| February 5, 2003
The Fragile X Syndrome Protein as
RNA Distribution Hub
New Technique Tracks RNAs Associated with The Protein
Responsible for Fragile X
PA) - The process of turning genes into protein makes
the insides of cells terribly crowded and complicated
places. Signals tell machinery to transcribe the DNA
of genes into messenger RNA (mRNA) whose translation
into protein has to be coordinated with everything else
that is happening within the cell. Fortunately, there
are RNA binding proteins to organize mRNAs. These proteins
are so critical that the loss of one particular RNA
binding protein, FMRP, leads to Fragile X syndrome,
the most common inherited forms of mental retardation.
Researchers based at the University of Pennsylvania
School of Medicine invented a technique called Antibody
Positioned RNA Amplification (APRA) to determine the
identity of RNA molecules associated with RNA binding
proteins. Their findings on FMRP, presented in the February
6th issue of the journal Neuron, further define
the complex basis of Fragile X syndrome.
Fragile X syndrome is the most common inherited cause
of mental retardation in both men and women. The disorder
causes mental abnormalities that range from slight learning
disabilities to severe mental retardation. The syndrome
is caused by a mutation in what has been termed the
Fragile X mental retardation-1 (Fmr1) gene, which
encodes FMRP, the Fragile X mental retardation protein.
"RNA-binding proteins regulate all aspects of RNA
synthesis, such as mRNA transcription, splicing and
editing, as well as translation of mRNA into protein,"
said James Eberwine, PhD, professor in Penn's
Department of Pharmacology. "The mRNAs held by
FMRP encode for proteins that assist in transmitting
signals within the brain. FMRP provides cellular mRNA
traffic control, and moves selected mRNAs to sites where
they can be translated. How FMRP knows where to move
these mRNAs and how these mRNAs are released from FMRP
is unclear at present."
To study how RNA binding proteins such as FMRP function,
Eberwine and his colleagues developed a technique to
identify specific mRNAs associated with a particular
binding protein. At its basis, APRA enables researchers
to analyze an RNA binding protein's cargo on a genome-wide
In practice, APRA works a bit like a homing beacon attached
to a photocopier: Eberwine connected an antibody that
specifically binds to FMRP to a DNA molecule that can
bind to the RNA near the FMRP protein. In the presence
of enzymes, the DNA molecule helps copy these RNAs into
cDNA (a term for DNA made from RNA).
After it is synthesized, the cDNA is amplified into
hundreds of thousands of RNA molecules by an amplification
procedure also developed in the Eberwine lab a few years
ago. These amplified RNA molecules can be screened against
a microarray to identify their corresponding genes.
In this bridging of genomics (the study of the genome)
and proteomics (global analysis of proteins), the specificity
of the antibody's attraction to FMRP induces the specificity
of the RNA analysis.
Given the nature of Fragile X syndrome - and the fact
that FMRP is found only in the tissues of the central
nervous system - the researchers were encouraged to
find that among the FMRP's cargo are mRNAs encoding
proteins involved in transmitting signals between neurons
and in neuron maturation.
As a research tool, the researchers believe that APRA
analysis has great potential for researchers who want
to target specific RNA binding proteins for analysis.
Given its specificity, ARPA can track down RNA binding
proteins that are only found in certain tissues and
examine those proteins under varying physiological conditions
or disease states.
"In that sense, APRA could mean to RNA studies
as much as DNA and RNA amplification techniques have
meant to studying the genome," said Eberwine. "It
is also part of the growing frontier of molecular biology
- somewhere between genomics and proteomics is the interplay
of RNA with RNA-binding proteins."
Researchers also involved in these findings include:
lead author Kevin Miyashiro of Penn's Department of
Pharmacology; Andrea Beckel-Mitchener, T. Patrick Purk,
Ivan Jeanne Wieler, Willam T. Greenough, of the Beckman
Institute at the University of Illinois; Lei Liu of
the W.M. Keck Center for Comparative and Functional
Genomics at the University of Illinois; Salvatore Carbonetto
of the Centre for Neuroscience Research at McGill University;
and Kevin G. Becker and Tanya Barret of the DNA Array
Unit of the National Institute on Aging.
This research was funded through grants from the National
Institute on Aging and the National Institute of Mental
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