RNA-sequencing based identification of microRNA-204 targets
Migliore, Chiara Maria
With the completion of the sequencing and annotation of hundreds of genomes, and the accumulation of data on the mammalian transcriptome, greater emphasis has been placed on elucidating the function of non-coding DNA and RNA sequences. It is well known that the non-coding portion of the genome can transcribe functional RNAs. Several categories of non-coding RNAs (ncRNAs) have been defined, such as transport RNAs (tRNAs) ribosomal RNAs (rRNAs), small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). A larger group of ncRNAs comprises the so-called microRNAs (miRNAs) and long non-coding RNAs serving key regulatory roles. It has been shown that miRNAs directly target a large number of genes, thus affecting significantly major pathways. In my project, I focused on miR-204, a microRNA that is highly conserved from zebrafish to human and located in the sixth intron of the human TRPM3 gene. I sought to identify mir-204 targets by using the Medaka fish (Oryzias latipes), where mir-204 is expressed at very low levels in the nervous system, as a model for perturbation of the mir-204 network. Transient transgenic Medaka fish were produced to knock down and over-express mir-204. Next-generation sequencing was used to sequence the Medaka transcriptome, dissect the putative targets of miR-204, and thus gain further insight about its function. Potential target genes of mir-204 were selected by choosing genes, which presented lower expression in the wild-type (wt) fish than in the knock down, a lower expression in the over-expression than in the wt and, finally, a higher expression in the knock down than in the over-expression. At the same time, I collected a list of putative miR-204 mouse and human targets using the prediction softwares miRanda, PicTar and TargetScan, obtained the Medaka orthologues and verified that the selected genes in Medaka had a statistically significant enrichment in miR-204 targets as compared to the complete set of genes obtained from the RNA-Sequencing approach. The combined RNA-Sequencing and bioinformatics analysis revealed 147 predicted targets of mir-204, which showed a significant enrichment for the axon guidance pathway. In order to confirm this data, real time quantitative PCR has been performed on total RNA from wt and morphant fish. Results showed a higher expression in the knock down fish for 15 out of 25 putative targets (Neo1, Trim71, Ddx3y, Prkar1a, MyoX, Sema3B, Sema3F, Ptprg, Slit2, Epha4, Epha7, Amot, Lpp, Odz4, Jarid2). I further validated these genes by both Q-PCR and luciferase assays. To this aim, I cloned five putative target sequences into the 3’UTR of a luciferase reporter vector (pGL3-TK-luc Promega) to use them in luciferase assays: co-transfection with miR-204 reduced the luciferase activity of Sema3F, belonging to the class of receptors involved upstream of the axon guidance pathway. These results indicate that mir-204 directly targets key genes involved in the axon guidance pathway such as Sema3F in the nervous system. Further validation of the disruption of axon guidance in the transgenic fish has been undertaken in vivo by our collaborators: the experiment demonstrated a clear role of this microRNA in axon path finding during retinal development.