Taiowa A. Montgomery

Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA Affects Developmental Timing and Patterning in Arabidopsis

MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) in plants form through distinct pathways, although they function as negative regulators of mRNA targets by similar mechanisms . Three ta-siRNA gene families (TAS1, TAS2, and TAS3) are known in Arabidopsis thaliana. Biogenesis of TAS3 ta-siRNAs, which target mRNAs encoding several AUXIN RESPONSE FACTORs (including ARF3/ETTIN and ARF4 ) involves miR390-guided processing of primary transcripts, conversion of a precursor to dsRNA through RNA-DEPENDENT RNA POLYMERASE6 (RDR6) activity, and sequential DICER-LIKE4 (DCL4)-mediated cleavage events. We show that the juvenile-to-adult phase transition is normally suppressed by TAS3 ta-siRNAs, in an ARGONAUTE7-dependent manner, through negative regulation of ARF3 mRNA. Expression of a nontargeted ARF3 mutant (ARF3mut) in a wild-type background reproduced the phase-change phenotypes detected in rdr6-15 and dcl4-2 mutants, which lose all ta-siRNAs. Expression of either ARF3 or ARF3mut in rdr6-15 plants, in which both endogenous and transgenic copies of ARF3 were derepressed, resulted in further acceleration of phase change and severe morphological and patterning defects of leaves and floral organs. In light of the functions of ARF3 and ARF4 in organ asymmetry, these data reveal multiple roles for TAS3 ta-siRNA-mediated regulation of ARF genes in developmental timing and patterning.

Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis

RNA interference pathways can involve amplification of secondary siRNAs by RNA-dependent RNA polymerases. In plants, RDR6-dependent secondary siRNAs arise from transcripts targeted by some microRNAs (miRNAs). Here, Arabidopsis thaliana secondary siRNAs from mRNA as well as trans-acting siRNAs are shown to be triggered through initial targeting by a 22-nucleotide (nt) miRNA that associates with AGO1. In contrast to canonical 21-nt miRNAs, 22-nt miRNAs primarily arise from foldback precursors containing asymmetric bulges. Using artificial miRNA constructs, conversion of asymmetric foldbacks to symmetric foldbacks resulted in the production of 21-nt forms of miR173, miR472 and miR828. Both 21- and 22-nt forms associated with AGO1 and guided accurate slicer activity, but only 22-nt forms were competent to trigger RDR6-dependent siRNA production from target RNA. These data suggest that AGO1 functions differentially with 21- and 22-nt miRNAs to engage the RDR6-associated amplification apparatus.

Pattern formation via small RNA mobility

MicroRNAs and trans-acting siRNAs (ta-siRNAs) have important regulatory roles in development. Unlike other developmentally important regulatory molecules, small RNAs are not known to act as mobile signals during development. Here, we show that low-abundant, conserved ta-siRNAs, termed tasiR-ARFs, move intercellularly from their defined source of biogenesis on the upper (adaxial) side of leaves to the lower (abaxial) side to create a gradient of small RNAs that patterns the abaxial determinant AUXIN RESPONSE FACTOR3. Our observations have important ramifications for the function of small RNAs and suggest they can serve as mobile, instructive signals during development.

AGO1-miR173 complex initiates phased siRNA formation in plants

MicroRNA (miRNA)-guided cleavage initiates entry of primary transcripts into the transacting siRNA (tasiRNA) biogenesis pathway involving RNA-DEPENDENT RNA POLYMERASE6, DICER-LIKE4, and SUPPRESSOR OF GENE SILENCING3. Arabidopsis thaliana TAS1 and TAS2 families yield tasiRNA that form through miR173-guided initiation–cleavage of primary transcripts and target several transcripts encoding pentatricopeptide repeat proteins and proteins of unknown function. Here, the TAS1c locus was modified to produce synthetic (syn) tasiRNA to target an endogenous transcript encoding PHYTOENE DESATURASE and used to analyze the role of miR173 in routing of transcripts through the tasiRNA pathway. miR173 was unique from other miRNAs in its ability to initiate TAS1c-based syn-tasiRNA formation. A single miR173 target site was sufficient to route non-TAS transcripts into the pathway to yield phased siRNA. We also show that miR173 functions in association with ARGONAUTE 1 (AGO1) during TAS1 and TAS2 tasiRNA formation, and we provide data indicating that the miR173–AGO1 complex possesses unique functionality that many other miRNA–AGO1 complexes lack.

Functional Analysis of Three Arabidopsis ARGONAUTES Using Slicer-Defective Mutants

Wild-type and active-site defective forms of several Arabidopsis thaliana ARGONAUTE proteins involved in posttranscriptional silencing were used to examine several ARGONAUTE functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary siRNA formation, and antiviral activity. In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.

Computational and analytical framework for small RNA profiling by high-throughput sequencing

The advent of high-throughput sequencing (HTS) methods has enabled direct approaches to quantitatively profile small RNA populations. However, these methods have been limited by several factors, including representational artifacts and lack of established statistical methods of analysis. Furthermore, massive HTS data sets present new problems related to data processing and mapping to a reference genome. Here, we show that cluster-based sequencing-by-synthesis technology is highly reproducible as a quantitative profiling tool for several classes of small RNA from Arabidopsis thaliana. We introduce the use of synthetic RNA oligoribonucleotide standards to facilitate objective normalization between HTS data sets, and adapt microarray-type methods for statistical analysis of multiple samples. These methods were tested successfully using mutants with small RNA biogenesis (miRNA-defective dcl1 mutant and siRNA-defective dcl2 dcl3 dcl4 triple mutant) or effector protein (ago1 mutant) deficiencies. Computational methods were also developed to rapidly and accurately parse, quantify, and map small RNA data.

Identification of MIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing

Transacting siRNA (tasiRNA) biogenesis in Arabidopsis is initiated by microRNA (miRNA) –guided cleavage of primary transcripts. In the case of TAS3 tasiRNA formation, ARGONAUTE7 (AGO7)–miR390 complexes interact with primary transcripts at two sites, resulting in recruitment of RNA-DEPENDENT RNA POLYMERASE6 for dsRNA biosynthesis. An extensive screen for Arabidopsis mutants with specific defects in TAS3 tasiRNA biogenesis or function was done. This yielded numerous ago7 mutants, one dcl4 mutant, and two mutants that accumulated low levels of miR390. A direct genome sequencing-based approach to both map and rapidly identify one of the latter mutant alleles was developed. This revealed a G-to-A point mutation (mir390a-1) that was calculated to stabilize a relatively nonpaired region near the base of the MIR390a foldback, resulting in misprocessing of the miR390/miR390* duplex and subsequent reduced TAS3 tasiRNA levels. Directed substitutions, as well as analysis of variation at paralogous miR390-generating loci (MIR390a and MIR390b), indicated that base pair properties and nucleotide identity within a region 4–6 bases below the miR390/miR390* duplex region contributed to the efficiency and accuracy of precursor processing.

PIWI associated siRNAs and piRNAs specifically require the Caenorhabditis elegans HEN1 ortholog henn-1.

Small RNAs—including piRNAs, miRNAs, and endogenous siRNAs—bind Argonaute proteins to form RNA silencing complexes that target coding genes, transposons, and aberrant RNAs. To assess the requirements for endogenous siRNA formation and activity in Caenorhabditis elegans, we developed a GFP-based sensor for the endogenous siRNA 22G siR-1, one of a set of abundant siRNAs processed from a precursor RNA mapping to the X chromosome, the X-cluster. Silencing of the sensor is also dependent on the partially complementary, unlinked 26G siR-O7 siRNA. We show that 26G siR-O7 acts in trans to initiate 22G siRNA formation from the X-cluster. The presence of several mispairs between 26G siR-O7 and the X-cluster mRNA, as well as mutagenesis of the siRNA sensor, indicates that siRNA target recognition is permissive to a degree of mispairing. From a candidate reverse genetic screen, we identified several factors required for 22G siR-1 activity, including the chromatin factors mes-4 and gfl-1, the Argonaute ergo-1, and the 3′ methyltransferase henn-1. Quantitative RT–PCR of small RNAs in a henn-1 mutant and deep sequencing of methylated small RNAs indicate that siRNAs and piRNAs that associate with PIWI clade Argonautes are methylated by HENN-1, while siRNAs and miRNAs that associate with non-PIWI clade Argonautes are not. Thus, PIWI-class Argonaute proteins are specifically adapted to associate with methylated small RNAs in C. elegans.

mut-16 and other mutator class genes modulate 22G and 26G siRNA pathways in Caenorhabditis elegans

Argonaute-associated siRNAs and Piwi-associated piRNAs have overlapping roles in silencing mobile genetic elements in animals. In Caenorhabditis elegans, mutator (mut) class genes mediate siRNA-guided repression of transposons as well as exogenous RNAi, but their roles in endogenous RNA silencing pathways are not well-understood. To characterize the endogenous small RNAs dependent on mut class genes, small RNA populations from a null allele of mut-16 as well as a regulatory mut-16(mg461) allele that disables only somatic RNAi were subjected to deep sequencing. Additionally, each of the mut class genes was tested for a requirement in 26G siRNA pathways. The results indicate that mut-16 is an essential factor in multiple endogenous germline and somatic siRNA pathways involving several distinct Argonautes and RNA-dependent RNA polymerases. The results also reveal essential roles for mut-2 and mut-7 in the ERGO-1 class 26G siRNA pathway and less critical roles for mut-8, mut-14, and mut-15. We show that transposons are hypersusceptible to mut-16–dependent silencing and identify a requirement for the siRNA machinery in piRNA biogenesis from Tc1 transposons. We also show that the soma-specific mut-16(mg461) mutant allele is present in multiple C. elegans laboratory strains.

MUT-16 promotes formation of perinuclear Mutator foci required for RNA silencing in the C. elegans germline

RNA silencing can be initiated by endogenous or exogenously delivered siRNAs. In Caenorhabditis elegans, RNA silencing guided by primary siRNAs is inefficient and therefore requires an siRNA amplification step involving RNA-dependent RNA polymerases (RdRPs). Many factors involved in RNA silencing localize to protein- and RNA-rich nuclear pore-associated P granules in the germline, where they are thought to surveil mRNAs as they exit the nucleus. Mutator class genes are required for siRNA-mediated RNA silencing in both germline and somatic cells, but their specific roles and relationship to other siRNA factors are unclear. Here we show that each of the six mutator proteins localizes to punctate foci at the periphery of germline nuclei. The Mutator foci are adjacent to P granules but are not dependent on core P-granule components or other RNAi pathway factors for their formation or stability. The glutamine/asparagine (Q/N)-rich protein MUT-16 is specifically required for the formation of a protein complex containing the mutator proteins, and in its absence, Mutator foci fail to form at the nuclear periphery. The RdRP RRF-1 colocalizes with MUT-16 at Mutator foci, suggesting a role for Mutator foci in siRNA amplification. Furthermore, we demonstrate that genes that yield high levels of siRNAs, indicative of multiple rounds of siRNA amplification, are disproportionally affected in mut-16 mutants compared with genes that yield low levels of siRNAs. We propose that the mutator proteins and RRF-1 constitute an RNA processing compartment required for siRNA amplification and RNA silencing.