286 research outputs found
Cytokinin and auxin intersection in root meristems
Cytokinin promotes cell differentiation in plant roots by repressing auxin transport and responses at the meristem boundary
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Endogenous and antiviral RNA silencing pathways in Arabidopsis
RNA silencing pathways are required for a wide variety of processes in most
eucaryotes. In plants, small-interfering RNA (siRNA) arising from transposons and other
repetitive sequences is associated with heterochromatin formation and maintenance.
MicroRNAs and trans-acting siRNAs encoded at discrete loci function as negative regulators of
gene expression by triggering cleavage or translational repression of mRNA transcripts with
base complementarity to the small RNA. siRNA processed from viral RNA directs antiviral
silencing that represses virus accumulation in plants and other organisms. Together, these
pathways serve numerous functions in plants including genome maintenance, developmental
timing and patterning and antiviral defense.
Virus-encoded RNA silencing suppressor proteins are viral pathogenicity factors and
inhibit the antiviral silencing response through interaction with small RNA intermediates. In
this work, small RNA duplex binding was demonstrated for unrelated suppressors from multiple
viruses using molecular biology and biochemistry techniques. Sequestration of virus-derived
siRNA and microRNA/microRNA* duplexes, inhibition of microRNA methylation, and
perturbation of Arabidopsis development was demonstrated for several suppressors using
transgenic approaches and molecular techniques. Suppressor inhibition of antiviral RNA
silencing and endogenous microRNA pathways indicates that small RNA binding is a common
strategy used by unrelated viruses, and suggests that interference with miRNA-directed
processes may be a general feature contributing to pathogenicity of many viruses.
Finally, small RNA preparation and high-throughput sequencing procedures were
developed for profiling small RNA populations in Arabidopsis. Genome-wide profiles of small
RNA from wild-type Arabidopsis thaliana and silencing pathway mutants revealed dynamic
changes in expression of some microRNA families as well as genome-wide distribution patterns
of small RNA in plants. These results establish high-throughput sequencing as a small RNA
profiling tool and provide a comprehensive description of the major small RNA pathways in
Arabidopsis. Together, the results presented here provide a basic understanding of the breadth
of small RNA pathways in plants and show how interference with these pathways by viruses
contributes to virus disease
Preparation of multiplexed small RNA libraries from plants
[EN] High-throughput sequencing is a powerful tool for exploring small RNA populations in
plants. The ever-increasing output from an Illumina Sequencing System allows for multiplexing
multiple samples while still obtaining sufficient data for small RNA discovery and characterization.
Here we describe a protocol for generating multiplexed small RNA libraries for sequencing up to
12 samples in one lane of an Illumina HiSeq System single-end, 50 base pair run. RNA ligases
are used to add the 3¿ and 5¿ adaptors to purified small RNAs; ligation products that lack a small
RNA molecule (adaptor-adaptor products) are intentionally depleted. After cDNA synthesis, a
linear PCR step amplifies the DNA fragments. The 3¿ PCR primers used here include unique 6-
nucleotide sequences to allow for multiplexing up to 12 samples.The original version of this protocol was described in Carbonell et al. (2012). The updated version of the protocol was described in Carbonell et al. (2014). This work was supported by grants from the National Science Foundation (MCB-0956526, MCB-1231726) and National Institutes of Health (AI043288)Gilbert, KB.; Fahlgren, N.; Kasschau, KD.; Chapman, EJ.; Carrington, JC.; Carbonell, A. (2014). Preparation of multiplexed small RNA libraries from plants. Bio-protocol. 4(21):1-17. https://doi.org/10.21769/BioProtoc.1275S11742
Hypocotyl Transcriptome Reveals Auxin Regulation of Growth-Promoting Genes through GA-Dependent and -Independent Pathways
Many processes critical to plant growth and development are regulated by the hormone auxin. Auxin responses are initiated through activation of a transcriptional response mediated by the TIR1/AFB family of F-box protein auxin receptors as well as the AUX/IAA and ARF families of transcriptional regulators. However, there is little information on how auxin regulates a specific cellular response. To begin to address this question, we have focused on auxin regulation of cell expansion in the Arabidopsis hypocotyl. We show that auxin-mediated hypocotyl elongation is dependent upon the TIR1/AFB family of auxin receptors and degradation of AUX/IAA repressors. We also use microarray studies of elongating hypocotyls to show that a number of growth-associated processes are activated by auxin including gibberellin biosynthesis, cell wall reorganization and biogenesis, and others. Our studies indicate that GA biosynthesis is required for normal response to auxin in the hypocotyl but that the overall transcriptional auxin output consists of PIF-dependent and -independent genes. We propose that auxin acts independently from and interdependently with PIF and GA pathways to regulate expression of growth-associated genes in cell expansion
Genome-Wide Profiling and Analysis of Arabidopsis siRNAs
Eukaryotes contain a diversified set of small RNA-guided pathways that control genes, repeated sequences, and viruses at the transcriptional and posttranscriptional levels. Genome-wide profiles and analyses of small RNAs, particularly the large class of 24-nucleotide (nt) short interfering RNAs (siRNAs), were done for wild-type Arabidopsis thaliana and silencing pathway mutants with defects in three RNA-dependent RNA polymerase (RDR) and four Dicer-like (DCL) genes. The profiling involved direct analysis using a multiplexed, parallel-sequencing strategy. Small RNA-generating loci, especially those producing predominantly 24-nt siRNAs, were found to be highly correlated with repetitive elements across the genome. These were found to be largely RDR2- and DCL3-dependent, although alternative DCL activities were detected on a widespread level in the absence of DCL3. In contrast, no evidence for RDR2-alternative activities was detected. Analysis of RDR2- and DCL3-dependent small RNA accumulation patterns in and around protein-coding genes revealed that upstream gene regulatory sequences systematically lack siRNA-generating activities. Further, expression profiling suggested that relatively few genes, proximal to abundant 24-nt siRNAs, are regulated directly by RDR2- and DCL3-dependent silencing. We conclude that the widespread accumulation patterns for RDR2- and DCL3-dependent siRNAs throughout the Arabidopsis genome largely reflect mechanisms to silence highly repeated sequences
Specificity of ARGONAUTE7-miR390 Interaction and Dual Functionality in TAS3 Trans-Acting siRNA Formation
SummaryTrans-acting siRNA form through a refined RNAi mechanism in plants. miRNA-guided cleavage triggers entry of precursor transcripts into an RNA-DEPENDENT RNA POLYMERASE6 pathway, and sets the register for phased tasiRNA formation by DICER-LIKE4. Here, we show that miR390-ARGONAUTE7 complexes function in distinct cleavage or noncleavage modes at two target sites in TAS3a transcripts. The AGO7 cleavage, but not the noncleavage, function could be provided by AGO1, the dominant miRNA-associated AGO, but only when AGO1 was guided to a modified target site through an alternate miRNA. AGO7 was highly selective for interaction with miR390, and miR390 in turn was excluded from association with AGO1 due entirely to an incompatible 5′ adenosine. Analysis of AGO1, AGO2, and AGO7 revealed a potent 5′ nucleotide discrimination function for some, although not all, ARGONAUTEs. miR390 and AGO7, therefore, evolved as a highly specific miRNA guide/effector protein pair to function at two distinct tasiRNA biogenesis steps
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Visuo-spatial cognition in Williams syndrome: Reviewing and accounting for the strengths and weaknesses in performance
Individuals with Williams syndrome typically show relatively poor visuo-spatial abilities in comparison to stronger verbal skills. However, individuals' level of performance is not consistent across all visuo-spatial tasks. The studies assessing visuo-spatial functioning in Williams syndrome are critically reviewed, in order to provide a clear pattern of the relative difficulty of these tasks. This prompts a possible explanation of the variability in performance seen which focuses on the processing demands of some of these tasks. Individuals with Williams syndrome show an atypical processing style on tests of construction, which does not affect tests of perception
No Detectable Fertility Benefit from a Single Additional Mating in Wild Stalk-Eyed Flies
Background: Multiple mating by female insects is widespread, and the explanation(s) for repeated mating by females has been the subject of much discussion. Females may profit from mating multiply through direct material benefits that increase their own reproductive output, or indirect genetic benefits that increase offspring fitness. One particular direct benefit that has attracted significant attention is that of fertility assurance, as females often need to mate multiply to achieve high fertility. This hypothesis has never been tested in a wild insect population.Methodology/Principal Findings: Female Malaysian stalk-eyed flies (Teleopsis dalmanni) mate repeatedly during their lifetime, and have been shown to be sperm limited under both laboratory and field conditions. Here we ask whether receiving an additional mating alleviates sperm limitation in wild females. In our experiment one group of females received a single additional mating, while a control group received an interrupted, and therefore unsuccessful, mating. Females that received an additional mating did not lay more fertilised eggs in total, nor did they lay proportionately more fertilised eggs. Female fertility declined significantly through time, demonstrating that females were sperm limited. However, receipt of an additional mating did not significantly alter the rate of this decline.Conclusions/Significance: Our data suggest that the fertility consequences of a single additional mating were small. We discuss this effect (or lack thereof), and suggest that it is likely to be attributed to small ejaculate size, a high proportion of failed copulations, and the presence of X-linked meiotic drive in this species
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Genome-Wide Profiling and Analysis of Arabidopsis siRNAs
Eukaryotes contain a diversified set of small RNA-guided pathways that control genes, repeated sequences, and viruses at the transcriptional and posttranscriptional levels. Genome-wide profiles and analyses of small RNAs, particularly the large class of 24-nucleotide (nt) short interfering RNAs (siRNAs), were done for wild-type Arabidopsis thaliana and silencing pathway mutants with defects in three RNA-dependent RNA polymerase (RDR) and four Dicer-like (DCL) genes. The profiling involved direct analysis using a multiplexed, parallel-sequencing strategy. Small RNA-generating loci, especially those producing predominantly 24-nt siRNAs, were found to be highly correlated with repetitive elements across the genome. These were found to be largely RDR2- and DCL3-dependent, although alternative DCL activities were detected on a widespread level in the absence of DCL3. In contrast, no evidence for RDR2-alternative activities was detected. Analysis of RDR2- and DCL3-dependent small RNA accumulation patterns in and around protein-coding genes revealed that upstream gene regulatory sequences systematically lack siRNA-generating activities. Further, expression profiling suggested that relatively few genes, proximal to abundant 24-nt siRNAs, are regulated directly by RDR2- and DCL3-dependent silencing. We conclude that the widespread accumulation patterns for RDR2- and DCL3-dependent siRNAs throughout the Arabidopsis genome largely reflect mechanisms to silence highly repeated sequences
Recommendations for the use of common outcome measures in pediatric traumatic brain injury research
This article addresses the need for age-relevant outcome measures for traumatic brain injury (TBI) research and summarizes the recommendations by the inter-agency Pediatric TBI Outcomes Workgroup. The Pediatric Workgroup\u27s recommendations address primary clinical research objectives including characterizing course of recovery from TBI, prediction of later outcome, measurement of treatment effects, and comparison of outcomes across studies. Consistent with other Common Data Elements (CDE) Workgroups, the Pediatric TBI Outcomes Workgroup adopted the standard three-tier system in its selection of measures. In the first tier, core measures included valid, robust, and widely applicable outcome measures with proven utility in pediatric TBI from each identified domain including academics, adaptive and daily living skills, family and environment, global outcome, health-related quality of life, infant and toddler measures, language and communication, neuropsychological impairment, physical functioning, psychiatric and psychological functioning, recovery of consciousness, social role participation and social competence, social cognition, and TBI-related symptoms. In the second tier, supplemental measures were recommended for consideration in TBI research focusing on specific topics or populations. In the third tier, emerging measures included important instruments currently under development, in the process of validation, or nearing the point of published findings that have significant potential to be superior to measures in the core and supplemental lists and may eventually replace them as evidence for their utility emerges
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