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Summary: Paula Grabowski uses biochemical and genetic approaches to study alternative RNA splicing in the mammalian nervous system. She applies information from model systems to understand how splicing responds to different cell types and stages of development, and the underlying rationale for splicing errors in inherited disease.

Paula Grabowski uses biochemical and genetic approaches to study alternative RNA splicing in the mammalian nervous system. She applies information from model systems to understand how splicing responds to different cell types and stages of development, and the underlying rationale for splicing errors in inherited disease.

We are interested in understanding alternative RNA splicing, a flexible, post-transcriptional mechanism that joins exons together in different arrangements to generate multiple mRNAs from a single gene. This genetic process generates proteomic diversity in the nervous system, where it has roles in establishing the functions of neurotransmitter receptors, ion channels, and transporters at the synapse. A variety of neurological diseases and cancers are associated with errors in splicing, but their underlying mechanisms are poorly understood.

	Tissue-specific control mechanisms... Alternative splicing control...

Our strategy has been to dissect the functional architecture of the RNA control regions of neurotransmitter receptor transcripts. We then apply this information to determine the roles of protein factors and the influence of cellular context. Mechanisms studied in detail include the neuron-specific exon of the GABAA receptor and the NI and CI cassette exons of the N-methyl-D-aspartate (NMDA) R1 receptor.

We have learned that RNA control regions are complex entities capable of engaging in positive and negative control. The neuron-specific exon of the GABAA receptor transcript is modulated in part by an intronic splicing silencer region and the polypyrimidine tract RNA-binding protein (PTB). PTB is also a silencing factor in the mechanism involving the NI cassette exon of the NMDA R1 receptor. For the most part, PTB is widely expressed, but in the rat brain its expression levels change dramatically from fetal to adult development. These results suggest that PTB plays an important role in the silencing of certain neuron-specific transcripts early in brain development, and that silencing diminishes as development proceeds.

Our recent studies have focused on the RNA-binding protein NAPOR/CUGBP2 (neuroblastoma- and apoptosis-related RNA-binding protein/CUG-binding protein 2), which is involved in the control of the NI and CI cassette exons. These splicing mechanisms are specific to regions of the brain, and NAPOR/CUGBP2 is implicated as a controlling factor in the forebrain, where its expression is enriched. In this case, an RNA-binding protein appears to engage in either silencing or enhancement, depending on the nature of the RNA control region. Our current experiments are aimed at understanding how NAPOR/CUGBP2 can exhibit dual modes of control. The protein structure contains three RNA-binding domains (RRMs), with a linker region separating the second and third RRMs. Mutations have been generated throughout the protein structure to test for their effects on alternative splicing of the NI and CI exons. One hypothetical scenario that we are testing is that neighboring protein factors bound to the RNA control region define the function of NAPOR/CUGBP2.

One such factor, PTB, binds in the neighborhood of NAPOR/CUGBP2 in the intronic splicing silencer near the NI exon. A prediction of this model, that PTB and NAPOR/CUGBP2 should interact, has been demonstrated.

The expression of NAPOR/CUGBP2 is enriched in the rat forebrain, where it is likely to have additional targets of regulation at the level of RNA splicing. To test our working model and to determine how alternative splicing is affected in a global sense, we are constructing transgenic mice lacking NAPOR/CUGBP2. These mice will also be used to study the effects of the knockout on nervous system development, behavior, and memory.

Last updated July 11, 2003