Alternative pre-mRNA splicing is prevalent throughout vertebrate genomes where an individual gene can be diversified into hundreds or even thousands of related mRNA isoforms 
. Functional consequences of alternative splicing can involve changes to a subset of the protein's biochemical properties or subcellular localization. These are powerful mechanisms used to regulate protein functions across different cell types, during development, or in response to extracellular signals 
. One of the major challenges in postgenome biology is to understand how alternative splicing, which involves a high degree of inherent flexibility, can achieve the specificity needed to select the correct set of target transcripts for regulation.
The spliceosome is the functional context for regulation, since this is the macromolecular machinery that guides intron removal and exon joining. It is assembled from the dynamic associations of five small nuclear ribonucleoprotein particles (snRNPs) and hundreds of accessory factors 
. Initially, U1 snRNP and U2AF (U2 snRNP auxiliary factor) recognize the 5′ and 3′ splice sites of the exon, respectively, and U2 snRNP base pairs with the branch site region thereby designating the adenosine to be used as the branchpoint. The association of U456 tri-snRNP and various RNA rearrangements then activate the first step of catalysis, which generates the 5′ exon and lariat intron-3′ exon intermediate. As catalysis advances to the second step, the lariat intron is excised and the 5′ and 3′ exons are ligated. The overall pattern of exon inclusion/skipping depends on the ability of the spliceosome to recognize each splice site signal, which is a reflection of the inherent strength of the site as well as the regulatory effects of splicing factors acting from sequence motifs nearby 
. Exon definition, which involves the interactions of U1 snRNP bound to the 5′ splice site and U2AF bound to the 3′ splice site across an exon is a particularly sensitive mechanism to specify alternative splicing patterns 
Two families of RNA binding proteins known to regulate alternative splicing by direct recognition of RNA sequence motifs include the arginine-serine rich (SR) and hnRNP splicing factors 
. SR splicing factors most commonly recognize exonic splicing enhancers, whereas hnRNP proteins recognize intronic or exonic splicing silencers and enhancers. These regulatory motifs are typically short and degenerate making it difficult to reliably predict the target exons of a splicing factor based upon sequence inspection alone. CUG Binding Protein 2, or CUGBP2 (also called NAPOR, CELF-2, ETR-3, or BRUNOL3) is a member of the larger family of CUGBP and ETR-3-like (CELF) RNA binding proteins, which have been shown to regulate alternative splicing through UG-rich motifs in accordance with their tissue-specific expression patterns 
. CELF proteins have been shown to play important roles in heart development, whereas their misregulation has been implicated in the pathogenesis of myotonic dystrophy 
We previously reported a close correlation between the distribution of protein expression patterns of CUGBP2 (called NAPOR in the previous study) and splicing patterns of the NI and CI cassette exons of the NMDA R1 receptor transcript (GRIN1
gene) in the rat brain 
. In particular, high levels of CUGBP2 protein in the forebrain were associated with skipping of the NI exon and inclusion of the CI exon, and these splicing patterns reversed in the hindbrain where CUGBP2 was deficient. In vivo
splicing reporter assays confirmed dual functional roles for CUGBP2 as a splicing silencer of the NI exon and as an enhancer of the CI exon. These dual roles are thought to be important in directing the brain region-specific distribution of GRIN1 mRNA isoforms for fine-tuning of receptor functions at the synapse 
. Additional splicing factors have been shown to exhibit dual roles in enhancement and silencing depending upon the context of the target exons, but these mechanisms are, at present, poorly understood 
In this study, we focus on the silencing face of CUGBP2's dual character to understand how it recognizes the NI target exon and the mechanism used for splicing silencing. A variety of intronic UG-rich motifs can be found within several hundred nucleotides of the NI and CI exons by sequence inspection, but functionally significant motifs in these regions have not yet been identified. We initially used a chemical-based RNA footprinting approach to detect RNA-protein interactions at nucleotide-level resolution. Here we identify the direct contact sites of CUGBP2 in the 3′ splice site region of the NI exon, and establish that this arrangement of binding sites plays a mechanistic role in silencing a group of branch sites in between. We show the significance of this mechanism by demonstrating its involvement in the regulation of other skipped exons, most notably exon 6 of the CUGBP2 transcript itself.