Validation of CAGE predicted miRNA target sites and target genes in an in vivo vertebrate embryo model

Tyrrell, Christopher Peter (2015). Validation of CAGE predicted miRNA target sites and target genes in an in vivo vertebrate embryo model. University of Birmingham. M.Res.

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Abstract

MicroRNAs (miRNAs) are small, non-coding regulatory RNAs that regulate gene expression. miRNAs exert inhibitory effects on gene expression via complementary binding to cognate messenger RNA (mRNA) transcripts, and subsequent degradation of the targeted transcripts. miRNAs are abundant and have many thousands of potential gene targets – only a few of which are true targets. This project involves testing a potential enhancement in the prediction of miRNA target sites via cap analysis gene expression (CAGE) tags, possibly conferring increased specificity in miRNA predictions. To test this potential prediction tool, unique CAGE predicted miRNA target sites are identified and 1 cell stage Danio rerio embryos are injected with RNA constructs containing the predicted miRNA target site linked to a fluorescent probe. Both ‘wild type’ and ‘mutant’ target sites are injected, and expression patterns of the target gene observed to confirm the existence of a ‘true’ predicted miRNA target site. Two CAGE predicted target sites were tested (cxcr7b and nploc4) and both validated as true target sites, indicated by differential gene expression patterns seen between WT and mutant target sites (significantly reduced expression is seen in WT embryos due to miRNA mediated degradation). These results show a potential for future use of CAGE tags in miRNA prediction.

Type of Work:

Thesis (Masters by Research > M.Res.)

Award Type:

Masters by Research > M.Res.

Supervisor(s):