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Analysis of RNA associated with the RISC complex (Ago2) in four day old mice carrying a mutation in miR-96

ABSTRACT: The diminuendo mouse carries a mutation in the seed region of miR-96. Homozygotes are deaf and exhibit vestibular dysfunction, heterozygotes display rapidly progressive hearing loss. Some targets of miR-96 are known, but it is likely that many still remain to be discovered. MicroRNAs function by recruiting the RISC complex to their mRNA targets, so one way to examine the mRNAs being controlled by microRNAs in a cell is to immunoprecipitate Ago2, one of the RISC proteins, and extract the RNA bound to it. This should detect all the mRNAs being targetted by microRNAs in the tissue. We extracted mRNA bound to Ago2 from the organs of Corti and olfactory bulbs of diminuendo homozygote and wildtype littermates at four days old, and carried out microarray analysis.

INSTRUMENT(S): Beadarray reader

ORGANISM(S): Mus musculus

SUBMITTER: Morag Lewis

PROVIDER: E-MTAB-3737 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Exploring regulatory networks of miR-96 in the developing inner ear.

Lewis Morag A MA Buniello Annalisa A Hilton Jennifer M JM Zhu Fei F Zhang William I WI Evans Stephanie S van Dongen Stijn S Enright Anton J AJ Steel Karen P KP

Scientific reports 20160318

Mutations in the microRNA Mir96 cause deafness in mice and humans. In the diminuendo mouse, which carries a single base pair change in the seed region of miR-96, the sensory hair cells crucial for hearing fail to develop fully and retain immature characteristics, suggesting that miR-96 is important for coordinating hair cell maturation. Our previous transcriptional analyses show that many genes are misregulated in the diminuendo inner ear and we report here further misregulated genes. We have ch ...[more]

PMID: 26988146

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Remodeling of Ago2-mRNA interactions upon cellular stress reflects miRNA complementarity and correlates with altered translation rates (part 3)

Project description:When adapting to environmental stress, cells attenuate and reprogram their translational output. In part, these altered translation profiles are established through changes in the interactions between RNA-binding proteins and mRNAs. The Ago2/microRNA machinery has been shown to participate in stress-induced translational upregulation of a particular mRNA, CAT-1; however, a detailed, transcriptome-wide understanding of the involvement of Ago2 in the process has been lacking. Here, we profiled the overall changes in Ago2-mRNA interactions upon arsenite stress by CLIP-seq. Ago2 displayed a significant remodeling of its transcript occupancy, with the majority of 3` UTR and CDS sites exhibiting stronger interaction. Interestingly, target sites that were destined for release from Ago2 upon stress were depleted in miRNA complementarity signatures, suggesting an alternative mode of interaction. To compare the changes in Ago2 binding patterns across transcripts with changes in their translational states, we measured mRNA profiles on ribosome/polysome gradients by RNA-seq. Increased Ago2 occupancy correlated with stronger repression of translation for those mRNAs, as evidenced by a shift toward lighter gradient fractions upon stress, while release of Ago2 was associated with the limited number of transcripts that remained translated. Taken together, these data point to a role for Ago2 and the mammalian microRNAs in mediating the translational component of the stress response. In this sub-series, CLIP and RNAseq data from the hippuristanol treatment experiment are presented. Experiments on 293S cells +/- hippuristanol treatment, in 2 biological replicates. Here, for each treatment/replicate sample, an aliquot was used for RNA-seq, and the rest was split into three aliquots to perform 3 parallel CLIP-seq protocols with different antibodies. So, each RNA-seq dataset here corresponds to 3 CLIP-seq datasets.

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