Genome-wide identification of miRNA targets by PAR-CLIP

ABSTRACT RNA silencing pathways are conserved gene regulation mechanisms that elicit decay and/or translational repression of mRNAs complementary to short interfering RNAs and microRNAs (miRNAs). The fraction of the transcriptome regulated by these pathways is not known, but it is thought that each miRNA may have hundreds of targets. To identify transcripts regulated by silencing pathways at the genomic level, we examined mRNA expression profiles in Drosophila melanogaster cells depleted of four Argonaute paralogs (i.e., AGO1, AGO2, PIWI, or Aubergine) that play essential roles in RNA silencing. We also profiled cells depleted of the miRNA-processing enzyme Drosha. The results reveal that transcripts differentially expressed in Drosha-depleted cells have highly correlated expression in the AGO1 knockdown and are significantly enriched in predicted and validated miRNA targets. The levels of a subset of miRNA targets are also regulated by AGO2. Moreover, AGO1 and AGO2 silence the expression of a common set of mobile genetic elements. Together, these results indicate that the functional overlap between AGO1 and AGO2 in Drosophila is more important than previously thought.

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Genome-wide identification of miRNAs targets involved in cold response in cassava

Plant Omics ◽

10.21475/poj.13.01.20.p2337 ◽

2020 ◽

pp. 57-64

Author(s):

Shuxia Li ◽

Zhihao Cheng ◽

Ming Peng

Keyword(s):

Stress Response ◽

Cold Stress ◽

Stress Responses ◽

High Throughput Sequencing ◽

Target Genes ◽

Cold Response ◽

Mirna Targets ◽

Genome Wide ◽

A Genome ◽

Growth Development

MicroRNAs (miRNAs) are recognized as essential transcriptional or post-transcriptional regulators, and play versatile roles in plants growth, development and stress responses. Cassava (Manihot esculenta) is a major root crop widely grown worldwide. Cold stress seriously affects cassava plants growth, development and yield. MiRNAs and their targets have been extensively studied in model plants, but a genome-wide identification of miRNAs’ targets is still lacking in cassava. In this study, two degradome libraries were constructed using cold-treated and control cassava seedlings to identify the roles of miRNAs and their targets in response to cold stress. Following high-throughput sequencing and comparing with miRNA database, degradome data allowed us to identify a total of 151 non-redundant miRNA-target pairs. We revealed that ~ 42% of miRNA targets are conserved across plant species. However, 83 novel miRNA targets were identified in the two libraries. Gene ontology analyses showed that many target genes involved in cellular and metabolic process. In addition, 12 miRNAs and 31 corresponding targets of them were further found to be involved in cold stress response. Particularly, miR159, 164 and 396 participated in cold stress response by up-regulating certain transcription factors that were involved in the regulation of downstream gene expression. The work helps identifing cold-responsive miRNA targets in cassava and increases the number of novel targets involved in cold stress response. Furthermore, the findings of this study might provide valuable reference and new insights for understanding the functions of miRNA in stress response in plants.

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Genome-Wide Function Analysis of lincRNAs as miRNA Targets or Decoys in Plant

Plant Epigenetics - RNA Technologies ◽

10.1007/978-3-319-55520-1_8 ◽

2017 ◽

pp. 149-162 ◽

Cited By ~ 1

Author(s):

Guanglin Li ◽

Zhiqiang Hao ◽

Chunyan Fan ◽

Xianmiao Wu

Keyword(s):

Function Analysis ◽

Mirna Targets ◽

Genome Wide

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MicroRNA Screening and the Quest for Biologically Relevant Targets

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057115578837 ◽

2015 ◽

Vol 20 (8) ◽

pp. 1003-1017 ◽

Cited By ~ 20

Author(s):

Ana Eulalio ◽

Miguel Mano

Keyword(s):

Gene Expression ◽

Large Fraction ◽

Low Complexity ◽

Biologically Relevant ◽

Mirna Targets ◽

Genome Wide ◽

Eukaryotic Gene ◽

Experimental Approaches ◽

Interfering Rna

MicroRNAs (miRNAs) are a class of genome-encoded small RNAs that post-transcriptionally regulate gene expression by repressing target transcripts containing partially or fully complementary binding sites. Despite their relatively low number, miRNAs have been shown to directly regulate a large fraction of the transcriptome. In agreement with their pervasive role in the regulation of eukaryotic gene expression, miRNAs have been implicated in virtually all biological processes, including different pathologies. The use of screening technologies to systematically analyze miRNA function in cell-based assays offers a unique opportunity to gain new insights into complex biological and disease-relevant processes. Given the low complexity of the miRNome and the similarities to small interfering RNA (siRNA) screening experimental approaches, phenotypic screening using genome-wide libraries of miRNA mimics or inhibitors is not, per se, technically challenging. The identification of miRNA targets and, more importantly, the characterization of their mechanisms of action through the identification of the key targets underlying observed phenotypes remain the major challenges of this approach. This article provides an overview of cell-based screenings for miRNA function that were performed in different biological contexts. The advantages and limitations of computational and experimental approaches commonly used to identify miRNA targets are also discussed.

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Genome-Wide Analysis of miRNA targets in Brachypodium and Biomass Energy Crops

10.2172/1209217 ◽

2015 ◽

Author(s):

Pamela J. Green

Keyword(s):

Energy Crops ◽

Biomass Energy ◽

Genome Wide Analysis ◽

Mirna Targets ◽

Genome Wide

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Profiling direct mRNA-microRNA interactions using synthetic biotinylated microRNA-duplexes

10.1101/005439 ◽

2014 ◽

Cited By ~ 10

Author(s):

Shivangi Wani ◽

Nicole Cloonan

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

False Positive ◽

Negative Regulators ◽

Specific Detection ◽

High Quality ◽

Protein Coding ◽

Mirna Targets ◽

Genome Wide ◽

Genomic Scale

MicroRNAs (miRNAs) are predominantly negative regulators of gene expression that act through the RNA-induced Silencing Complex (RISC) to suppress the translation of protein coding mRNAs. Despite intense study of these regulatory molecules, the specific molecular functions of most miRNAs remain unknown, largely due to the challenge of accurately identifying miRNA targets. Reporter gene assays can determine direct interactions, but are laborious and do not scale to genome-wide screens. Genomic scale methods such as HITS-CLIP do not preserve the direct interactions, and rely on computationally derived predictions of interactions that are plagued by high false positive rates. Here we describe a protocol for the isolation of direct targets of a mature miRNA, using synthetic biotinylated miRNA duplexes. This approach allows sensitive and specific detection of miRNA-mRNA interactions, isolating high quality mRNA suitable for analysis by microarray or RNAseq.

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Identification of miRNAs associated with the follicular–luteal transition in the ruminant ovary

Reproduction ◽

10.1530/rep-12-0025 ◽

2012 ◽

Vol 144 (2) ◽

pp. 221-233 ◽

Cited By ~ 118

Author(s):

D McBride ◽

W Carré ◽

S D Sontakke ◽

C O Hogg ◽

A Law ◽

...

Keyword(s):

Granulosa Cells ◽

Expression Profiles ◽

Oestrous Cycle ◽

Corpora Lutea ◽

Follicular Cells ◽

Mirna Targets ◽

Genome Wide ◽

Ovulatory Follicles

Little is known about the involvement of microRNAs (miRNAs) in the follicular–luteal transition. The aim of this study was to identify genome-wide changes in miRNAs associated with follicular differentiation in sheep. miRNA libraries were produced from samples collected at defined stages of the ovine oestrous cycle and representing healthy growing follicles, (diameter, 4.0–5.5 mm), pre-ovulatory follicles (6.0–7.0 mm), early corpora lutea (day 3 post-oestrus) and late corpora lutea (day 9). A total of 189 miRNAs reported in sheep or other species and an additional 23 novel miRNAs were identified by sequencing these libraries. miR-21, miR-125b, let-7a and let-7b were the most abundant miRNAs overall, accounting for 40% of all miRNAs sequenced. Examination of changes in cloning frequencies across development identified nine different miRNAs whose expression decreased in association with the follicular–luteal transition and eight miRNAs whose expression increased during this transition. Expression profiles were confirmed by northern analyses, and experimentally validated targets were identified using miRTarBase. A majority of the 29 targets identified represented genes known to be actively involved in regulating follicular differentiation in vivo. Finally, luteinisation of follicular cells in vitro resulted in changes in miRNA levels that were consistent with those identified in vivo, and these changes were temporally associated with changes in the levels of putative miRNA targets in granulosa cells. In conclusion, this is the first study to characterise genome-wide miRNA profiles during different stages of follicle and luteal development. Our data identify a subset of miRNAs that are potentially important regulators of the follicular–luteal transition.

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