Specificity of plant microRNA target MIMICs: cross-targeting of miR159 and miR319

Plant microRNA (miRNA) target MIMICs (MIMs) are non-coding RNA transcripts that can inhibit endogenous miRNAs, as they contain a miRNA binding site that forms a three nucleotide (nt) mismatch loop opposite the miRNA cleavage site upon miRNA binding. This loop renders the MIMs non-cleavable, presumably leading to sequestration of the miRNA and thus enabling the endogenous targets to be deregulated. Arabidopsis miR319 and miR159 are two closely related but distinct miRNA families, as they are functionally specific for two different sets of targets, TCP and MYB genes, respectively. Being offset by one nt, MIM319 and MIM159 should have specificity to their respective miRNA families. However, MIM319 and MIM159 plants appear indistinguishable, having highly similar developmental defects reminiscent of a loss-of-function mir159 mutant. In both MIM319 and MIM159 plants, miR159 and miR319 levels are reduced, and correspondingly, both MYB and TCP mRNA levels are elevated, implying that these MIMs are inhibiting both miR159 and miR319. These data demonstrate that MIMs are able to inhibit closely related miRNAs, including those with cleavage sites not opposite the three nt loop. This highlights that MIMs can have unintended off-target effects and that their use should include corresponding molecular analysis to investigate their impact on closely related miRNAs.This research was supported by an Australian Research Council grant DP130103697 and an International ANU PhD scholarship to M.R

Allelic effects on starch structure and properties of six starch biosynthetic genes in a rice recombinant inbred line population

BACKGROUND: The genetic diversity of six starch biosynthetic genes (Wx, SSI, SSIIa, SBEI, SBEIIa and SBEIIb) in indica and japonica rices opens an opportunity to produce a new variety with more favourable grain starch quality. However, there is limited information about the effects of these six gene allele combinations on starch structure and properties. A recombinant inbred line population from a cross between indica and japonica varieties offers opportunities to combine specific alleles of the six genes. RESULTS: The allelic (indica vs japonica) effects of six starch biosynthetic genes on starch structure, functional properties, and abundance of granule bound proteins in rice grains were investigated in a common genetic background using a recombinant inbred line population. The indica Wx (Wxi) allele played a major role while indica SSI (SSIi), japonica SSIIa (SSIIaj) and indica SBEI (SBEIi) alleles had minor roles on the increase of amylose content. SSIIaj and japonica SBEIIb (SBEIIbj) alleles had a major and a minor role on high ratio of ∑DP ≤ 10 to ∑DP ≤ 24 fractions (RCL10/24), respectively. Both major alleles (Wxi and SSIIaj) reduced peak viscosity (PV), onset, peak and end gelatinization temperatures (GTs) of amylopectin, and increased amylose-lipid complex dissociation enthalpy compared with their counterpart-alleles, respectively. SBEIIai and SBEIIbj decreased PV, whereas SSIi and SBEIIbj decreased FV. SBEIi reduced setback viscosity and gelatinization enthalpy. RCL10/24 of chain length distribution in amylopectin is negatively correlated with PV and BD of paste property and GTs of thermal properties. We also report RILs with superior starch properties combining Wxi, SSIj, SSIIaj, SBEIi and SBEIIbj alleles. Additionally, a clear relation is drawn to starch biosynthetic gene alleles, starch structure, properties, and abundance of granule bound starch biosynthetic enzymes inside starch granules. CONCLUSIONS: Rice Wxi and SSIIaj alleles play major roles, while SSIi, SBEIi, SBEIIai and SBEIIbj alleles have minor roles in the determination of starch properties between indica and japonica rice through starch structural modification. The combination of these alleles is a key factor for starch quality improvement in rice breeding programs. RCL10/24 value is critical for starch structure and property determination.Jixun Luo was supported by CSC (Chinese Scholarship Council) and Australian National University scholarships. This work was funded by CSIRO Food Future National Research Flagship

Expression of human ARGONAUTE 2 inhibits endogenous microRNA activity in Arabidopsis

Plant and animal microRNA (miRNA) pathways share many analogous components, the ARGONAUTE (AGO) proteins being foremost among them. We sought to ascertain the degree of functional conservation shared by Homo sapiens ARGONAUTE 2 (HsAGO2) and Arabidopsis thaliana ARGONAUTE 1 (AtAGO1), which are the predominant AGO family members involved with miRNA activity in their respective species. Transgenic Arabidopsis plants expressing HsAGO2 were indistinguishable from counterparts over-expressing AtAGO1, each group exhibiting the morphological and molecular hallmarks of miRNA-pathway loss-of-function alleles. However, unlike AtAGO1, HsAGO2 was unable to rescue the ago1-27 allele. We conclude that, despite the evolutionary gulf between them, HsAGO2 is likely capable of interacting with some component/s of the Arabidopsis miRNA pathway, thereby perturbing its operation, although differences have arisen such that HsAGO2 alone is insufficient to confer efficient silencing of miRNA targets in planta

MicroR159 regulation of most conserved targets in Arabidopsis has negligible phenotypic effects

BACKGROUND A current challenge of microRNA (miRNA) research is the identification of biologically relevant miRNA:target gene relationships. In plants, high miRNA:target gene complementarity has enabled accurate target predictions, and slicing of target mRNAs has facilitated target validation through rapid amplification of 5' cDNA ends (5'-RACE) analysis. Together, these approaches have identified more than 20 targets potentially regulated by the deeply conserved miR159 family in Arabidopsis, including eight MYB genes with highly conserved miR159 target sites. However, genetic analysis has revealed the functional specificity of the major family members, miR159a and miR159b is limited to only two targets, MYB33 and MYB65. Here, we examine the functional role of miR159 regulation for the other potential MYB target genes. RESULTS For these target genes, functional analysis failed to identify miR159 regulation that resulted in any major phenotypic impact, either at the morphological or molecular level. This appears to be mainly due to the quiescent nature of the remaining family member, MIR159c. Although its expression overlaps in a temporal and spatial cell-specific manner with a subset of these targets in anthers, the abundance of miR159c is extremely low and concomitantly a mir159c mutant displays no anther defects. Examination of potential miR159c targets with conserved miR159 binding sites found neither their spatial or temporal expression domains appeared miR159 regulated, despite the detection of miR159-guided cleavage products by 5'-RACE. Moreover, expression of a miR159-resistant target (mMYB101) resulted predominantly in plants that are indistinguishable from wild type. Plants that displayed altered morphological phenotypes were found to be ectopically expressing the mMYB101 transgene, and hence were misrepresentative of the in vivo functional role of miR159. CONCLUSIONS This study presents a novel explanation for a paradox common to plant and animal miRNA systems, where among many potential miRNA-target relationships usually only a few appear physiologically relevant. The identification of a quiescent miR159c:target gene regulatory module in anthers provides a likely rationale for the presence of conserved miR159 binding sites in many targets for which miR159 regulation has no obvious functional role. Remnants from the demise of such modules may lead to an overestimation of miRNA regulatory complexity when investigated using bioinformatic, 5'-RACE or transgenic approaches.RSA was funded by an ANU postgraduate scholarship and by a CSIRO Emerging Science Initiative. JL is the recipient of an ANU international student postgraduate scholarship. This research was supported by an Australian Research Council grant DP0773270

Ubiquitous miR159 repression of MYB33/65 in Arabidopsis rosettes is robust and is not perturbed by a wide range of stresses

Morphological impact of TuMV infection. Representative classification of symptom severities among TuMV-infected rosettes. (PPTX 1545 kb

Investigating Anomalous Photochemistry in the Inner Wind of IRC+10216 Through ALMA Observations of HC3_3N

In recent years, many questions have arisen regarding the chemistry of photochemical products in the carbon-rich winds of evolved stars. To address them, it is imperative to constrain the distributions of such species through high angular resolution interferometric observations covering multiple rotational transitions. We used archival ALMA observations to map rotational lines involving high energy levels of cyanoacetylene (HC$_3$N) toward the inner envelope (radius <8"/1000 AU) of the carbon star IRC+10216. The observed lines include the J=28-27, J=30-29, and J=38-37, transitions of HC$_3$N in its ground vibrational state. In contrast to previous observations of linear carbon chains toward this AGB star which show extended, hollow emission at 15"-20" radii (e.g. C$_4$H, C$_6$H, HC$_5$N), the maps of the HC$_3$N lines here show compact morphologies comprising various arcs and density enhancements, with significant emission from gas clumps at an angular distance of ~3" (350 AU) from the central AGB star. We compared visibility sampled non-LTE radiative transfer models with the observed brightness distributions, and derive a fractional abundance with respect to H$_2$ of $10^{-8}$ for HC$_3$N at the radii probed by these lines. These results are consistent with enhanced photochemistry occurring in warm (~200 K) regions of the circumstellar envelope. After application of a specialized chemical model for IRC+10216, we find evidence that the enhanced HC$_3$N abundances in the inner wind are most likely due to a solar-type binary companion initiating photochemistry in this region.Comment: 17 pages, 9 figures, 2 tables. Accepted for publication in Ap

Facile mutant identification via a single parental backcross method and application of whole genome sequencing based mapping pipelines

Forward genetic screens have identified numerous genes involved in development and metabolism, and remain a cornerstone of biological research. However, to locate a causal mutation, the practice of crossing to a polymorphic background to generate a mapping population can be problematic if the mutant phenotype is difficult to recognize in the hybrid F2 progeny, or dependent on parental specific traits. Here in a screen for leaf hyponasty mutants, we have performed a single backcross of an Ethane Methyl Sulphonate (EMS) generated hyponastic mutant to its parent. Whole genome deep sequencing of a bulked homozygous F2 population and analysis via the Next Generation EMS mutation mapping pipeline (NGM) unambiguously determined the causal mutation to be a single nucleotide polymorphisim (SNP) residing in HASTY, a previously characterized gene involved in microRNA biogenesis. We have evaluated the feasibility of this backcross approach using three additional SNP mapping pipelines; SHOREmap, the GATK pipeline, and the samtools pipeline. Although there was variance in the identification of EMS SNPs, all returned the same outcome in clearly identifying the causal mutation in HASTY. The simplicity of performing a single parental backcross and genome sequencing a small pool of segregating mutants has great promise for identifying mutations that may be difficult to map using conventional approaches.This work was funded by an Australian Research Council Discovery Grant DP1097150

A Search for Hydroxylamine (NH2OH) toward Select Astronomical Sources

Observations of 14 rotational transitions of hydroxylamine (NH2OH) using the NRAO 12 m Telescope on Kitt Peak are reported towards IRC+10216, Orion KL, Orion S, Sgr B2(N), Sgr B2(OH), W3IRS5, and W51M. Although recent models suggest the presence of NH2OH in high abundance, these observations resulted in non-detection. Upper limits are calculated to be as much as six orders of magnitude lower than predicted by models. Possible explanations for the lower than expected abundance are explored.Comment: 18 pages, 3 figures, 3 table