Examples of the discovery and characterization of alternative splicing events in circadian genes and identification and validation of the I4R events in LHY and CCA1 . (A) Potential alternative splicing events were suggested by the distribution of RNA-seq reads density along gene features. The screen shots of the CCA1 and LHY loci are from the Arabidopsis RNA-seq GBrowse. Histograms represent the density of coverage of gene features by Illumina reads (GBrowse tracks of HTS reads) under different abiotic stress conditions (e.g., salt, drought, and cold treatments). The tracks "WT control" and "upf1-1" represent read density in the wild-type control and in the upf1-1 NMD mutant, respectively. The fourth introns in both the CCA1 and LHY transcripts display an increase in read coverage (bracketed) under specific treatments suggesting that these introns may be retained more often under certain conditions. The I9R event in LHY is also represented by an increase in read coverage on the 3' UTR distal portion (bracketed) in the upf1-1 NMD mutant. The numbers of LHY introns 4 and 9 correspond to the TAIR10 gene models AT1G01060.2 and AT1G01060.3, respectively. The CCA1 intron 4 corresponds to AT2G46830.1. All three IR events were confirmed by RT-PCR and qRT-PCR (Figures 2, 3 and 5, Table 1). The scale of the read depth histogram is limited to a maximum of 50 reads. (B) Primer design strategy developed for validation of the I4R events in CCA1 and LHY transcripts. Dark boxes depict protein-coding exons. The exons in the 5' and 3' UTRs are indicated by light boxes. The oligonucleotide primers used for the amplification of the constitutive and alternative splicing events are indicated by green and red arrows, respectively. The I4R events are depicted by dashed boxes. A small nested intron 4a in CCA1 is indicated by a dashed line. Normal and premature termination codons are shown by top and bottom star symbols, respectively. The expected sizes of RT-PCR products are given in base pairs (bp). The gene models are not drawn to scale. The inset in (B) shows the RT-PCR confirmation of the LHY and CCA1 transcripts with spliced and retained intron 4. Expected RT-PCR product sizes for the LHY transcript when intron 4 is spliced correctly is 264 bp; the size of the I4R isoform (if intron 4 is retained and introns 2 and 3 spliced out) is 224 bp. For CCA1 the predicted product sizes are 226 bp (introns 4/4a and 5 spliced out) and 225 bp for the I4R isoform (introns 4a and 5 are spliced out but intron 4 is not). RT-PCR products were separated on a 2 % agarose gels and stained with ethidium bromide. ‘M’ - molecular size markers (markers correspond from bottom to top to 100, 200, and 300 bp).