LRCFS/GSR_Paper: Complete release to accompany publication

FInal version following feedback from reviewers</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

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

<p>(<i>A</i>) Histogram of log₂ fold change profiles for protein-coding genes differentially expressed (DE) between wild-type (WT) and <i>fpa-7</i>. (<i>B</i>) Reads mapping to the locus encoding SO<i>C1</i>. The <i>SOC1</i> gene is orientated 5′–3′. (<i>C</i>) Reads mapping to the locus encoding <i>FLC</i>. Normalised reads are presented for WT and <i>fpa</i>. The top panel displays the reads corresponding to the <i>FLC</i> asRNAs and corresponds to the (+) strand while the bottom panel displays the reads corresponding to <i>FLC</i> mRNA and corresponds to the (−) strand. The <i>FLC</i> gene is orientated 3′–5′, while the reads corresponding to <i>FLC</i> asRNAs are orientated 5′–3′. Proximally polyadenylated ‘P’ <i>FLC</i> asRNAs were detected in <i>fpa-7</i> and WT by the low number of reads at surrounding sites, while the distal ‘D’ cleavage sites were clearly defined. 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

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

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

<p>(<i>A</i>) H3K4me3 Chromatin ImmunoPrecipitation (ChIP) analysis of genomic regions at the <i>PIF5</i> locus. γ RNAs result from a shift in the transcription start site. Black lines depict the chromatin regions analysed by qPCR. Histograms show means ± SEM obtained for enrichment calculated by percentage input normalised against actin for three PCR amplifications. <i>*</i>, <i>P</i><0.05; Student's t-test. (<i>B</i>) RT-qPCR analysis of <i>PIF5–PA03</i> chimeric RNAs in <i>fpa</i> and <i>upf1</i> mutants. Data are the means ± SEM obtained for three independent PCR amplifications of three biological replicates. The y-axis shows the fold change relative to wild-type (WT; (WT set to 1) after normalisation to <i>UBC21</i> gene expression. Location of the RT-qPCR amplicon is displayed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003867#pgen.1003867.s007" target="_blank">Figure S7</a><i>D</i>. <i>*</i>, <i>P</i><0.05; Student's t-test. (<i>C</i>) RNA gel blot analysis of <i>PIF5–PA03</i> chimeric RNAs in <i>fpa</i> and <i>upf1</i> mutants.</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

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

Additional file 2: Figure S3. of Improved annotation with de novo transcriptome assembly in four social amoeba species

Annotated domain diagrams for 16 D. discoideum developmentally relevant proteins which have had their protein sequence altered. (PDF 367 kb

Additional file 3: Table S1. of Improved annotation with de novo transcriptome assembly in four social amoeba species

D. discoideum GFF file of genomic positions with introns < 5 bp. (TXT 9 kb