Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and <i>FLC</i> expression.

<p><b>(A)</b> RNA gel blot analysis of WT <i>A</i>. <i>thaliana</i> accession Columbia (Col-0) plants <i>fpa-8</i> and <i>fpa-8</i> mutants expressing <i>FPA</i>::<i>FPA R477A;Y515A</i> using poly(A)+ purified mRNAs. Black arrows indicate the proximally and distally polyadenylated <i>FPA</i> mRNAs. A probe corresponding to the 5’UTR region of <i>FPA</i> mRNA was used to detect <i>FPA</i> specific mRNAs. RNA size (kb) marker (Ambion). <i>TUBULIN</i> was detected as an internal control. The ratio of distal:proximal polyadenylated forms is given under each lane. <b>(B)</b>. qRT-PCR analysis was performed with total RNA purified from Col-0, <i>fpa-8</i>, and <i>FPA</i>::<i>FPA R477A;Y515A</i> plants. Transcript levels were normalized to the control <i>UBC</i>. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.</p

A conserved surface patch of FPA SPOC domain.

<p><b>(A)</b>. Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs. Purple: most conserved; cyan: least conserved. <b>(B)</b>. Residues in the conserved surface patch of FPA SPOC domain. The side chains of the residues are shown in stick models, colored orange in the first sub-patch and green in the second. <b>(C)</b>. Molecular surface of FPA SPOC domain colored based on electrostatic potential. Blue: positively charged; red: negatively charged.</p

Structural homologs of the FPA SPOC domain.

<p><b>(A)</b>. Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray). The bound position of a doubly-phosphorylated peptide from SMRT is shown in magenta. <b>(B)</b>. Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray). Ku80 contains a homologous domain (green), which forms a hetero-dimer with that in Ku70. The two domains, and inserted segments on them, mediate the binding of dsDNA (orange). The red rectangle highlights the region of contact between the two β-barrel domains. <b>(C)</b>. Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray). The binding partner of Chp1, Tas3, is shown in green. The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (<b>D</b>). Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).</p

Crystal structure of the SPOC domain of <i>A</i>. <i>thaliana</i> FPA.

<p><b>(A)</b>. Schematic drawing of the structure of FPA SPOC domain, colored from blue at the N terminus to red at the C terminus. The view is from the side of the β-barrel. The disordered segment (residues 460–465) is indicated with the dotted line. (<b>B</b>). Structure of the FPA SPOC domain, viewed from the end of the β-barrel, after 90° rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>).</p

Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA.

<p>RNA gel blot analysis of WT <i>A</i>. <i>thaliana</i> accession Columbia (Col-0) plants <i>fpa-8</i> and <i>fpa-8</i> mutants expressing either <i>FPA</i>::<i>FPA R477A</i> <b>(A)</b>, or <i>FPA</i>::<i>FPA Y515A</i> <b>(B)</b> using poly(A)+ purified mRNAs. A probe corresponding to the 5’UTR region of <i>FPA</i> mRNA was used to detect <i>FPA</i> specific mRNAs. RNA size (kb) marker (Ambion). <i>TUBULIN</i> was detected as an internal control. Proximally and distally polyadenylated <i>FPA</i> transcripts are marked with arrows. The ratio of distal:proximal polyadenylated forms is given under each lane. <b>(C,D)</b> Impact of individual FPA SPOC domain mutations on <i>FLC</i> transcript levels. qRT-PCR analysis was performed with total RNA purified from Col-0, <i>fpa-8</i>, <i>35S</i>::<i>FPA</i>:<i>YFP</i> and <i>FPA</i>::<i>FPA R477A</i> <b>(C)</b>, <i>FPA</i>::<i>FPA Y515A</i> <b>(D)</b> plants. Transcript levels were normalized to the control <i>UBC</i>. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.</p

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

<p>(<i>A</i>) Reads mapping to the locus encoding <i>FPA</i>. 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 <i>fpa-7</i>. (<i>B, C</i>) Nucleotide composition profiles around cleavage sites within annotated genes (<i>B</i>) and at intergenic sites (<i>C</i>) 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003867#pgen.1003867-Jan1" target="_blank">[58]</a>; DSE, downstream sequence element. (<i>D–F</i>) Example of intergenic DRS reads mapping antisense to a coding gene. Normalised reads mapping to the At1g29520–At1g29530 loci are displayed in (<i>D</i>). 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. (<i>E</i>) 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 <i>fpa</i>-7. (<i>F</i>) 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003867#pgen.1003867-Nicol1" target="_blank">[55]</a> and correspond to combined reads from the three sequenced biological replicates for each genotype.</p

Distribution of DRS reads.

<p>(<i>A</i>) Genome-wide distribution after re-annotation in wild-type (WT) and <i>fpa-7</i>. (<i>B</i>) Distribution of DRS reads mapping to protein-coding genes after re-annotation.</p

Differentially expressed transposons between wild-type and <i>fpa-7</i>.

<p>(<i>A</i>) Differential expression of the transposable element gene (At5g10670) in <i>fpa-7</i>. (<i>B</i>) Read-through contiguous RNAs were validated by RT-PCR (red dashed line). Three biological replicates (1, 2 and 3) were used for each genotype: wild-type (WT) and <i>fpa</i>-7. UBIQUITIN <i>LIGASE 21</i> (<i>UBC21</i>) was used as a control. RT-PCR products were separated on agarose gels and stained with ethidium bromide. (<i>C</i>) 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 rectangles represent the 3′UTR specific to the read-through transcript and red lines represent 3′UTR introns. (<i>D</i>) Differential expression of the transposable element gene (At5g35935) in <i>fpa-7</i>. Recent re-annotation of At5g35935 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003867#pgen.1003867-Maher1" target="_blank">[12]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003867#pgen.1003867-Kannan1" target="_blank">[13]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003867#pgen.1003867-Garcia1" target="_blank">[28]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003867#pgen.1003867-Pontier1" target="_blank">[29]</a> defines two transcription units within it: the recently arisen pseudogene <i>psORF</i> and the transposon At5TE50260. DRS data reveal that silencing of <i>psORF</i> is lost in <i>fpa-7</i>. (<i>E</i>) RT-qPCR analysis of <i>psORF</i> in <i>fpa-7</i> and <i>fpa-8</i> mutant alleles. Silencing of <i>psORF</i> (p2 and p2b) is lost in <i>fpa-7</i> but not in <i>fpa-8</i>. Data are the means ± SEM obtained for three independent PCR amplifications of three biological replicates. The y-axis shows the fold change relative to WT (WT set to 1) after normalisation to <i>UBC21</i> gene expression. Location of the RT-qPCR amplicon is displayed on the left panel. <i>*</i>, <i>P</i><0.05; Student's t-test. Normalised reads mapping to the different loci are presented for WT and <i>fpa</i>. Genes are orientated 5′–3′; exons are denoted by rectangles, UTRs by adjoining narrower rectangles and introns by lines. Images of normalised read alignments were made using the Integrated Genome Browser <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003867#pgen.1003867-Nicol1" target="_blank">[55]</a> and correspond to combined reads from the three sequenced biological replicates for each genotype.</p

Characterisation of intergenic read-through RNAs in <i>fpa</i> and <i>dc11</i> mutants.

<p>(<i>A–C</i>) Characterisation of <i>FCA</i> intergenic read-through RNAs. (<i>A</i>) Normalised reads mapping to the locus encoding <i>FCA</i>. (<i>B</i>) Identification of R1 and R2 contiguous RNAs. Three biological replicates (1, 2 and 3) were used for each genotype: wild-type (WT), <i>fpa</i>-7 and <i>dc11–11</i>. (<i>C</i>) Description of the R1 and R2 read-through transcripts. (<i>D–F</i>) Characterisation of At1g51390 read-through RNAs. (<i>D</i>) Normalised reads mapping to At1g51390–At1g51402–At1g51400. (<i>E</i>) Identification of R1 and R2 contiguous RNAs. Three biological replicates (1, 2 and 3) were used for each genotype: WT, <i>fpa</i>-7 and <i>dc11–11</i>. (<i>F</i>) Description of the R1 and R2 read-through transcripts. The red dashed lines indicate the regions amplified by RT-PCR on reverse-transcribed poly(A)+ RNAs. Red narrower rectangles represent 3′UTR parts specific to the read-through transcript and red lines the 3′UTR introns. RT-PCR products were separated on agarose gels and stained with ethidium bromide. The purple line indicates the location and strand detected by the probe, which was used previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003867#pgen.1003867-Zhang1" target="_blank">[24]</a>. The image of normalised read alignments was made using the Integrated Genome Browser <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003867#pgen.1003867-Nicol1" target="_blank">[55]</a> and corresponds to combined reads from the three sequenced biological replicates for each genotype. Exons are denoted by coloured rectangles, UTRs by adjoining narrower rectangles and introns by lines.</p

<i>PIF5–PA03</i>, an example of chimeric RNA formation controlled by FPA.

<p>(<i>A</i>) Normalised reads mapping to the locus encoding <i>PIF5–PA03</i>. Exons are denoted by coloured rectangles, UTRs by adjoining narrower rectangles and introns by lines. The image of normalised read alignments was made using the Integrated Genome Browser <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003867#pgen.1003867-Nicol1" target="_blank">[55]</a> and corresponds to combined reads from the three sequenced biological replicates for each genotype. (<i>B</i>) Location of RNA gel blot probes are indicated by numbers (P1–P6) and the tested fusion region by a dotted line. (<i>C</i>) RT-PCR analysis of a contiguous RNA between <i>PIF5</i> and <i>PA03</i> detected in <i>fpa-8</i>. <i>UBIQUITIN LIGASE 21</i> (<i>UBC</i>) was used as a control. (<i>D,E</i>) RNA gel blot analysis of <i>PIF5–PA03</i> chimeric RNAs in wild-type (WT) and <i>fpa-8</i>. P, <i>PIF5</i>; C, <i>PA03</i> transcripts. <i>β-TUBULIN</i> (<i>β-TUB.</i>) was used as an internal control. Probes used are shown in (<i>B</i>). (<i>F</i>) 5′RACE analysis of the γ and γ′ RNAs with or without tobacco acid pyrophosphatase (TAP) treatment. PCR products were separated on an agarose gel and stained with ethidium bromide. (<i>G</i>) Schematic representation of the different RNAs expressed at the <i>PIF5</i> locus in <i>fpa</i> mutants. The splicing event occurring in the β chimeric RNA is shown by a red line.</p