g., Tanc2, Ppp1r12a, Add1) or adult exon skipping (e.g., Kcnma1, Csnk1d, Cacna1d). Some of these developmental splicing defects were regional ( Figures 5B and S3B). For example, enhanced skipping of Ndrg4 exon 14 was observed throughout the P6 brain, while the fetal pattern for Kcnma1 exon 25a was enhanced skipping in the forebrain but an increase in inclusion in the hindbrain ( Figure 5B). These results demonstrate that Mbnl2

regulates a distinct set of exons to promote adult splicing patterns during postnatal brain development and this regulation varies in different regions of the brain. Since both Mbnl2 heterozygous and homozygous knockouts developed seizures upon PTZ induction, AZD5363 chemical structure we selected genes and gene families from the splicing microarray or RNA-seq data sets ( Tables S1 and S2) that had been previously linked to epilepsy ( Klassen et al., 2011) to determine whether any of these pre-mRNAs showed splicing dysregulation in heterozygous knockouts. Of eight genes assayed (Mbnl2 targets Tanc2 and Csnk1d were included as controls), two (Cacna1d, Ryr2) showed a significant switch to the fetal pattern in adult Mbnl2+/ΔE2 hippocampus. The Cacna1d (CaV1.3)

voltage-gated L-type calcium channel subunit was the most profoundly affected ( Figures 5C and S3C). Missplicing of these pre-mRNAs in Mbnl2 knockouts was particularly interesting since CUGexp RNAs have the greatest impact on the expression of genes involved Gefitinib concentration in calcium signaling and homeostasis ( Osborne et al., 2009). We next used HITS-CLIP to detect target RNAs containing direct binding sites for Mbnl2 in vivo. After immunopurification of crosslinked RNA-protein complexes from mouse hippocampi, extensive RNase A digestion resulted in the appearance of a major band at ∼42 kDa in wild-type that was absent in Mbnl2 knockouts ( Figure 6A). At a lower RNase concentration, this distinct band was replaced with a more heterogeneous mixture of RNA-Mbnl2 complexes

from which RNA was isolated and subsequently sequenced ( Licatalosi et al., 2008). Three CLIP libraries were prepared from independent biological replicates. After quality filtering, genomic mapping, and removal of potential PCR duplicates, 3-mercaptopyruvate sulfurtransferase we obtained a stringent set of 703,431 unique CLIP tags that represent independent protein-RNA interactions for further analysis (Table S2). Approximately half (51%) of the unique Mbnl2 CLIP tags were located on annotated 3′ UTRs, making Mbnl2 distinct from other splicing factors such as Nova and PTB, which primarily bind within introns (Figure 6B) (Licatalosi et al., 2008; Llorian et al., 2010; Xue et al., 2009). In addition, there was a substantial number of intron targets (23%), consistent with a role for Mbnl2 in splicing. To identify sites of robust Mbnl2-RNA interaction, we clustered overlapping CLIP tags and conservatively determined 10,408 peaks, whose peak height is significantly above gene-specific background expected from uniform random distribution (p < 0.