Figure 3.

PIRL6 undergoes unproductive alternative splicing outside of gametophytes. PIRL6 transcripts were amplified from polyadenylated RNA from wild-type and spl mutant homozygotes using primers designed to amplify the full-length coding region. A, RT-PCR products from wild-type leaves (L), roots (R), and flowers (F). Sequencing confirmed that the predominant flower cDNA was derived from the translatable, spliced PIRL6 mRNA. Larger alternative products are evident in leaves and roots; the largest band corresponds in size to the unspliced transcript (1,468 bp). B, RT-PCR of PIRL6 transcripts from wild-type (WT) and sterile spl mutant flowers, showing the predominance of alternative transcripts in flowers that lack gametophytes. To detect transcripts present in spl, the spl lane was loaded with 10× the volume of the reaction product than the wild-type lane. C, Structures of alternative polyadenylated PIRL6 transcripts sequenced from roots and leaves. Gel-resolved leaf and root RT-PCR products between approximately 1.1 and 1.4 kb were cloned in Escherichia coli, and 17 independent cDNAs were sequenced. Black triangles indicate primer positions used for PCR following cDNA synthesis primed with oligo(dT). Functional mRNA represents the flower RT-PCR product, corresponding to the annotated spliced PIRL6 mRNA. Transcripts A to F are alternative mRNA species. Numbers in brackets indicate the number of independent cDNA clones obtained for each transcript species. Lowercase letters indicate residual unspliced nucleotides from intron I; cross-hatched regions indicate unspliced introns. Red triangles mark the positions of premature termination codons. The dashed line marks the position of the normal 5′ end of exon II. The sizes of RT-PCR products corresponding to each mRNA species are indicated in bp at far right. D, qPCR showing increased accumulation of PIRL6 alternative transcripts in the NMD-deficient mutant upf3. qPCR was carried out on leaf RNA using an intron II-specific forward primer, allowing the detection of alternative transcripts A, C, D, E, and F. Values are means of two biological replicates, with three reaction replicates for each; se is shown.