Traffic into silence: endomembranes and post-transcriptional RNA silencing.

microRNAs (miRNAs) and small interfering RNAs (siRNAs) are small RNAs that repress gene expression at the post-transcriptional level in plants and animals. Small RNAs guide Argonaute-containing RNA-induced silencing complexes to target RNAs in a sequence-specific manner, resulting in mRNA deadenylation followed by exonucleolytic decay, mRNA endonucleolytic cleavage, or translational inhibition. Although our knowledge of small RNA biogenesis, turnover, and mechanisms of action has dramatically expanded in the past decade, the subcellular location of small RNA-mediated RNA silencing still needs to be defined. In contrast to the prevalent presumption that RNA silencing occurs in the cytosol, emerging evidence reveals connections between the endomembrane system and small RNA activities in plants and animals. Here, we summarize the work that uncovered this link between small RNAs and endomembrane compartments and present an overview of the involvement of the endomembrane system in various aspects of RNA silencing. We propose that the endomembrane system is an integral component of RNA silencing that has been long overlooked and predict that a marriage between cell biology and RNA biology holds the key to a full understanding of post-transcriptional gene regulation by small RNAs

Single-Molecule Imaging Reveals that Argonaute Reshapes the Binding Properties of Its Nucleic Acid Guides

SummaryArgonaute proteins repress gene expression and defend against foreign nucleic acids using short RNAs or DNAs to specify the correct target RNA or DNA sequence. We have developed single-molecule methods to analyze target binding and cleavage mediated by the Argonaute:guide complex, RISC. We find that both eukaryotic and prokaryotic Argonaute proteins reshape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridization—a small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Argonautes distinguish substrates from targets with similar complementarity. Mouse AGO2, for example, binds tighter to miRNA targets than its RNAi cleavage product, even though the cleaved product contains more base pairs. By re-writing the rules for nucleic acid hybridization, Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than of RNA or DNA

Dissecting Small RNA Loading Pathway in \u3cem\u3eDrosophila melanogaster\u3c/em\u3e: A Dissertation

In the preceding chapters, I have discussed my doctoral research on studying the siRNA loading pathway in Drosophila using both biochemical and genetic approaches. We established a gel shift system to identify the intermediate complexes formed during siRNA loading. We detected at least three complexes, named complex B, RISC loading complex (RLC) and RISC. Using kinetic modeling, we determined that the siRNA enters complex B and RLC early during assembly when it remains double-stranded, and then matures in RISC to generate Argonaute bearing only the single-stranded guide. We further characterized the three complexes. We showed that complex B comprises Dcr-1 and Loqs, while both RLC and RISC contain Dcr-2 and R2D2. Our study suggests that the Dcr-2/R2D2 heterodimer plays a central role in RISC assembly. We observed that Dcr-1/Loqs, which function together to process pre-miRNA into mature miRNA, were also involved in siRNA loading. This was surprising, because it has been proposed that the RNAi pathway and miRNA pathway are separate and parallel, with each using a unique set of proteins to produce small RNAs, to assemble functional RNA-guided enzyme complexes, and to regulate target mRNAs. We further examined the molecular function of Dcr-1/Loqs in RNAi pathway. Our data suggest that, in vivo and in vitro, the Dcr-1/Loqs complex binds to siRNA. In vitro, the binding of the Dcr-1/Loqs complex to siRNA is the earliest detectable step in siRNA-triggered Ago2-RISC assembly. Futhermore, the binding of Dcr-1/Loqs to siRNA appears to facilitate dsRNA dicing by Dcr-2/R2D2, because the dicing activity is much lower in loqslysate than in wild type. Long inverted repeat (IR) triggered white silencing in fly eyes is an example of endogenous RNAi. Consistent with our finding that Dcr-1/Loqs function to load siRNA, less white siRNA accumulates in loqs mutant eyes compared to wild type. As a result, loqs mutants are partially defective in IR trigged whitesilencing. Our data suggest considerable functional and genetic overlap between the miRNA and siRNA pathways, with the two sharing key components previously thought to be confined to just one of the two pathways. Based on our study on siRNA loading pathway, we also elucidated the molecular function of Armitage (Armi) protein in RNAi. We showed that armi is required for RNAi. Lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes suggests that armi mutants support early steps in the RNAi pathway, i.e., the formation of complex B and RLC, but are defective in the production of the RISC

Artificially induced phased siRNAs promote virus resistance in transgenic plants

We previously developed transgenic tobacco plants that were resistant to two geminiviruses. We generated resistance using RNAi constructs that produced trans-acting siRNA (tasiRNA) like secondary siRNAs known as phased siRNA (phasiRNA) that targeted several regions of Tomato Leaf Curl New Delhi Virus (ToLCNDV) and Tomato Leaf Curl Gujarat Virus (ToLCGV) transcripts encoding the RNA silencing suppressor proteins AC2 and AC4. Here, we performed degradome analysis to determine the precise cleavage sites of RNA-RNA interaction between phasiRNA and viral transcripts. We then apply our RNAi technology in tomato, which is the natural host for ToLCNDV and ToLCGV. The relative ease of developing and using phasiRNA constructs represents a significant technical advance in imparting virus resistance in crops and/or important model systems

ROLES OF MICRORNAS IN PLANT ABIOTIC STRESS, DEVELOPMENT AND VIRAL INFECTION

Plant microRNAs play important roles in plant growth and development. Here we investigated the roles of miRNAs in the plant abiotic stress, development and viral infection. MicroRNA membrane array analysis using five different abiotic stress treatments resulted in the identification of 8 novel stress inducible miRNA-families. Functional studies on novel stress inducible miR168 revealed its functional relation with abiotic stress. Over expression of miR168 in Arabidopsis showed upregulation of four stress related miRNAs (miR163, miR167, miR398 and miR408). Analysis of 9 independent transgenic lines showed induction of miR398, an oxidative stress responsive miRNA with a corresponding down regulation of its target genes. Heavy metal oxidative stress tolerance bioassays confirmed the susceptibility of transgenics compared to the wild types indicating the fact that the miR168 is indirectly involved in plant abiotic stress by inducing other stress responsive miRNAs. MicroRNAs are highly conserved across the plant kingdom. A miRNA atlas was drafted for different tomato organs and fruit stages using the known miRNA sequences from different plants species. A large variation in both number and level of miRNA expression pattern was observed among different organs as well as among fruit stages. In the present investigation, we have found a window of expression for different miRNAs during the fruit development. A gradual decrease in the expression levels of miR160h, miR167a and miR399d and a gradual increase in miR164a have been noticed towards the fruit maturation while miR398b showed dual peaks during fruit development indicating a potential role of various miRNAs in fruit development and maturation. Sonchus yellow net virus (SYNV) infected Nicotinana benthamiana leaves showed severe disease symptoms at two weeks post infection (WPI) and gradually recovered from the SYNV infection after 4-5 WPI correlating with the overall miRNA levels. The miRNA array and northern analysis showed an overall reduction of miRNA biogenesis during 2WPI followed by restoration to normal levels supporting the idea that the SYNV indeed interfered with the host miRNA levels which caused the symptoms and recovery phenotypes. Overall studies on plant abiotic stress, development and viral infection showed important roles of miRNAs in different aspects of plant life

Biochemical Mechanism of RNA Interference in Higher Organisms: A Dissertation

RNA interference (RNAi) is an evolutionarily conserved, sequence-specific gene silencing pathway found in eukaryotes, in which 21-nucleotide, small interfering RNAs (siRNAs) guide destruction of a corresponding target mRNA. RNAi is a natural mechanism for both genome surveillance and gene regulation. Moreover, siRNAs can be transfected into cultured mammalian cells, causing the sequence-specific ‘knock down’ of an mRNA. My work in the Zamore lab has centered around the Drosophilain vitro system and cultured mammalian cells to study the RNA interference (RNAi) pathway. small interfering RNAs (siRNAs) are incorporated into the RNA-induced silencing complex (RISC), which culminates in the cleavage of a complementary target mRNA. Previous work proved that certain structural features of siRNAs are essential for RNAi in flies, including the requirement for 5´ phosphates and 3´ hydroxyl groups. In cultured mammalian cells, the requirement for a 5´ phosphate also holds true, but we found no evidence to support the necessity for 3´ hydroxyls in either system. In addition, siRNAs can act as single strands entering the pathway downstream of double-stranded siRNAs, both of which are competent in directing the cleavage of its cognate mRNA at a single site. While these key features are a requirement for functional siRNAs, alone they do not determine the efficiency to which an siRNA can enter the RISC. In fact, both strands of an siRNA can enter RISC to a different degree as determined by the stabilities of the 5´ ends of the siRNA strand, a phenomenon termed ‘functional asymmetry’. This characteristic is also reflected in another class of small RNAs involved in gene silencing known as microRNAs (miRNAs), which are processed from long hairpin RNA structures into mature, single-stranded non-coding RNAs. The asymmetric loading of siRNAs suggests that miRNAs are initially generated from siRNA-like duplexes cleaved from the stem of the hairpins. The strand whose 5´ end is less tightly paired will be processed into the mature miRNA, while the other strand is destroyed. By applying the rules of siRNA asymmetry it is possible to predict which side of the stem will be processed into the mature miRNA, a finding verified experimentally by our lab and others. This discovery also has additional implications in designing highly effective siRNAs and in reducing siRNA off-target effects. We used these results to design siRNAs that target the single nucleotide polymorphism in superoxide dismutase that causes the familial form of amyotrophic lateral sclerosis (ALS), but leave the wild-type mRNA intact and functional. Our experiments have helped define the ‘rules’ for creating SNP-specific siRNAs. In particular, we found that only siRNAs with a purine:purine mismatch to the allele not intended for destruction show good discrimination. The placement of the mismatch in a tiled set of siRNAs shows that mismatches located in the 5´ region of the siRNA, a region shown to be responsible for siRNA binding, can not discriminate between alleles. In contrast, mismatches in the 3´ region of the siRNA, the region contributing to catalysis, discriminate between wild-type and mutant alleles. This work is an important step in creating allele-specific siRNAs as therapeutics for dominant negative genetic diseases. But how does RISC cleave its target? By isolating both the 5´ and 3´ cleavage products produced by RISC in the Drosophila in vitro system, we discovered that RISC acts as a Mg2+-dependent endonuclease that cleaves a single phosphodiester bond in the mRNA target, leaving 5´ phosphate and 3´ hydroxyl groups. These findings were a critical step in the demonstration that Argonaute, a protein known to be a component of RISC, is the RNAi endonuclease

Genome-Wide Approaches To Study Rna Secondary Structure

The central hypothesis of molecular biology depicts RNA as an intermediary conveyor of genetic information. RNA is transcribed from DNA and translated to proteins, the molecular machines of the cell. However, many RNAs do not encode protein and instead function as molecular machines themselves. The most famous examples are ribosomal RNAs and transfer RNAs, which together form the core translational machinery of the cell. Many other non-coding RNAs have been discovered including catalytic and regulatory RNAs. In many cases RNA function is tightly linked to its secondary structure, which is the collection of hydrogen bonds between complimentary RNA sequences that drives these molecules into their three dimensional structure. Over the last decade, technology for determining the sequence of DNA and RNA has advanced rapidly, making transcriptome-wide expression profiling fast and widely available. In this dissertation, I discuss recent efforts to leverage this powerful technology to study, not just RNA expression, but several other aspects of RNA function. In particular, I focus on three tightly linked aspects of RNA biology: RNA-secondary structure, RNA cleavage, and regulatory small RNAs. I introduce a database for integrating, comparing, and contrasting techniques for determining RNA secondary structure including a technique developed in my dissertation laboratory. Additionally, I discuss a newly improved technology capable of detecting RNA cleavage events. Finally, I integrate RNA secondary structure probing and RNA cleavage detection to interrogate a family of genes important for eukaryotic small RNA-mediated silencing. These diverse analyses are just a few examples of the vast promises offered by adapting RNA-sequencing technology to probe RNA function across many cellular processes

Role of Hormonal and Developmental Signaling in Plant-Cyst Nematode Interaction

Plant-parasitic cyst nematodes are one of the most destructive root parasites that cause severe yield losses in many crop plants. These obligate parasites induce a specialized multi-nucleate feeding site called syncytium. This study was conducted to explore the roles of phytohormones particularly auxin and ethylene, and miRNA-mediated crosstalk between development and defense responses in establishing the compatible interaction between Arabidopsis and Heterodera schachtii. Using yeast two-hybrid assay, a complete protein-protein interaction map between Auxin/indole-3-acetic acid (Aux/IAA) proteins and auxin response factors (ARFs) was generated. In addition, gene co-expression profiles of ARFs and Aux/IAAs were incorporated with protein-protein interaction data. Together, these analyses revealed the biological significance of the ARFs and Aux/IAA interactions in the differentiation and development of various plant tissues and organs, including H. schachtii–induced syncytium. Our analyses also provided evidence for the roles of ETHYLENE RESPONSE 1 and ETHYLENE INSENSITIVE 3/ETHYLENE INSENSITIVE LIKE 1 in regulating Arabidopsis responses to H. schachtii infection. The role of miRNAs in mediating the coordination between developmental signaling and defense response is emerging. Functional characterization of miR858 and its MYB83 target gene pointed into novel cooperative regulatory functions of this regulatory module in syncytium transcriptome reprogramming during cyst nematode parasitism of Arabidopsis. We discovered that miR858-mediated silencing of MYB83 is tightly regulated through a feedback loop that may ensure proper expression levels of more than a thousand of MYB83-regulated genes in the syncytium. Finally, the direct targets of Growth Regulating Factor 1 (GRF1) and 3, master regulators of syncytium differentiation, were identified. Specific and the shared cis-binding elements of GRF1 and GRF3 were identified, providing unprecedented understanding of the mechanism of their functional redundancy. GRF1 and GRF3 directly target genes associated with cell cycle regulation, cytoskeleton organization, phytohormone biosynthesis and signaling, and defense responses, key cellular processes that determine the outcomes of plant-cyst nematode interactions. The analysis also provided intriguing evidence for the involvement of GRF1/3 in mediating the trade-off between plant growth and stress signaling. Understanding the molecular mechanisms underlying the coordinated interactions between plant growth and defense signaling will open new avenues for enhancing plant growth and stress response simultaneously

The role of REVOLUTA and KANADI1 in plant development and environmental responses

In plants, the establishment of organ patterning and polarity is mediated by the action of several transcription factors. Among them, KANADIs and HD-ZIPIIIs act antagonistically by which they play crucial roles in organ polarity. Using a combination of chromatin immunoprecipitation (ChIP-Seq) approach and tiling arrays, we could identify a set of potential direct target genes of KAN1. Further analysis showed that a number of KAN1 targets appear to regulate organ patterning or response to auxin. In addition, KAN1 shares a set of common direct targets with REV, implying that the REV/KAN1 module acts in organ patterning through opposite regulation of shared targets. As sessile organisms, plants have to continuously adjust growth and development to changing environmental conditions. During the final stage of growth, plants induce leaf senescence to reallocate nutrients and energy-rich substances from mature leaves to reproductive seeds, leading to increased reproductive success. Therefore leaf senescence is tightly coupled to the developmental age of the plant. In this study, we show that class III HD-ZIP transcription factors have an additional role in controlling the onset of leaf senescence in Arabidopsis. We report that acting as a redox-sensitive transcription factor, REV directly and positively regulates the expression of WRKY53, a senescence-related transcription factor. REV is required for the induction of WRKY53 in response to oxidative stress, and reducing the activity of HD-ZIP III genes strongly delays the onset of leaf senescence. Besides WRKY53, we also identified nine direct REV targets which are differentially expressed during senescence. Thus, a crosstalk between early and late stages of leaf development appears to contribute to reproductive success