Projects

1. Characterization of the role of the neuronal Hu RNA-binding proteins in the regulation of mRNA during the early development of the zebrafish.

We have developed a novel technique in the laboratory that allows the identification of genuine, in vivo RNA targets for any chosen RNA binding protein. In brief, the fundamental principle of the method is the use of UV photocrosslinking to “freeze” RNA-protein interactions in fresh brain tissue. These stable complexes can be purified and the RNA sequence surrounding the site of crosslinking identified by sequencing; thus both the identity of the mRNA and the site of interaction with the RNA binding protein are known. The laboratory is working to validate candidate mRNAs identified by our crosslinking method as interacting with the Hu proteins, and experiments will cover a wide variety of techniques in molecular biology, biochemistry and genetics.

We have used our novel RNA-protein crosslinking method to identify candidate mRNA regulated by the neuronal Hu proteins in early postnatal mouse brain tissue and in 24 hfp zebrafish neurons. Approximately 60% of the RNA “tags” identified by Hu-CLIP arose from message 3’ UTRs and a significant fraction of these 3’ UTR tags belonged to mRNAs whose proteins carry out essential functions at the neuronal growth cone and in dendrites. Surprisingly, these tags did not contain AU-rich elements previously shown to bind Hu; instead the tags showed high similarity to the known dendritic targeting elements in the 3’ UTRs of ?-CamKII and neurogranin. These preliminary data suggest that the neuronal Hu proteins may be key factors in targeting a functionally related subset of mRNAs to neurites. Several of the mRNAs identified by Hu-CLIP are known from published experimental evidence to be localized to growth cones and dendrites in neurons.

A current focus of the laboratory of is the validation of these potential targets of the neuronal Hu proteins. Research in the laboratory is broadly split into genetic experiments using the model organism Danio rerio (zebrafish), and biochemical and cell biological experiments using cultured neurons and a variety of in vitro systems.

Biochemical experiments will include binding assays and RNA mutagenesis experiments to determine the 3’ UTR sequence elements necessary for high affinity binding to Hu, and the construction of reporter expression constructs to determine the biological effects of particular 3’ UTR sequences on mRNA localization and protein expression. Cell Biology experiments include using an established rat hippocampal neuronal culture system and assaying, using a variety of reporters and Hu protein mutants, the regulatory contribution of the neuronal Hu proteins on our putative 3’ UTR targets. Genetic experiments will involve analyzing HuC and HuD zebrafish knockdowns. The HuC and HuD knockdown fish have distinct developmental defects and we are using whole mount immunoflorescence and FISH techniques to better understand how the loss of HuC and HuD affect known developmental markers. Also, we will soon begin to analyze how the loss of HuC and HuD affect the regulation of messages in which we have found CLIP tags.

It is strongly recommended that you make an appointment to speak with Kirk Jensen about the possibility of doing Honours in the lab, and about the exact nature of the projects available.

Funding Sources

NHMRC Project Grant 349540. Regulation of pre-mRNA and mRNA processing by the neuron specific Hu RNA binding proteins.

ARC Discovery Grant DP0559550. The role of the neuronal Hu proteins in the regulation of the BMP signalling pathway.

Member. ARC Special Research Centre, the Centre for the Molecular Genetics of Development.