Pubblicazioni Aperte DIgitali Sapienza > Biologia e Biotecnologie "Charles Darwin" > GENETICA E BIOLOGIA MOLECOLARE >

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Title: Control of muscle differentiation in normal and pathological condition: the role of dystrophin and non coding RNAs
Tutor: Bozzoni, Irene
Issue Date: 5-Feb-2014
Abstract: Muscle differentiation is an excellent system to study the mechanisms of transcriptional and post-transcriptional gene regulation in vertebrates. A regulatory circuitry in which competing endogenous RNAs (ceRNAs) act as a sponges to micro-RNAs was first demonstrated in in-vitro mouse myoblast differentiation for the muscle specific pro-myogenic long non coding RNA, linc-MD1. Here we characterized linc-hMD1, the human homologue of murine linc-MD1. We demonstrated that linc-hMD1 is down regulated in Dunchenne Muscular Dystrophy (DMD) myoblast and it is rescued towards wild type levels in exon skipping treated cells. We showed that it can act as a sponge for miR-133. One of the interesting features of linc-hMD1 is that it is also the host transcript for miR-133b and the biogenesis of these two non-coding RNAs is mutually exclusive. Towards this we showed that the alternative biogenesis of linc-hMD1 and miR-133b is regulated post transcriptionally through binding of HuR protein to pri-linc-hMD1 transcript. We studied the physiological relevance of this regulatory circuitry in human myoblast differentiation and showed that sponging activity of linc-hMD1 occurs during early stages of differentiation while at later stages,linc-hMD1 acts as a precursor for miR-133b.
Research interests: My general interest lies in the nucleic acid metabolism. I carried out my PhD research on role of non coding RNAs in normal muscle differentiation and in Duchenne Muscular Dystrophy. I am interested in gene regulation that goes inside the cell.
Skills short description: Molecular biology: Primer design, PCR, Digestion, Ligation, Transformation, Cloning, Mutagenesis, DNA extraction (Mini and Midi prep), quantification and gel analysis, RNA extraction, Reverse transcription, Rapid Amplification of cDNA Ends (RACE), Semi quantitative and Real time quantitative PCR , in-vitro transcription, shRNAs and siRNAs design for RNAi in mammalian cell. Biochemistry: Western blotting, Northern blotting, co-immunoprecipitation, RNA immunoprecipitation, Luciferase assay Cell biology and Microscopy: Grow, pass and collect C2C12 , Hela and human myoblast cells. Lipofectamine transfection. Nuclear and cytoplasmic fractionation of cellular protein and RNA. Light and Fluorescence microscopy. DAPI staining to visualize nucleus Analytical methods: Recombinant protein expression in bacteria and purification using Aktaprime, gel filtration chromatography, Spectrophotometry

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