osphyb mutant, but remained unchanged in the osphya and osphyc mutants. Thus, we conclude that OsCOL13 functions as a negative regulator downstream of OsphyB and upstream of Ehd1 in the photoperiodic flowering in rice.

OsCOL10, a CONSTANS-Like Gene, Functions as a Flowering Time Repressor Downstream of Ghd7 in Rice.

Flowering time, or heading date, is a critical agronomic trait that determines the cropping season and regional adaptability, and ultimately grain yield in rice. A number of genes involved in photoperiodic flowering have been cloned and their roles in modulating expression of the flowering genes have been characterized to a certain extent. However, much less is known about the pathway in transmitting the day length response signal(s) to induce transition to reproductive growth. Here, we report a constitutive flowering repressor OsCOL10, which encodes a member of the CONSTANS-like (COL) family. Transgenic rice plants overexpressing OsCOL10 (driven by a strong promoter or by fusing it to the activation domain of VP64) showed delayed flowering time under both short and long days.OsCOL10 is affected by the circadian clock and is preferentially expressed in leaf mesophyll cells; it is localized to the nucleus and has transcriptional activation activity. Further studies show that OsCOL10 represses the expression of theFT-like genes RFT1 and Hd3a through Ehd1. Transcripts of OsCOL10 are more abundant in plants carrying a functional Ghd7 allele or overexpressing Ghd7 than in Ghd7-deficient plants, thus placing OsCOL10 downstream of Ghd7.Taking these findings together, we conclude that OsCOL10 functions as a flowering time repressor that links Ghd7 and Ehd1 in rice.

The role of OsMSH4 in male and female gamete development in rice meiosis.

Meiosis is essential for gametogenesis in sexual reproduction in rice (Oryza sativa L.). We identified a MutS-homolog (MSH) family gene OsMSH4 in a trisomic plant. Cytological analysis showed that developments of both pollen and embryo sacs in an Osmsh4 mutant were blocked due to defective chromosome pairing. Compared with the wild type, the Osmsh4 mutant displayed a significant ~21.9% reduction in chiasma frequency, which followed a Poisson distribution, suggesting that class I crossover formation in the mutant was impaired. Temporal and spatial expression pattern analyses showed that OsMSH4 was preferentially expressed in meiocytes during their meiosis, indicating a critical role in gametogenesis. Subcellular localization showed that OsMSH4-green fluorescent protein was predominantly located in the nucleus. OsMSH4 could interact with another MSH member (OsMSH5) through the N-terminus and C-terminus, respectively. Direct physical interaction between OsMSH5, OsRPA1a, OsRPA2b, OsRPA1c, and OsRPA2c was identified by yeast two-hybrid assays and further validated by pull-down assays. Our results supported the conclusion that the OsMSH4/5 heterodimer plays a key role in regulation of crossover formation during rice meiosis by interaction with the RPA complex.

VLN2 Regulates Plant Architecture by Affecting Microfilament Dynamics and Polar Auxin Transport in Rice.

As a fundamental and dynamic cytoskeleton network, microfilaments (MFs) are regulated by diverse actin binding proteins (ABPs). Villins are one type of ABPs belonging to the villin/gelsolin superfamily, and their function is poorly understood in monocotyledonous plants. Here, we report the isolation and characterization of a rice (Oryza sativa) mutant defective in VILLIN2 (VLN2), which exhibits malformed organs, including twisted roots and shoots at the seedling stage. Cellular examination revealed that the twisted phenotype of the vln2 mutant is mainly caused by asymmetrical expansion of cells on the opposite sides of an organ. VLN2 is preferentially expressed in growing tissues, consistent with a role in regulating cell expansion in developing organs. Biochemically, VLN2 exhibits conserved actin filament bundling, severing and capping activities in vitro, with bundling and stabilizing activity being confirmed in vivo. In line with these findings, the vln2 mutant plants exhibit a more dynamic actin cytoskeleton network than the wild type. We show that vln2 mutant plants exhibit a hypersensitive gravitropic response, faster recycling of PIN2 (an auxin efflux carrier), and altered auxin distribution. Together, our results demonstrate that VLN2 plays an important role in regulating plant architecture by modulating MF dynamics, recycling of PIN2, and polar auxin transport.