Allison C. Mallory

Criteria for Annotation of Plant MicroRNAs

MicroRNAs (miRNAs) are ∼21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs play critical roles in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interactions with specific target mRNAs. miRNAs are not the only Dicer-derived small RNAs produced by plants: A substantial amount of the total small RNA abundance and an overwhelming amount of small RNA sequence diversity is contributed by distinct classes of 21- to 24-nucleotide short interfering RNAs. This fact, coupled with the rapidly increasing rate of plant small RNA discovery, demands an increased rigor in miRNA annotations. Herein, we update the specific criteria required for the annotation of plant miRNAs, including experimental and computational data, as well as refinements to standard nomenclature.

Functions of microRNAs and related small RNAs in plants

MicroRNAs (miRNAs) and short interfering RNAs (siRNAs), 20- to 27-nt in length, are essential regulatory molecules that act as sequence-specific guides in several processes in most eukaryotes (with the notable exception of the yeast Saccharomyces cerevisiae). These processes include DNA elimination, heterochromatin assembly, mRNA cleavage and translational repression. This review focuses on the regulatory roles of plant miRNAs during development, in the adaptive response to stresses and in the miRNA pathway itself. This review also covers the regulatory roles of two classes of endogenous plant siRNAs, ta-siRNAs and nat-siRNAs, which participate in post-transcriptional control of gene expression.

MicroRNA-Directed Regulation of Arabidopsis AUXIN RESPONSE FACTOR17 Is Essential for Proper Development and Modulates Expression of Early Auxin Response Genes

The phytohormone auxin plays critical roles during plant growth, many of which are mediated by the auxin response transcription factor (ARF) family. MicroRNAs (miRNAs), endogenous 21-nucleotide riboregulators, target several mRNAs implicated in auxin responses. miR160 targets ARF10, ARF16, and ARF17, three of the 23 Arabidopsis thaliana ARF genes. Here, we describe roles of miR160-directed ARF17 posttranscriptional regulation. Plants expressing a miRNA-resistant version of ARF17 have increased ARF17 mRNA levels and altered accumulation of auxin-inducible GH3-like mRNAs, YDK1/GH3.2, GH3.3, GH3.5, and DFL1/GH3.6, which encode auxin-conjugating proteins. These expression changes correlate with dramatic developmental defects, including embryo and emerging leaf symmetry anomalies, leaf shape defects, premature inflorescence development, altered phyllotaxy along the stem, reduced petal size, abnormal stamens, sterility, and root growth defects. These defects demonstrate the importance of miR160-directed ARF17 regulation and implicate ARF17 as a regulator of GH3-like early auxin response genes. Many of these defects resemble phenotypes previously observed in plants expressing viral suppressors of RNA silencing and plants with mutations in genes important for miRNA biogenesis or function, providing a molecular rationale for phenotypes previously associated with more general disruptions of miRNA function.

MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5¿ region

MicroRNAs (miRNAs) are ∼22‐nucleotide noncoding RNAs that can regulate gene expression by directing mRNA degradation or inhibiting productive translation. Dominant mutations in PHABULOSA (PHB) and PHAVOLUTA (PHV) map to a miR165/166 complementary site and impair miRNA‐guided cleavage of these mRNAs in vitro. Here, we confirm that disrupted miRNA pairing, not changes in PHB protein sequence, causes the developmental defects in phb‐d mutants. In planta, disrupting miRNA pairing near the center of the miRNA complementary site had far milder developmental consequences than more distal mismatches. These differences correlated with differences in miRNA‐directed cleavage efficiency in vitro, where mismatch scanning revealed more tolerance for mismatches at the center and 3′ end of the miRNA compared to mismatches to the miRNA 5′ region. In this respect, miR165/166 resembles animal miRNAs in its pairing requirements. Pairing to the 5′ portion of the small silencing RNA appears crucial regardless of the mode of post‐transcriptional repression or whether it occurs in plants or animals, supporting a model in which this region of the silencing RNA nucleates pairing to its target.

MicroRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and floral organs.

BACKGROUND MicroRNAs (miRNAs) are approximately 21 nucleotide (nt) RNAs that regulate gene expression in plants and animals. Most known plant miRNAs target transcription factors that influence cell fate determination, and biological functions of miRNA-directed regulation have been reported for four of 15 known microRNA gene families: miR172, miR159, miR165, and miR168. Here, we identify a developmental role for miR164-directed regulation of NAC-domain genes, which encode a family of transcription factors that includes CUP-SHAPED COTYLEDON1 (CUC1) and CUC2. RESULTS Expression of a miR164-resistant version of CUC1 mRNA from the CUC1 promoter causes alterations in Arabidopsis embryonic, vegetative, and floral development, including cotyledon orientation defects, reduction of rosette leaf petioles, dramatically misshapen rosette leaves, one to four extra petals, and one or two missing sepals. Reciprocally, constitutive overexpression of miR164 recapitulates cuc1 cuc2 double mutant phenotypes, including cotyledon and floral organ fusions. miR164 overexpression also leads to phenotypes not previously observed in cuc1 cuc2 mutants, including leaf and stem fusions. These likely reflect the misregulation of other NAC-domain mRNAs, including NAC1, At5g07680, and At5g61430, for which miR164-directed cleavage products were detected. CONCLUSIONS These results demonstrate that miR164-directed regulation of CUC1 is necessary for normal embryonic, vegetative, and floral development. They also show that proper miR164 dosage or localization is required for separation of adjacent embryonic, vegetative, and floral organs, thus implicating miR164 as a common regulatory component of the molecular circuitry that controls the separation of different developing organs and thereby exposes a posttranscriptional layer of NAC-domain gene regulation during plant development.

Partially Redundant Functions of Arabidopsis DICER-like Enzymes and a Role for DCL4 in Producing trans-Acting siRNAs

Arabidopsis encodes four DICER-like (DCL) proteins. DCL1 produces miRNAs, DCL2 produces some virus-derived siRNAs, and DCL3 produces endogenous RDR2-dependent siRNAs, but the role of DCL4 is unknown. We show that DCL4 is the primary processor of endogenous RDR6-dependent trans-acting siRNAs (tasiRNAs). Molecular and phenotypic analyses of all dcl double mutants also revealed partially compensatory functions among DCL proteins. In the absence of DCL4, some RDR6-dependent siRNAs were produced by DCL2 and DCL3, and in the absence of DCL3, some RDR2-dependent siRNAs were produced by DCL2 and DCL4. Consistent with partial redundancies, dcl2 and dcl3 mutants developed normally, whereas dcl4 and dcl3 dcl4 mutants had weak and severe rdr6 phenotypes, respectively, and increased tasiRNA target mRNA accumulation. After three generations, dcl3 dcl4 and dcl2 dcl3 mutants exhibited stochastic developmental phenotypes, some of which were lethal, likely owing to the accumulated loss of heterochromatic siRNA-directed marks. dcl1 dcl3 and dcl1 dcl4, but not dcl1 dcl2 mutants, had phenotypes more severe than dcl1 mutants, consistent with DCL1, DCL3, and DCL4 acting as the primary processors of the three respective classes of endogenous silencing RNAs and DCL2 acting to produce viral-derived siRNAs and as an alternative DCL for endogenous siRNA production.

HC-Pro Suppression of Transgene Silencing Eliminates the Small RNAs but Not Transgene Methylation or the Mobile Signal

Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation mechanism that is widespread in eukaryotic organisms. It is often associated with methylation of the transcribed region of the silenced gene and with accumulation of small RNAs (21 to 25 nucleotides) homologous to the silenced gene. In plants, PTGS can be triggered locally and then spread throughout the organism via a mobile signal that can cross a graft junction. Previously, we showed that the helper component–proteinase (HC-Pro) of plant potyviruses suppresses PTGS. Here, we report that plants in which PTGS has been suppressed by HC-Pro fail to accumulate the small RNAs associated with silencing. However, the transgene locus of these plants remains methylated. Grafting experiments indicate that HC-Pro prevents the plant from responding to the mobile silencing signal but does not eliminate its ability to produce or send the signal. These results demonstrate that HC-Pro functions downstream of transgene methylation and the mobile signal at a step preceding accumulation of the small RNAs.

A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco

Two major classes of small noncoding RNAs have emerged as important regulators of gene expression in eukaryotes, the short interfering RNAs (siRNAs) associated with RNA silencing and endogenous micro-RNAs (miRNAs) implicated in regulation of gene expression. Helper component-proteinase (HC-Pro) is a viral protein that blocks RNA silencing in plants. Here we examine the effect of HC-Pro on the accumulation of siRNAs and endogenous miRNAs. siRNAs were analyzed in transgenic tobacco plants silenced in response to three different classes of transgenes: sense-transgenes, inverted-repeat transgenes, and amplicon-transgenes. HC-Pro suppressed silencing in each line, blocking accumulation of the associated siRNAs and allowing accumulation of transcripts from the previously silenced loci. HC-Pro-suppression of silencing in the inverted-repeat- and amplicon-transgenic lines was accompanied by the apparent accumulation of long double-stranded RNAs and proportional amounts of small RNAs that are larger than the siRNAs that accumulate during silencing. Analysis of these results suggests that HC-Pro interferes with silencing either by inhibiting siRNA processing from double-stranded RNA precursors or by destabilizing siRNAs. In contrast to siRNAs, the accumulation of endogenous miRNAs was greatly enhanced in all of the HC-Pro-expressing lines. Thus, our results demonstrate that accumulation of siRNAs and miRNAs in plants can be differentially regulated by a viral protein. The fact that HC-Pro affects the miRNA pathway raises the possibility that this pathway is targeted by plant viruses as a means to control gene expression in the host.

Form, Function, and Regulation of ARGONAUTE Proteins

Both transcriptional (TGS) and posttranscriptional gene silencing (PTGS) are conserved eukaryotic gene regulatory mechanisms, integral for taming exogenous (viruses and bacteria) or endogenous (repetitive elements and transposons) invasive nucleic acids to minimize their impact on genome integrity and function. TGS and PTGS also are essential for controlling the expression of protein coding genes throughout development or in response to environmental stimuli. In plants and animals, at least one member of the conserved ARGONAUTE (AGO) protein family comprises the catalytic engine of the silencing complex, which is guided by sequence-specific small RNA to cognate RNA. In this review, we present general features of plant and animal AGO proteins and detail our knowledge on the 10 Arabidopsis thaliana AGOs.

Novel long non-protein coding RNAs involved in Arabidopsis differentiation and stress responses

Long non-protein coding RNAs (npcRNA) represent an emerging class of riboregulators, which either act directly in this long form or are processed to shorter miRNA and siRNA. Genome-wide bioinformatic analysis of full-length cDNA databases identified 76 Arabidopsis npcRNAs. Fourteen npcRNAs were antisense to protein-coding mRNAs, suggesting cis-regulatory roles. Numerous 24-nt siRNA matched to five different npcRNAs, suggesting that these npcRNAs are precursors of this type of siRNA. Expression analyses of the 76 npcRNAs identified a novel npcRNA that accumulates in a dcl1 mutant but does not appear to produce trans-acting siRNA or miRNA. Additionally, another npcRNA was the precursor of miR869 and shown to be up-regulated in dcl4 but not in dcl1 mutants, indicative of a young miRNA gene. Abiotic stress altered the accumulation of 22 npcRNAs among the 76, a fraction significantly higher than that observed for the RNA binding protein-coding fraction of the transcriptome. Overexpression analyses in Arabidopsis identified two npcRNAs as regulators of root growth during salt stress and leaf morphology, respectively. Hence, together with small RNAs, long npcRNAs encompass a sensitive component of the transcriptome that have diverse roles during growth and differentiation.