Noncoding rnas in neurons.

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Lab meeting - 28th October 2009

Main Project:
Secondary Project: Piwi proteins and piRNAs.

Rodrigo Louro

Characterization of neuronal noncoding RNA-protein complexes expressed during axonal injury

Rodrigo Louro

Aim

Neuronal ncRNPs in axonal injury

The designed approach aim to provide insights into the mechanism of action and biological role ncRNA-protein complexes in thenormal and injured mammalian brain. •

Isolate somal and axonal compartments - Microfluidics Chambers (Taylor et al. 2005; Park et al. 2006) to idependently perform gene expression analysis;



Identify using Solexa sequencing:
1- RNAs (ncRNAs and mRNAs) preferentially localized in axons (and in soma); 2- RNAs synthesized in response to axonal injury (and regeneration) to study theirinvolvement in neurodegenerative diseases.



Other biochemical techniques will identify RBP complement of a few ncRNAs.

Background •


Neuronal ncRNPs in axonal injury

Regulatory ncRNAs seem to be pivotal to the evolution of complex attributes – different species have a very similar set (with a similar number) of protein-coding genes;
ncRNAs control gene expression acting attranscriptional, post-transcriptional, and translational levels. Chromatin rearrangements (Plath et al. 2003; Rinn et al. 2007), promoter assembly interference (Martianov et al. 2007; Wang et al. 2008), alternative splicing (Yan et al. 2005; Hirose et al. 2006), and repression (Pillai et al. 2007) or activation of translation (Vasudevan et al. 2007); Cell diversity, environmental responsiveness, neuralconnections, and synaptic plasticity - the human/mammalian brain - the most extreme complexity among organs (Abrous et al. 2005); ncRNAs are overly represented in the nervous system (Mehler and Mattick 2006); Axons can be very long (e.g. ~1m single cell sciatic nerve) - post-transcriptional regulation in neurons must be highly compartmentalized and respond to local environment;



• •

•This regulation its not completely understood - RBPs regulate these steps (Ule and Darnell 2006) are known to be involved, and local mRNA translation has been demonstrated to occur during axon regeneration (Willis and Twiss 2006);
Regulatory roles could be performed by the RNA itself or by interaction with RNA binding proteins (RBPs) - ncRNA as a molecular adaptor – specificity (Amaral et al 2008);Such complexes are perturbed in neurological syndromes - FMRP (Fragile X Mental Retardation Protein) (Mehler and Mattick 2007).





Aim

Neuronal ncRNPs in axonal injury

The designed approach aim to provide insights into the mechanism of action and biological role ncRNA-protein complexes in the normal and injured mammalian brain. • •

Isolate somal and axonal compartments -Microfluidics Chambers (Taylor et al. 2005; Park et al. 2006) to idependently perform gene expression analysis; Identify using Solexa sequencing: 1- RNAs (ncRNAs and mRNAs) preferentially localized in axons (and in soma); 2- RNAs synthesized in response to axonal injury (and regeneration) to study their involvement in neurodegenerative diseases.



Other biochemical techniques will identify RBPcomplement of a few ncRNAs.

Microfluidics Chambers

Neuronal ncRNPs in axonal injury

http://www.jove.com/index/details.stp?ID=305

Neuronal Primary Culture

Neuronal ncRNPs in axonal injury

http://www.jove.com/index/Details.stp?ID=269

Methodology

Neuronal ncRNPs in axonal injury

First year

Neuronal Primary Culture (E18 mouse)

RNA extraction from each compartmentSolexa cDNA libraries construction

Data mining

Axotomy to mimic axonal injury

RNA extraction from soma – different periods

Solexa cDNA libraries construction

Data mining

Neuronal Primary Culture

Neuronal ncRNPs in axonal injury

Tiago Ferreira, Dr. Cornelius Gross (EMBL-Monterotondo) Agnès Belly and Gaëlle Lachenal, Rémy Sadoul (Grenoble Institut des Neurosciences) • Mouse...
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