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. 2021 Apr 27;10:e65537. doi: 10.7554/eLife.65537

Figure 2. FPA-dependent poly(A) site selection.

Loss of FPA function is associated with the preferential selection of distal poly(A) sites, whereas FPA overexpression leads to the preferential selection of proximal poly(A) sites. (A) Illumina RNA-Seq, Helicos DRS and Nanopore DRS reveal FPA-dependent RNA 3′ end processing changes at the FPA (AT2G43410) locus. The 35S::FPA:YFP construct has alternative transgene-derived untranslated regions, so mRNAs derived from the transgene do not align to the native FPA 5′UTR and 3′UTR. (B) Histograms showing change in mean RNA 3′ end position for significantly alternatively polyadenylated loci (EMD >25, FDR < 0.05) in fpa-8 (left panel) and 35S::FPA:YFP (right panel) compared with Col-0, as detected using Nanopore DRS. Orange and green shaded regions indicate sites with negative and positive RNA 3′ end position changes, respectively. (C) Effect size of significant proximal (orange) and distal (green) alternative polyadenylation events in fpa-8 (left panel) and 35S::FPA:YFP (right panel) compared with Col-0, as measured using the EMD. (D) Histograms showing change in mean RNA 3′ end position for significantly alternatively polyadenylated loci (EMD >25, FDR < 0.05) in fpa-8 (left panel) and 35S::FPA:YFP (right panel) compared with Col-0, as detected using Nanopore DRS. Orange and green shaded regions indicate sites with negative and positive RNA 3′ end position changes, respectively. (E) Effect size of significant proximal (orange) and distal (green) alternative polyadenylation events in fpa-8 (left panel) and 35S::FPA:YFP (right panel) compared with Col-0, as measured using the EMD. (F) Boxplots showing the effect size (absolute log2 fold change (logFC)) of alternatively processed loci identified using Illumina RNA-Seq in fpa-8 (left panel) and 35S::FPA:YFP (right panel) respectively. Down- and upregulated loci are shown in orange and green, respectively. For each locus, the region with the largest logFC was selected to represent the locus. Loci with both up- and downregulated regions contribute to both boxes. (G) Boxplots showing the effect size (absolute logFC) of loci with alternative splice junction usage identified using Illumina RNA-Seq in fpa-8 (left panel) and 35S::FPA:YFP (right panel), respectively. Down- and upregulated loci are shown in orange and green, respectively. For each locus, the junction with the largest logFC was selected to represent the locus. Loci with both up- and downregulated junctions contribute to both boxes.

Figure 2—source data 1. Nanopore StringTie assembly [Linked to Figure 2A–B].
Figure 2—source data 2. Differential 3′ processing results for fpa-8 vs Col-0, as identified by Nanopore DRS [Linked to Figure 2B–C].
Figure 2—source data 3. Differential 3′ processing results for 35S::FPA:YFP vs Col-0, as identified by Nanopore DRS [Linked to Figure 2B–C].
Figure 2—source data 4. Differential 3′ processing results for fpa-8 vs Col-0, as identified by Helicos DRS [Linked to Figure 2D–E].
Figure 2—source data 5. Differential 3′ processing results for 35S::FPA:YFP vs Col-0, as identified by Helicos DRS [Linked to Figure 2D–E].
Figure 2—source data 6. Differentially expressed regions results for fpa-8 vs Col-0, as identified by Illumina RNA-Seq [Linked to Figure 2F].
Figure 2—source data 7. Differentially expressed regions results for 35S::FPA:YFP vs Col-0, as identified by Illumina RNA-Seq [Linked to Figure 2F].
Figure 2—source data 8. Differential splice junction usage results for fpa-8 vs Col-0, as identified by Illumina RNA-Seq [Linked to Figure 2G].
Figure 2—source data 9. Differential splice junction usage results for 35S::FPA:YFP vs Col-0, as identified by Illumina RNA-Seq [Linked to Figure 2G].

Figure 2.

Figure 2—figure supplement 1. Nanopore and Helicos DRS reveal FPA-dependent RNA 3′ end processing changes.

Figure 2—figure supplement 1.

(A) Comparison of RNA 3′ ends identified in Nanopore and Helicos DRS datasets in fpa-8 and 35S::FPA:YFP (compared with Col-0). Bar size indicates the number of alternatively polyadenylated loci common to an intersection (highlighted using circles below). Bars indicating loci that are identified as alternatively polyadenylated in a single condition (fpa-8 or 35S::FPA:YFP) using a single technique (Nanopore or Helicos DRS) are presented in black; bars indicating loci identified as distally polyadenylated in fpa-8 using both Nanopore and Helicos DRS, in orange; bars indicating loci identified as proximally polyadenylated in 35S::FPA:YFP using both Nanopore and Helicos DRS, in green; and bars indicating loci identified as reciprocally regulated by FPA (distal polyadenylation in fpa-8, proximal in 35S::FPA:YFP) using at least one technique, in yellow.
Figure 2—figure supplement 2. Splicing alterations in fpa-8 can be explained by changes in RNA 3′ end formation.

Figure 2—figure supplement 2.

Gene track showing chimeric RNA formation at the PIF5 gene locus, as detected with Illumina RNA-Seq, Helicos DRS, and Nanopore DRS.
Figure 2—figure supplement 3. FPA does not affect global mRNA m6A methylation.

Figure 2—figure supplement 3.

Box plot showing the m6A/A ratio, as analysed using LC-MS/MS.
Figure 2—figure supplement 3—source data 1. m6A : A ratios for Col-0, fpa-8, 35S::FPA:YFP and vir-1, as detected by LC-MS/MS [Linked to Figure 2—figure supplement 3].
Figure 2—figure supplement 4. FPA-dependent control of NLR expression is independent of IBM1.

Figure 2—figure supplement 4.

Venn diagram showing genes with altered H3K9me2 levels in ibm1–four mutants, in yellow (Inagaki et al., 2017) and orange (Lai et al., 2020); and genes with altered poly(A) site choice in 35S::FPA:YFP, in green.
Figure 2—figure supplement 4—source data 1. Differential H3K9me2 results for ibm1–four vs Col-0 [Linked to Figure 2—figure supplement 4].