Studying miRNA mediated effect on regulation of the MECP2 gene expression

Summary

In this project the possible role of microRNAs on the pathogenesis of Rett syndrome will be studied.

Supervisor(s)

Professor John Christodoulou

Research Location

The Children's Hospital at Westmead Clinical School

Program Type

PHD

Synopsis

MicroRNAs (miRNA) are a large family of non-coding regulatory RNA molecules. They are approximately 21-23 nucleotides long with important roles in regulating post-transcriptional gene expression by reducing the level of protein production from specific target mRNAs. miRNAs have important roles in diverse developmental, biological, and physiological processes. In mammals, miRNAs expression is tissue-specific and may possess functions specific for organs or cell types. Inappropriate regulation of miRNA expression has been linked to many types of cancer, and recent evidence suggests multiple modes of miRNA-mediated regulation, including translational inhibition, increased mRNA de-adenylation and degradation or sequestration. Rett syndrome a neurodevelopmental disorder, with most patients having mutations in X-linked MECP2 gene coding for the methyl-CpG-binding protein 2 (MeCP2).   MECP2 mRNA, specifically the 10 kb isoform, is abundant in the mouse brain and the protein is highly expressed in the hippocampus, cerebellum, and the cortex. The 3’UTR of the MECP2 mRNA is a likely target for miRNA regulation. miRNAs such as 29:has-miR-22, 77:has-miR-30a-3P, 70:has-miR-425, 7256:has-miR-197 and 7292:has-miR-197 potentially specifically target the MECP2 3’UTR. Preliminary data has identified several previously undescribed transcripts that appear to be miRNA.  This project will also involve identifying and characterisation novel RNA produced from the MECP2 locus. This project will focus on exploring the potential role that miRNAs play in the pathogenesis of Rett syndrome.  Research studies will include:

1. Identification and characterization of novel RNA molecules transcribed from the MECP2 locus
2. assessment of the expression of miRNA and novel MeCP2 transcripts in brain, heart, lung, and other tissues by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using the TaqMan and or Whole mount in situ hybridization.
3. Using DNA from Rett syndrome patients who are negative for mutations in the MECP2 coding region, the MECP2 3’UTR region containing the sequences specific for the above miRNAs will be examined for sequence variations which could interfere with miRNA – MECP2 interactions.
4. Where such sequence variations are identified, patient cell lines will be used to determine the levels of the relevant miRNA, and the effect on MeCP2 protein synthesis will be assessed by quantitative Western blot or ELISA system. In addition, experiments will be designed to examine the effect on of these 3’UTR sequence variations on protein synthesis by constructing a reporter vector containing the variant 3’-UTR sequence.

These studies provide valuable information about the role of miRNA in regulating the level of MeCP2 expression, and may lead to the identification of potentially pathogenic sequence variations in the 3’UTR of the MECP2 gene.

Additional Information

Techniques used in the project: A range of molecular genetic techniques including high resolution melt analysis and mutation screening

Current PhD topics: Studying mutations in CDKL5/STK9 and its consequences in humans

• Development and study of mouse models for CDKL5 deficiency
• Structural and Functional Studies of Unusual MECP2 Sequence Variations in Rett Syndrome
• Mutation Screening in Rett Syndrome and Related Clinical Disorders

Keywords

Rett syndrome, X-linked disorders, mental retardation, neurodevelopment, brain, genetic disorder, neurogenetics, developmental brain disorders, methyl CpG-binding protein 2, MECP2, mutation, microRNA, pathogenic mechanisms, brain development, neural regulation, Brain & nervous system disorders, Genetic disorders, Infertility & developmental problems, Genes in biology & medicine, Neuroscience & psychology, Reproduction & development

Opportunity ID

The opportunity ID for this research opportunity is: 51

Other opportunities with Professor John Christodoulou

• Molecular and functional studies in the mitochondrial respiratory chain disorders
• The Role of CDKL5 in the pathophysiology of Rett syndrome and related disorders