Fig 1.
Comparative expression analysis of Chlamydomonas carbonic anhydrase (CAH) genes.
Cells grown in two different media types with and without acetate (TP–Tris-Phosphate, TAP–Tris-Phosphate-Acetate, HS–High-Salt, HAS–High-Salt-Acetate) were profiled for poly(A) site choice via construction of PATs followed by mapping of the tags to the annotated Chlamydomonas genome. Total PAT counts for each gene were normalized using the values from triplicate samples grown in the four different media types and presented as tags per million (tpm) for each of the genes shown. Phytozome 10.3 gene IDs for the CAHs used are as follows: CAH1 –Cre04.g223100, CAH2 –Cre04.g223050, CAH4 –Cre05.g248400, CAH5 –Cre05.g248450, CAH6 –Cre12.g485050, CAH8 –Cre09.g405750.
Fig 2.
Chlamydomonas genes with multiple poly(A) sites.
(A) Fraction of all genes with 1, 2, 3, 4, or >5 individual poly(A) sites (PAS). Total number of PAS = 41,048. (B) Fraction of all genes with 1, 2, 3, 4, 5, or >5 poly(A) site clusters (PACs). Total number of PACs = 19,674.
Fig 3.
Position-by-position base composition of poly(A) sites for different genic regions in Chlamydomonas genes.
The panels shown depict the following genic regions: (A) 3’-UTRs, (B) 5’-UTRs, (C) introns, and (D) protein-coding regions. Y-axis values are the fractional nucleotide content at each position (plotted along the x-axis) with individual traces color-coded as indicated in the legend. On the x-axis, “0” denotes the actual cleavage/polyadenylation site–negative values represent positions that are 5’ (upstream) of the poly(A) site and positive values are positions 3’ (downstream) of the poly(A) site. Number of poly(A) sites used for each analysis are as follows: A = 39,412, B = 343, C = 311, and D = 840.
Fig 4.
Motif analysis of regions surrounding 3’-UTR-situated poly(A) sites in Chlamydomonas.
The occurrences of 5 nt motifs was determined using SignalSleuth2 [51]. The relative position of the motif is given on the x-axis, with the poly(A) site being set as “0”; in these plots, regions extending from 80 nts 5’ (upstream) to 80 nts 3’ (downstream” from the poly(A) sites are shown. The numerical count of each motif is given on the y-axis. The plot shows the distributions of the 50 most-abundant motifs, which are listed in the legend embedded on the right. The number of sites used for this analysis was 39,415. (A) Profiles of the 10 most abundant motifs. The individual motifs are noted in the caption in the upper right corner. (B) Profiles of the next 40 most abundant motifs. The individual motifs are noted in the caption on the right. Note that the y-axis scale for panel B is 10% of that for panel A.
Fig 5.
Box plot comparing the changes in expression of genes affected by alternative polyadenylation (“APA”) with the changes of all genes expressed in at least one of the four treatments (“all”).
Numbers of genes: for “all” = 16,784, for “APA” = 172. Values on the y-axis denote the absolute value for the maximum fold-change for a given gene in the pairwise comparisons that were performed.
Fig 6.
Genome browser views of PAT mappings to the Chlamydomonas chloroplast and mitochondrial genomes.
Panels A and B depict the results of mappings to the complete chloroplast and mitochondrial genomes, respectively, while C and D show mappings to the rRNA-encoding regions in the chloroplast and mitochondria, respectively (in the case of panel C, only one of the two chloroplast rRNA-encoding regions is shown). In panels A and B, the uppermost plots denote the locations of annotated genes, the middle plots show the locations of annotated protein-coding regions, and the lower plots are bar graphs representing the tag abundance across the genome. In panels C and D, the uppermost plots depict details of the gene annotations, the middle line (“consensus”) shows the overall extent of contiguous tag mappings, and the lower plots shows the relative tag abundances across the depicted genomic regions.
Fig 7.
Details of poly(A) tracts for previously described Chlamydomonas chloroplast genes.
(A) Genome browser representation of tag mapping to a selected portion of the chloroplast genome that omits the rRNA-encoding regions. The uppermost plots depict details of the gene annotations, the middle line (“consensus”) shows the overall extent of contiguous tag mappings, and the lower plot shows the mappings of individual tags (green or red tics). Red and green tics denote tags oriented in the left-to-right (5’-3’) or right-to-left (5’-3’) directions. (B) Close-up browser view of reads that map to the chloroplast trnD gene. The sequences of the individual reads are visible; lettering in light shaded fonts denote extraneous sequence that does not map to the genome, but rather represents the mixed heteropolymeric tract as noted in the text. (D) Close-up browser view of reads that map to the chloroplast atpH gene. The sequences of the individual reads are visible; lettering in light shaded fonts denote extraneous sequence that does not map to the genome, but rather represents the mixed homopolymer as noted in the text.