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The Regulation of Neuronal Gene Expression through Alternative Pre-mRNA Splicing
Summary: Douglas Black is interested in the regulation of pre-mRNA splicing in differentiated cells, particularly neurons.
Our lab is interested in the regulation of pre-mRNA splicing and the biochemical mechanisms that control changes in splice sites. The sequences of metazoan genomes, with their relatively low gene numbers, have highlighted the question of how protein number can be expanded beyond the gene number for a complex organism. Alternative splicing, which allows the production of multiple mRNAs and hence multiple proteins from a single gene, is a major contributor to protein diversity. However, despite its key role in gene expression, this process is poorly understood mechanistically.
Alternative splicing is particularly common in genes expressed in the mammalian nervous system, where many proteins important for neuronal differentiation and function are made in diverse isoforms through controlled changes in splicing. Our lab works on a range of projects related to the control of pre-mRNA splicing in neurons. We aim to determine the mechanisms of action of splicing regulators, as well as to understand their roles in neural development and mature neuronal function.
We are focused on four regulatory factors: polypyrimidine tract–binding protein (PTB), neuronal PTB (nPTB), Fox-1 (also called A2BP1), and Fox-2 (also called RBM9). PTB and nPTB are primarily splicing repressors, while Fox-1 and Fox-2 act to enhance splicing. Each of these proteins alters the splicing of a specific set of exons within the genome. In mechanistic studies, we examine the RNA-binding properties of these proteins and analyze how they can alter spliceosome assembly. In a second effort, we use cell culture models and conditional knockout mice to understand how these proteins affect neuronal development. In a final area of study, we focus on the effect of cell excitation on the splicing of ion channel transcripts and the role of this splicing in neuronal plasticity.
Mechanisms of Splicing Repression and Derepression
Our earlier work used the neuron-specific N1 exon of the c-src gene as a model for a tissue-specific splicing event (Figure 1). Analyses of the cis-acting RNA elements controlling the exon demonstrated that the tissue specificity of N1 derives from a combination of enhancer and repressor sequences in the surrounding RNA. This combination of positive and negative regulation has proved to be a common feature in systems of tissue-specific splicing.
We use an in vitro splicing system developed in my lab to study the molecules that regulate N1 splicing. Splicing of N1 is repressed in nuclear extracts of nonneuronal HeLa cells. In contrast, splicing proceeds in extracts of WERI-1 retinoblastoma cells. Using this system, we showed that the repression of N1 exon splicing in nonneural cells requires CUCUCU repressor elements that are bound by PTB. PTB-binding sites both upstream and downstream from N1 are needed for splicing repression, and the presence of the downstream sites stabilizes PTB binding to the upstream sites. This implies that PTB assembles a cooperative repressor complex. We are analyzing the structure of this complex and its interactions with the RNA. Frédéric Allain's lab (ETH, Zurich) has shown by NMR (nuclear magnetic resonance) spectroscopy that the four RNA-binding domains of PTB each bind a pyrimidine triplet (Figure 2). The orientation of two of these domains indicates that the protein will form an RNA loop, which fits well with the placement of the repressor elements surrounding N1.
We are also using proteomic approaches to understand PTB-mediated splicing repression (Figure 3). The large pre-mRNP complexes that assemble on the N1 exon under different regulatory conditions have been purified and their components identified by mass spectrometry. Under conditions of PTB-dependent splicing repression, we find that the U1 snRNP still assembles onto the 5' splice site of the N1 exon. The essential splicing factor U2AF bound at the downstream 3' splice site is, however, blocked from assembling with components at the N1 5' splice site. These results indicate that PTB may interact with a specific spliceosomal target in repressing splicing.
PTB is largely absent from neurons, but in its place a close homolog, nPTB, is expressed (Figure 4). Neuronal PTB is 74 percent identical to PTB in sequence but is much more limited in expression: it is found in testis, some muscle cells, and in neurons in the brain. In contrast, PTB is excluded from neurons but is expressed in neuronal progenitor cells and in glia. Neuronal PTB binds to the same CUCUCU regulatory elements as PTB, but has different regulatory properties. Using splicing-sensitive microarrays designed in collaboration with Manuel Ares (UC Santa Cruz), we have identified large target exon sets for PTB and nPTB. We find that during neuronal differentiation, the switch in expression from PTB to nPTB alters the splicing of a large set of transcripts differentially dependent on these two proteins. We are interested in the biological role of this widespread change in the splicing program, and in how the choice is made during neuronal differentiation to express PTB or nPTB (Figure 5).
The Fox Proteins Are Cell-Type-Specific Splicing Enhancers
The most significant element for the enhancer activity of the region downstream of the N1 exon is the hexanucleotide UGCAUG, which is also essential for other splicing enhancers and is found downstream of a whole set of neuronally regulated exons. This element is recognized by several mammalian homologs of the C. elegans Fox-1 protein. These RNA-binding proteins are strong activators of splicing for certain neuronal exons. Through the use of multiple promoters and alternative splicing events, the mouse Fox-1 and Fox-2 genes each encode a large family of related proteins that are expressed in muscle, heart, and neurons. The human homologs of Fox-1 (A2BP1) and Fox-2 (RBM-9) have been implicated in various forms of neurological disease, implying that aspects of these disorders may arise from problems of splicing control. Thus, in addition to their mechanisms of splicing enhancement, we are interested in the cellular roles of the Fox proteins and how Fox-regulated exons might affect neuronal development and function. Using approaches similar to those used with PTB and nPTB, we are studying the Fox proteins in vitro, in cell culture, and in conditional knockout mice.
The Regulation of Ion Channel Alternative Splicing by Cell Stimulation
A poorly understood aspect of splicing regulation is how extracellular stimuli and cell signaling pathways direct changes in splicing. This is especially interesting in the nervous system, where many proteins that determine neuronal excitation are modulated by alternative splicing. To examine how specific signaling cascades alter splicing patterns, we are studying the regulation of exons in the calcium-activated potassium channel transcript (BK channel), the L-type calcium channel, and the NMDA receptor R1. We found that culturing excitable cells in depolarizing media leads to the repression of specific exons in these and other transcripts. This reduction in exon inclusion depends on the calcium/calmodulin-dependent protein kinase (CaMK) pathway and L-type calcium channels, implicating calcium signaling in this splicing repression. As a model for this regulation, we express the regulated exons from transfected minigenes, where they can be repressed by a cotransfected constitutive CaMK IV. This approach allowed us to map specific RNA regulatory elements in these transcripts that are needed to respond to CaMK signals. We identified calcium-responsive RNA elements (CaRRE-1 and CaRRE-2) that can confer CaMK-dependent repression on a heterologous exon. We are interested in the mechanism of this repression and how it responds to culture conditions. Our goal is to understand how the information of an extracellular stimulus can be transmitted to the cell nucleus to alter splicing.
Alternative splicing alters the activity of many other proteins that mediate the induction and propagation of action potentials (Figure 6). If exons are controlled by cell excitation, then these splicing events are likely one component of the plastic changes in cell activity that underlie many aspects of neurophysiology.
Last updated: August 20, 2007
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