, 1994). We identified a large and diverse group of dendritically localized CIRTs by microarray and Illumina sequencing of mRNA from isolated dendrites and in situ hybridization. Computational analysis of the retained intron sequence revealed the enrichment of ID elements. see more Individual intronic ID elements from different genes were cloned, exogenously expressed in primary neurons, and shown by in situ hybridization to be capable of targeting mRNA to dendrites. Normal dendritic localization
of ID-containing transcripts is disrupted when ID-containing transgenes compete for the dendritic targeting machinery, thus showing that ID-mediated localization is an endogenous mechanism. Our findings represent an example of a general dendritic targeting mechanism for multiple transcripts from different genes. To determine whether CIRTs are present
in dendritic mRNA populations, we focused on a set of 33 candidate genes with mRNA previously found to localize to dendrites in rat ABT263 (Eberwine et al., 2002). Three batches of dendritic mRNA, each consisting of 150–300 individually dissected dendrites from primary rat hippocampal neurons, were independently aRNA amplified (Miyashiro et al., 1994) and analyzed by using a custom-built microarray consisting of probes generated from the 5′ ends of selected introns from each gene of interest. Three additional batches were subjected to Illumina NextGen sequencing. Sequencing allows us to recover minor, variably expressed CIRTs in the different RNA pools, while
microarrays provide additional evidence for a smaller set of hypothesized CIRTs that may escape detection by sequencing because of low-read depth or systematic biases such as nucleotide content (Harismendy et al., 2009). By using these methods, many CIRTs were detected (Table 1). A wide range of expression was observed across the arrays, with intronic loci from CAMK2B and FMR1 among others consistently showing high signal (Figure S1A, available not online, and Table S1). A similar pattern of intron retention was present in the sequencing data, supported by uniquely aligning end pairs to nonrepetitive intronic regions (Figure S1B, Table S2). For some genes such as ADCY4 and GRIK1, sequence reads spanned intron-exon boundaries. Retention of intronic sequence appears to be regulated, as some intronic loci consistently show retention while others do not. Some genes such as CAMK2A and SNCB lacked intron retention despite the confirmed presence of exonic regions in the RNA pool.