FPA affects intronic cleavage site selection and intergenic read-through.

(A) Reads mapping to the locus encoding FPA. Promoter proximal ‘P’ and distal ‘D’ alternative poly(A) sites are indicated, as are the poly(A) sites ‘T’ resulting from the T-DNA insertion in fpa-7. (B, C) Nucleotide composition profiles around cleavage sites within annotated genes (B) and at intergenic sites (C) display alternating A- and U-rich sequences. USE, upstream sequence element; PAS, poly(A) signal; Fip1, the U-rich sequence upstream of the cleavage site is the proposed binding site of FIP1 [58]; DSE, downstream sequence element. (D–F) Example of intergenic DRS reads mapping antisense to a coding gene. Normalised reads mapping to the At1g29520–At1g29530 loci are displayed in (D). The upper panel shows reads mapping to the (+) strand 3′ end of At1g29520, while the lower panel shows reads mapping to the (−) strand 3′ end of At1g29530. (E) R1 and R2 contiguous RNAs were validated by RT-PCR (red dashed lines) with poly(A)+ RNA. RT-PCR products were separated on agarose gels and stained with ethidium bromide. Three biological replicates (1, 2 and 3) were used for each genotype: wild-type (WT) and fpa-7. (F) Transcripts are either cleaved and polyadenylated in the annotated 3′UTR or at the intergenic sites, as determined by sequencing the cloned RT-PCR products. Red narrower rectangles represent regions specific to the read-through transcript and red lines the 3′UTR introns. Images of normalised read alignments were made using the Integrated Genome Browser [55] and correspond to combined reads from the three sequenced biological replicates for each genotype.