Additionally, the pattern of sequence-read coverage is inconsistent with these sources of contamination, as we found no significant enrichment in intergenic reads, nor did we find any systematic pattern of intron presence or absence across all genes. We also analyzed each retained intronic locus by using base composition properties

and public annotations and found that the majority had no evidence for unannotated alternate exons or overlapping genes (see Supplemental Text). Based on the retained intronic loci detected in the initial screen, we selected several candidates to visualize by using in situ hybridization to confirm retention and localization patterns. We assayed intronic probes designed to target microarrayed sequences from RNAs showing varying degrees of intronic sequence retention. Antisense riboprobes were generated and ISRIB purchase used for in situ hybridization to E18 rat neurons in primary cell culture. Cells were costained for

MAP2 protein to indicate dendrito-somatic regions of neurons (Figure 1, insets). All sequences tested showed dendritic in situ hybridization signals consistent with microarray results (Figure 1A). In situ hybridization of exonic probes confirmed the dendritic localization patterns find protocol of the intron-containing transcripts (Figure S2). Further, oligo probes to intron-exon junctions with sequencing support successfully confirmed that each region was within the dendritic compartment by in situ hybridization (Figure 1B). Interestingly, GRIK1 shows a higher dendritic signal for intron 16 joined with an alternate exon than with the canonical exon 17, suggesting an interaction between intronic sequence and the isoforms of the transcript in localization (Bell et al., 2010). Given the widespread occurrence of CIRTs, we hypothesized

that sequence elements important for mRNA regulation may be embedded in the retained intronic sequences and searched for putative regulatory sequences. We found Linifanib (ABT-869) several sets of sequences shared among different introns, including a large number of BC1 RNA-like ID elements. While ID elements are not unique to retained introns, many are found in the dendritic introns detected by microarray and sequencing. Among these intronic ID elements, we found that many retain motifs previously identified as BC1 localization signals that confer targeting to microinjected mRNA (Muslimov et al., 2006) as evidenced by their predicted secondary structures (Figure 2A). A total of 308 blocks of ID-derived sequence were found. Of these, 70 elements appearing in 46 introns across 23 genes were determined to possess mRNA targeting potential: these occurred in the sense orientation and forming a hairpin structure with a basal-medial unbranched helix, a uracil at position 22, and at least 90% sequence identity to the BC1 5′ domain (Table S3). Sequencing data provided evidence that many of these ID-containing loci were present in the dendritic RNA pools.