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Weill Medical College Team Studies Role of miRNAs in Fragile X Syndrome

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Name: Samie Jaffrey
Position: Associate professor, pharmacology, Weill Medical College of Cornell University
  

 
Samie Jaffrey’s lab at Weill Medical College of Cornell University focuses on the molecular mechanisms underlying neuronal growth, development, and synaptogenesis. As part of that work, Jaffrey and his colleagues investigate the role of abnormal axonal growth and synapse formation in autism and other mental disorders.
 
Last year, the National Institutes of Health issued a call for research projects investigating the role of microRNAs and other non-coding RNAs in mental disorders (see RNAi News, 5/18/2006).
 
In response to this, Jaffrey proposed a research effort looking at the role of miRNAs in Fragile X syndrome, a common cause of inherited mental retardation. Early this month, the NIH awarded him a five-year grant, worth $399,000 in its first year, to investigate this link.
 
This week, RNAi News spoke with Jaffrey about his lab and the NIH-funded project.
 
Let’s start with an overview of your lab.
 
The focus of my laboratory is on the role of RNA and mRNA in axons and developing axons. …
 
There is a considerable amount of evidence that [axonal] processes are affected in people who have epilepsy, mental retardation, autism, and other neuro-developmental disorders. One of the recent discoveries over the last five years has been that the ability of axons to perform [their] functions is dependent on the presence of messenger RNAs inside the axon.
 
Normally, messenger RNAs are thought to exist in the cell bodies and be translated directly to proteins. If an axon needs a protein, those proteins are thought to be shipped from the cell body to the axon. However, it is now becoming clear that in some cases there are mRNAs that are resident within axons, and those are translated into proteins … often in response to the exposure of the axon to certain environmental stimuli, [such as] growth factors or a variety of [other] molecules.
 
So the axons are able to detect molecules in their environment and transduce those signals into specific mRNA translation events. Those events seem to be critical for [axonal processes].
 
Our lab [is addressing] several questions, including, “What are the mRNAs in axons, why are they there, how do extracellular signals regulate the translation of mRNAs, what … happens when this translation process is affected … and what are the mechanisms by which these mRNAs are regulated?”
 
Recently, we’ve found evidence that there are microRNAs directly within axons, and that the machinery that microRNAs utilize to regulate mRNA translation is also present within axons. The finding of both microRNAs and microRNA machinery suggests that many of the processes within axons might utilize microRNA-dependent regulation. One area of the lab is to understand that process.
 
That is where the recent grant comes in.
 
The grant we were recently awarded has as one of its parts [the goal of understanding] microRNAs within axons [and their impact on] axon functioning. We imagine that understanding how microRNAs regulate mRNA translation in axons will give us insight into axonal pathfinding and synaptogenesis abnormalities, and may [shed light] on a variety of neurodegenerative disorders such as epilepsy, autism, and mental retardation.
 
The second thing our lab is interested in is Fragile X syndrome, [which] is a form of mental retardation. There is evidence that neuronal and synaptic abnormalities [are associated with] this disorder, and one of the questions [we hope to answer], is whether or not defective microRNA regulation within axons accounts for some of the abnormalities seen in these patients.
 
Fragile X is the most common form of mental retardation [and] is due to an inherited gene defect. … The protein that is mutated [in the disorder, called fragile X mental retardation protein] … is known to associate with microRNA machinery. These patients are missing that protein, and it’s very possible that defects in the ability of microRNAs to be processed or transported or to regulate their target mRNAs underlie the pathogenesis of this disorder.
 
This disease may intrinsically be a disease of impaired microRNA-dependent regulation. One of the major goals of our research is to determine the role of the FMRP protein in microRNA-dependent translational regulation.
 
Another facet of Fragile X syndrome … is that it is due to a trinucleotide repeat — a CGG repeated many times. In people who have more than 200 copies of the repeat, they get silencing of the gene [encoding FMRP]. … A critical finding came a few years ago … [with the demonstration] that RNAs that contain these CGG repeats are able to form secondary structures that can be processed by Dicer. … In this case, it looks like Dicer can, albeit with reduced efficiency compared to endogenous microRNA hairpins, process this mRNA into small, CGG-rich small RNAs.
 
Some recent findings have demonstrated that microRNAs that elicit transcript silencing can also elicit methylation of the promoter from which the microRNA derives. This mechanism is not completely understood, but it raises the possibility that the microRNAs that are derived from these CGG-repeat sequences due to Dicer-dependent processing might actually mediate the methylation seen in the disease.
 
The critical question in Fragile X syndrome is, “How does CGG-repeat expansion in the mRNA result in methylation of the promoter?” One possibility is that small RNAs that are generated from the CGG repeats are a critical component of the pathway that mediates silencing of the Fragile X promoter. That is another critical aspect of this grant. …
 
This project is particularly important because, in principle, reversal of the methylation status of the promoter could normalize FMRP expression levels, and that could presumably result in normalization of the cognitive defects of this disease.
 
If we can understand how these CGG repeats regulate methylation of the promoter, it could be very critical for this disease. Our questions [include], “Is Dicer required for the ability of the expanded CGG repeats to silence the promoter? Are microRNA proteins required?” [Answers to] those sorts of questions could help us determine if the microRNA pathway is responsible for the methylation of [the FMRP] promoter.
 
Where do you begin to address these issues? Do you start looking for possible microRNAs bioinformatically? What is the first step?
 
The first step is to do a total analysis of all the microRNAs present in the axon. We’re using a microRNA-profiling approach in collaboration with … Azim Surani. He is at the Gurdon Institute in the UK and has developed techniques to profile microRNAs.
 
The other thing is we know of certain mRNAs within axons that are critical for axonal function during [their] development. … Some of those mRNAs have an unusually high enrichment of microRNA binding sites. One question we have is, “If we re-engineer the neurons so that the endogenous transcripts no longer express, but a heterologous transcript that is deficient in these microRNA binding sites is expressed in their place, do axons exhibit abnormal properties?”
 
We can start from both a screen-based approach where we look at the complement of microRNAs that are present within axons, and we can also [take] an approach where we look at specific mRNAs that we think are likely to be regulated by microRNAs and examine how the properties of the axons change in the absence of those microRNAs. To confirm that, we can do microRNA-specific knockdown.
 
Lastly, we are collaborating with Brian Harfe at the University of Florida to look at mice that contain a floxed Dicer allele in order to determine if their axonal guidance properties and synaptogenesis properties are impaired in the absence of Dicer activity.

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