Characterization of the Small RNA Transcriptome of the Marine Coccolithophorid, Emiliania huxleyi

Small RNAs (smRNAs) control a variety of cellular processes by silencing target genes at the transcriptional or post-transcription level. While extensively studied in plants, relatively little is known about smRNAs and their targets in marine phytoplankton, such as Emiliania huxleyi (E. huxleyi). Deep sequencing was performed of smRNAs extracted at different time points as E. huxleyi cells transition from logarithmic to stationary phase growth in batch culture. Computational analyses predicted 18 E. huxleyi specific miRNAs. The 18 miRNA candidates and their precursors vary in length (18-24 nt and 71-252 nt, respectively), genome copy number (3-1,459), and the number of genes targeted (2-107). Stem-loop real time reverse transcriptase (RT) PCR was used to validate miRNA expression which varied by nearly three orders of magnitude when growth slows and cells enter stationary phase. Stem-loop RT PCR was also used to examine the expression profiles of miRNA in calcifying and non-calcifying cultures, and a small subset was found to be differentially expressed when nutrients become limiting and calcification is enhanced. In addition to miRNAs, endogenous small RNAs such as ra-siRNAs, ta-siRNAs, nat-siRNAs, and piwiRNAs were predicted along with the machinery for the biogenesis and processing of si-RNAs. This study is the first genome-wide investigation smRNAs pathways in E. huxleyi. Results provide new insights into the importance of smRNAs in regulating aspects of physiological growth and adaptation in marine phytoplankton and further challenge the notion that smRNAs evolved with multicellularity, expanding our perspective of these ancient regulatory pathways

Enhancing microRNA167A expression in seed decreases the α-linolenic acid content and increases seed size in Camelina sativa.

Despite well established roles of microRNAs in plant development, few aspects have been addressed to understand their effects in seeds especially on lipid metabolism. In this study, we showed that overexpressing microRNA167A (miR167OE) in camelina (Camelina sativa) under a seed-specific promoter changed fatty acid composition and increased seed size. Specifically, the miR167OE seeds had a lower α-linolenic acid with a concomitantly higher linoleic acid content than the wild-type. This decreased level of fatty acid desaturation corresponded to a decreased transcriptional expression of the camelina fatty acid desaturase3 (CsFAD3) in developing seeds. MiR167 targeted the transcription factor auxin response factor (CsARF8) in camelina, as had been reported previously in Arabidopsis. Chromatin immunoprecipitation experiments combined with transcriptome analysis indicated that CsARF8 bound to promoters of camelina bZIP67 and ABI3 genes. These transcription factors directly or through the ABI3-bZIP12 pathway regulate CsFAD3 expression and affect α-linolenic acid accumulation. In addition, to decipher the miR167A-CsARF8 mediated transcriptional cascade for CsFAD3 suppression, transcriptome analysis was conducted to implicate mechanisms that regulate seed size in camelina. Expression levels of many genes were altered in miR167OE, including orthologs that have previously been identified to affect seed size in other plants. Most notably, genes for seed coat development such as suberin and lignin biosynthesis were down-regulated. This study provides valuable insights into the regulatory mechanism of fatty acid metabolism and seed size determination, and suggests possible approaches to improve these important traits in camelina

In silico identification of conserved microRNAs in large number of diverse plant species

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are recently discovered small non-coding RNAs that play pivotal roles in gene expression, specifically at the post-transcriptional level in plants and animals. Identification of miRNAs in large number of diverse plant species is important to understand the evolution of miRNAs and miRNA-targeted gene regulations. Now-a-days, publicly available databases play a central role in the in-silico biology. Because, at least ~21 miRNA families are conserved in higher plants, a homology based search using these databases can help identify orthologs or paralogs in plants.</p> <p>Results</p> <p>We searched all publicly available nucleotide databases of genome survey sequences (GSS), high-throughput genomics sequences (HTGS), expressed sequenced tags (ESTs) and nonredundant (NR) nucleotides and identified 682 miRNAs in 155 diverse plant species. We found more than 15 conserved miRNA families in 11 plant species, 10 to14 families in 10 plant species and 5 to 9 families in 29 plant species. Nineteen conserved miRNA families were identified in important model legumes such as <it>Medicago</it>, <it>Lotus </it>and soybean. Five miRNA families – miR319, miR156/157, miR169, miR165/166 and miR394 – were found in 51, 45, 41, 40 and 40 diverse plant species, respectively. miR403 homologs were found in 16 dicots, whereas miR437 and miR444 homologs, as well as the miR396d/e variant of the miR396 family, were found only in monocots, thus providing large-scale authenticity for the dicot- and monocot-specific miRNAs. Furthermore, we provide computational and/or experimental evidence for the conservation of 6 newly found Arabidopsis miRNA homologs (miR158, miR391, miR824, miR825, miR827 and miR840) and 2 small RNAs (small-85 and small-87) in <it>Brassica spp</it>.</p> <p>Conclusion</p> <p>Using all publicly available nucleotide databases, 682 miRNAs were identified in 155 diverse plant species. By combining the expression analysis with the computational approach, we found that 6 miRNAs and 2 small RNAs that have been identified only in Arabidopsis thus far, are also conserved in <it>Brassica spp</it>. These findings will be useful for tracing the evolution of small RNAs by examining their expression in common ancestors of the <it>Arabidopsis</it>-<it>Brassica </it>lineage.</p

Unique and conserved MicroRNAs in wheat chromosome 5D revealed by next-generation sequencing

MicroRNAs are a class of short, non-coding, single-stranded RNAs that act as post-transcriptional regulators in gene expression. miRNA analysis of Triticum aestivum chromosome 5D was performed on 454 GS FLX Titanium sequences of flow sorted chromosome 5D with a total of 3,208,630 good quality reads representing 1.34x and 1.61x coverage of the short (5DS) and long (5DL) arms of the chromosome respectively. In silico and structural analyses revealed a total of 55 miRNAs; 48 and 42 miRNAs were found to be present on 5DL and 5DS respectively, of which 35 were common to both chromosome arms, while 13 miRNAs were specific to 5DL and 7 miRNAs were specific to 5DS. In total, 14 of the predicted miRNAs were identified in wheat for the first time. Representation (the copy number of each miRNA) was also found to be higher in 5DL (1,949) compared to 5DS (1,191). Targets were predicted for each miRNA, while expression analysis gave evidence of expression for 6 out of 55 miRNAs. Occurrences of the same miRNAs were also found in Brachypodium distachyon and Oryza sativa genome sequences to identify syntenic miRNA coding sequences. Based on this analysis, two other miRNAs: miR1133 and miR167 were detected in B. distachyon syntenic region of wheat 5DS. Five of the predicted miRNA coding regions (miR6220, miR5070, miR169, miR5085, miR2118) were experimentally verified to be located to the 5D chromosome and three of them : miR2118, miR169 and miR5085, were shown to be 5D specific. Furthermore miR2118 was shown to be expressed in Chinese Spring adult leaves. miRNA genes identified in this study will expand our understanding of gene regulation in bread wheat

Target RNAs strike back on MicroRNAs

MicroRNAs are extensively studied regulatory non-coding small RNAs that silence animal genes throughout most biological processes, typically doing so by binding to partially complementary sequences within target RNAs. A plethora of studies has described detailed mechanisms for microRNA biogenesis and function, as well as their temporal and spatial regulation during development. By inducing translational repression and/or degradation of their target RNAs, microRNAs can contribute to achieve highly specific cell-or tissue-specific gene expression, while their aberrant expression can lead to disease. Yet an unresolved aspect of microRNA biology is how such small RNA molecules are themselves cleared from the cell, especially under circumstances where fast microRNA turnover or specific degradation of individual microRNAs is required. In recent years, it was unexpectedly found that binding of specific target RNAs to microRNAs with extensive complementarity can reverse the outcome, triggering degradation of the bound microRNAs. This emerging pathway, named TDMD for Target RNA-Directed MicroRNA Degradation, leads to microRNA 3′-end tailing by the addition of A/U non-templated nucleotides, trimming or shortening from the 3′ end, and highly specific microRNA loss, providing a new layer of microRNA regulation. Originally described in flies and known to be triggered by viral RNAs, novel endogenous instances of TDMD have been uncovered and are now starting to be understood. Here, we review our current knowledge of this pathway and its potential role in the control and diversification of microRNA expression patterns.Fil: Fuchs Wightman, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Giono, Luciana Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Fededa, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: de la Mata, Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Evolution of MIR168 paralogs in Brassicaceae

<p>Abstract</p> <p>Background</p> <p>In plants, expression of ARGONAUTE1 (AGO1), the catalytic subunit of the RNA-Induced Silencing Complex responsible for post-transcriptional gene silencing, is controlled through a feedback loop involving the miR168 microRNA. This complex auto-regulatory loop, composed of miR168-guided AGO1-catalyzed cleavage of <it>AGO1 </it>mRNA and AGO1-mediated stabilization of miR168, was shown to ensure the maintenance of AGO1 homeostasis that is pivotal for the correct functioning of the miRNA pathway.</p> <p>Results</p> <p>We applied different approaches to studying the genomic organization and the structural and functional evolution of <it>MIR168 </it>homologs in Brassicaeae. A whole genome comparison of Arabidopsis and poplar, phylogenetic footprinting and phylogenetic reconstruction were used to date the duplication events originating <it>MIR168 </it>homologs in these genomes. While orthology was lacking between Arabidopsis and poplar <it>MIR168 </it>genes, we successfully isolated orthologs of both loci present in Arabidopsis (<it>MIR168a </it>and <it>MIR168b</it>) from all the Brassicaceae species analyzed, including the basal species <it>Aethionema grandiflora</it>, thus indicating that (1) independent duplication events took place in Arabidopsis and poplar lineages and (2) the origin of <it>MIR168 </it>paralogs predates both the Brassicaceae radiation and the Arabidopsis alpha polyploidization. Different phylogenetic footprints, corresponding to known functionally relevant regions (transcription starting site and double-stranded structures responsible for microRNA biogenesis and function) or for which functions could be proposed, were found to be highly conserved among <it>MIR168 </it>homologs. Comparative predictions of the identified microRNAs also indicate extreme conservation of secondary structure and thermodynamic stability.</p> <p>Conclusion</p> <p>We used a comparative phylogenetic footprinting approach to identify the structural and functional constraints that shaped <it>MIR168 </it>evolution in Brassicaceae. Although their duplication happened at least 40 million years ago, we found evidence that both <it>MIR168 </it>paralogs have been maintained throughout the evolution of Brassicaceae, most likely functionally as indicated by the extremely high conservation of functionally relevant regions, predicted secondary structure and thermodynamic profile. Interestingly, the expression patterns observed in Arabidopsis indicate that <it>MIR168b </it>underwent partial subfunctionalization as determined by the experimental characterization of its expression pattern provided in this study. We found further evolutionary evidence that pre-miR168 lower stem (the RNA-duplex structure adjacent to the miR-miR* stem) is significantly longer than animal lower stems and probably plays a relevant role in multi-step miR168 biogenesis.</p

Evidence for the rapid expansion of microRNA-mediated regulation in early land plant evolution

BACKGROUND: MicroRNAs (miRNAs) are regulatory RNA molecules that are specified by their mode of action, the structure of primary transcripts, and their typical size of 20–24 nucleotides. Frequently, not only single miRNAs but whole families of closely related miRNAs have been found in animals and plants. Some families are widely conserved among different plant taxa. Hence, it is evident that these conserved miRNAs are of ancient origin and indicate essential functions that have been preserved over long evolutionary time scales. In contrast, other miRNAs seem to be species-specific and consequently must possess very distinct functions. Thus, the analysis of an early-branching species provides a window into the early evolution of fundamental regulatory processes in plants. RESULTS: Based on a combined experimental-computational approach, we report on the identification of 48 novel miRNAs and their putative targets in the moss Physcomitrella patens. From these, 18 miRNAs and two targets were verified in independent experiments. As a result of our study, the number of known miRNAs in Physcomitrella has been raised to 78. Functional assignments to mRNAs targeted by these miRNAs revealed a bias towards genes that are involved in regulation, cell wall biosynthesis and defense. Eight miRNAs were detected with different expression in protonema and gametophore tissue. The miRNAs 1–50 and 2–51 are located on a shared precursor that are separated by only one nucleotide and become processed in a tissue-specific way. CONCLUSION: Our data provide evidence for a surprisingly diverse and complex miRNA population in Physcomitrella. Thus, the number and function of miRNAs must have significantly expanded during the evolution of early land plants. As we have described here within, the coupled maturation of two miRNAs from a shared precursor has not been previously identified in plants

Shade delays flowering in Medicago sativa

Shade intolerant plants respond to the decrease in the red (R) to far-red light (FR) ratio (R:FR) occurring under shade by elongating stems and petioles and re-positioning leaves, in a race to out-compete neighbors for the sunlight resource. In some annual species, these shade-avoidance responses (SAS) are accompanied by the early induction of flowering. Anticipated flowering is viewed as a strategy to set seeds before the resources become severely limiting. Little is known about the molecular mechanisms of SAS in perennial forage crops like alfalfa (Medicago sativa). To study SAS in alfalfa, we exposed alfalfa plants to simulated shade by supplementing with FR. Low R:FR produced a classical SAS, such as increased internode and petiole length but, unexpectedly, delayed flowering. To understand the molecular mechanisms involved in uncoupling SAS from early flowering, we used a transcriptomic approach. SAS were likely mediated by increased expression of msPIF3 and msHB2 in low R:FR. Constitutive expression of these genes in Arabidopsis led to SAS, including early flowering, strongly suggesting their roles are conserved. Delayed flowering was likely to be mediated by the downregulation of msSPL3, which promotes flowering in both Arabidopsis and alfalfa. Shade-delayed flowering in alfalfa may be important to extend the vegetative phase under sub-optimal light conditions and thus assure the accumulation of reserves necessary to resume growth after the next season. This article is protected by copyright. All rights reserved.Fil: Lorenzo, Christian Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Iserte, Javier Alonso. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Sanchez Lamas, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Antonietti, Mariana Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Garcia Gagliardi, Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Hernando, Carlos Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Dezar, Carlos Alberto Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Agrobiotecnología de Rosario; ArgentinaFil: Vazquez, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Agrobiotecnología de Rosario; ArgentinaFil: Casal, Jorge José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; ArgentinaFil: Yanovsky, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Cerdan, Pablo Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin