Genome-scale spatiotemporal analysis of Caenorhabditis elegans microRNA promoter activity

Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Genome Research (Impact Factor: 14.63). 12/2008; 18(12):2005-15. DOI: 10.1101/gr.083055.108
Source: PubMed


The Caenorhabditis elegans genome encodes more than 100 microRNAs (miRNAs). Genetic analyses of miRNA deletion mutants have only provided limited insights into miRNA function. To gain insight into the function of miRNAs, it is important to determine their spatiotemporal expression pattern. Here, we use miRNA promoters driving the expression of GFP as a proxy for miRNA expression. We describe a set of 73 transgenic C. elegans strains, each expressing GFP under the control of a miRNA promoter. Together, these promoters control the expression of 89 miRNAs (66% of all predicted miRNAs). We find that miRNA promoters drive GFP expression in a variety of tissues and that, overall, their activity is similar to that of protein-coding gene promoters. However, miRNAs are expressed later in development, which is consistent with functions after initial body-plan specification. We find that miRNA members belonging to families are more likely to be expressed in overlapping tissues than miRNAs that do not belong to the same family, and provide evidence that intronic miRNAs may be controlled by their own, rather than a host gene promoter. Finally, our data suggest that post-transcriptional mechanisms contribute to differential miRNA expression. The data and strains described here will provide a valuable guide and resource for the functional analysis of C. elegans miRNAs.

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    • "However, these methods can only be applied to intronic miRNAs and only to those miRNAs whose host gene expression is a good surrogate for their activity. Many host gene expression levels are not good surrogates [63,74-76]. "
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    ABSTRACT: Popular miRNA target prediction techniques use sequence features to determine the functional miRNA target sites. These techniques commonly ignore the cellular conditions in which miRNAs interact with their targets in vivo. Gene expression data are rich resources that can complement sequence features to take into account the context dependency of miRNAs. We introduce BayMiR, a new computational method, that predicts the functionality of potential miRNA target sites using the activity level of the miRNAs inferred from genome-wide mRNA expression profiles. We also found that mRNA expression variation can be used as another predictor of functional miRNA targets. We benchmarked BayMiR, the expression variation, Cometa, and the TargetScan ``context scores'' on two tasks: predicting independently validated miRNA targets and predicting the decrease in mRNA abundance in miRNA overexpression assays. BayMiR performed better than all other methods in both benchmarks and, surprisingly, the variation index performed better than Cometa and some individual determinants of the TargetScan context scores. Furthermore, BayMiR predicted miRNA target sets are more consistently annotated with GO and KEGG terms than similar sized random subsets of genes with conserved miRNA seed regions. BayMiR gives higher scores to target sites residing near the poly(A) tail which strongly favors mRNA degradation using poly(A) shortening. Our work also suggests that modeling multiplicative interactions among miRNAs is important to predict endogenous mRNA targets. We develop a new computational method for predicting the target mRNAs of miRNAs. BayMiR applies a large number of mRNA expression profiles and successfully identifies the mRNA targets and miRNA activities without using miRNA expression data. The BayMiR package is publicly available and can be readily applied to any mRNA expression data sets.
    BMC Genomics 08/2013; 14(1):592. DOI:10.1186/1471-2164-14-592 · 3.99 Impact Factor
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    • "Let-7, miR-15 and miR-133 families possessed at least two members, more interestingly, these miRNAs shared similar expression profile (up-regulated). Consistent with numerous studies [36], [37], miRNAs in the same family tend to share the same expression pattern. In the present study, miR-133b and miR-206 located closely on chromosome 7, and separated by only 3766 bp fragment. "
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    ABSTRACT: Hypothalamic gonadotropin-releasing hormone (GnRH) is a major regulator of follicle-stimulating hormone (FSH) secretion in gonadotrope cell in the anterior pituitary gland. microRNAs (miRNAs) are small RNA molecules that control gene expression by imperfect binding to the 3'-untranslated region (3'-UTR) of mRNA at the post-transcriptional level. It has been proven that miRNAs play an important role in hormone response and/or regulation. However, little is known about miRNAs in the regulation of FSH secretion. In this study, primary anterior pituitary cells were treated with 100 nM GnRH. The supernatant of pituitary cell was collected for FSH determination by enzyme-linked immunosorbent assay (ELISA) at 3 hours and 6 hours post GnRH treatment respectively. Results revealed that GnRH significantly promoted FSH secretion at 3 h and 6 h post-treatment by 1.40-fold and 1.80-fold, respectively. FSHβ mRNA at 6 h post GnRH treatment significantly increased by 1.60-fold. At 6 hours, cells were collected for miRNA expression profile analysis using MiRCURY LNA Array and quantitative PCR (qPCR). Consequently, 21 up-regulated and 10 down-regulated miRNAs were identified, and qPCR verification of 10 randomly selected miRNAs showed a strong correlation with microarray results. Chromosome location analysis indicated that 8 miRNAs were mapped to chromosome 12 and 4 miRNAs to chromosome X. Target and pathway analysis showed that some miRNAs may be associated with GnRH regulation pathways. In addition, In-depth analysis indicated that 10 up-regulated and 3 down-regulated miRNAs probably target FSHβ mRNA 3'-UTR directly, including miR-361-3p, a highly conserved X-linked miRNA. Most importantly, functional experimental results showed that miR-361-3p was involved in FSH secretion regulation, and up-regulated miR-361-3p expression inhibited FSH secretion, while down-regulated miR-361-3p expression promoted FSH secretion in pig pituitary cell model. These differentially expressed miRNAs resolved in this study provide the first guide for post-transcriptional regulation of pituitary gonadotrope FSH secretion in pig, as well as in other mammals.
    PLoS ONE 02/2013; 8(2):e57156. DOI:10.1371/journal.pone.0057156 · 3.23 Impact Factor
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    • "Strains were maintained as asynchronous well-fed populations for at least one full generation prior to imaging by using standard propagation methods (Stiernagle, 2006). Strains were as described in Martinez et al. (2008), Murray et al. (2012) (see Supplemental Table 1 for details). Young embryos at or before the four-cell stage were collected and mounted using a bead-slurry approach (Bao and Murray, 2011; Murray et al., 2006). "
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    ABSTRACT: The invariant lineage ofCaenorhabditis elegans has powerful potential for quantifying developmental variability in normal and stressed embryos. Previous studies of division timing by automated lineage tracing suggested that variability in cell cycle timing is low in younger embryos, but manual lineage tracing of specific lineages suggested that variability may increase for later divisions. We developed improved automated lineage tracing methods that allowroutine lineage tracing through the last round of embryonic cell divisions and we applied these methods to trace the lineage of 18 wild-type embryos. Cell cycle lengths, division axes and cell positions are remarkably consistent among these embryos at all stages, with only slight increases in variability later in development. The resulting quantitative 4-dimensional model of embryogenesis provides a powerful reference dataset to identify defects in mutants or in embryos that have experienced environmental perturbations. We also traced the lineages of embryos imaged at higher temperatures to quantify the decay in developmental robustness under temperature stress. Developmental variability increases modestly at 25°C compared with 22°C and dramatically at 26°C, and we identify homeotic transformations in a subset of embryos grown at 26°C. The deep lineage tracing methods provide a powerful tool for analysis of normal development, gene expression and mutants and we provide a graphical user interface to allow other researchers to explore the average behavior of arbitrary cells in a reference embryo.
    Developmental Biology 12/2012; 374(1). DOI:10.1016/j.ydbio.2012.11.034 · 3.55 Impact Factor
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