|Effects of Alternative Splicing at FMR1 Exon 15 on Understanding Fragile X Syndrome
by Robert Denman, 9/1/2008
Alternative splicing and
post-translational modification are two cellular
processes that subtly modify protein function.
Alternative splicing modifies the three-dimensional
structure of proteins by making small additions or
deletions to its primary structure. Post-translational
modification marks proteins with unique chemical tags
(methyl, phosphate sugar groups etc.) The fragile X
mental retardation protein, FMRP, is affected by both
processes; however, the consequence each has on FMRP's
function is not well-known.
The focus of our present studies is the exon 15-encoded
residues of FMRP, which are alternatively spliced and multiply modified post-translationally.
We have recently found that Fmr1 alternative splicing at both exon 12 and exon 15 are developmentally regulated and change predictably during neuronal differentiation. We have also determined that post-translational methylation of FMRP’s RG-rich region is modulated by exon 15 alternative splicing and are currently investigating the effect a potential "modifier" protein, MSP58, plays in controlling these processes.
As our objective is to relate both alternative splicing and arginine methylation to fundamental processes in neurons we have recently begun to determine whether particular arginine methyltransferases are found in dendritic processes and whether arginine methylation can be locally modulated.
Completing these studies will further our understanding of the role FMRP plays in normal cellular metabolism and may lead to new targets for future drug treatments.
Xie, W., Dolzhanskaya, N., La Fauci, G., Dobkin, C. and Denman, R. B.: Tissue-specific and Developmental Expression of Fmr1 Exon 12 and 15 Isoform mRNAs (2009) Neurobiology of Disease 35, 52-62.
by Robert Denman, 11/2003
All cells are composed of a variety of specialized structures and compartments. These structures and compartments perform the myriad of functions that we define as living. The master plan for each cell, that is, the general instructions that must be carried out for the cell to "live" and the auxiliary instructions it must carry out to perform specialized functions, is encoded in the cell's genetic information (DNA). For cells to perform these tasks, the genetic information is copied into messages (mRNA) that are in turn translated into proteins, which actually do the functioning.
We have identified some of the messages that may be responsible for the diverse behavioral features of fragile X syndrome. These messages bind to the protein FMRP, the fragile X mental retardation protein, which is absent in people with this syndrome. We call these messages the targets of FMRP.
We have gone on to show in several select cases that FMRP acts as a restrictor valve on the cell's protein synthesis machinery to regulate the normal levels of the proteins produced from these messages. When FMRP is present, the messages are fed slowly through the restrictor, and the corresponding proteins are made in low to moderate amounts. In the absence of FMRP, much greater amounts of the proteins are produced. Because protein levels are finely balanced, or tuned, in cells to orchestrate the many functions they perform, too much or too little of a particular protein may not be beneficial. FMRP alone may regulate more than 500 messages; therefore, its loss causes a significant disturbance to the normal balance of proteins.
We are attempting to answer the questions:
- How does FMRP bind particular
messages? Since not all messages bind to FMRP, what
enables this protein to bind one message and not
- Do the interactions between
FMRP and these messages change over time, and if they
do, what is responsible for these changes?
- How do the answers to the above two questions add to our understanding of the role FMRP plays in cells? Can we make a model of how FMRP works that will allow us to predict what is going wrong in individuals with fragile X syndrome?
We have found that two proteins (called PRMT and CK2) that communicate signals from outside the cell to information processing centers inside the cell can modify one of FMRP's message-binding sites and, in so doing, influence its ability to regulate the messages it binds.
No one before has ever modified FMRP in cells and found that doing so changes its basic properties.
We have shown that altering one of FMRP’s message binding sites dictates whether a particular message binds to it. Our results provide a framework for understanding how FMRP functions, i.e., how specific messages interact and are regulated.
Now that we know that PRMT and CK2 can potentially modify several sites in FMRP, we must identify which of these sites is the major site of modification and determine whether it changes when cells are perturbed in different ways.
Sung, Y-J., Dolzhanskaya, N., Nolin, S., Brown, W. T., and Denman, R.B.: The Fragile X Mental Retardation Protein FMRP Binds Elongation Factor lA mRNA and Negatively Regulates its Translation in vivo. (2003) Journal of Biological Chemistry 278, 15669-15678.
Dolzhanskaya, N., Merz, G., Aletta, J., and Denman, R. B.: Methylation Regulates its Intracellular Protein-Protein and Protein RNA Interactions of FMRP. (2006) Journal of Cell Science 119, 1933-1946.
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