Multiple Sequence Alignment of the <i>RMRP</i> Promoter Region

<p>The sequence alignment of the <i>RMRP</i> promoter region of nine mammalian species (from nucleotide g.−80 to the transcription initiation site of the human sequence) is shown. Polymorphisms and rare variants are indicated as blue boxes (single nucleotide changes); red arrows indicate pathogenic insertions, and red lines indicate pathogenic duplications and triplications. Conservation of nucleotides was analyzed for the single nucleotide substitutions (putative polymorphisms) included in the alignment interval (from g.−80 to g.−1). Positions were considered as conserved if eight out of nine species had the same nucleotide.</p

RACEfrag transcription map statistics.

<p><b>A- Distribution of RACEfrags among annotated genomic domains.</b> The proportion of RACEfrags overlapping different annotated genic features is represented in this histogram. Blue: intronic RACEfrags; Light orange: exonic RACEfrags; Light grey: intergenic RACEfrags. The three categories on the X axis are, from left to right: (1) - external genic RACEfrags (i.e. RACEfrags falling within the boundaries of a gene not interrogated by RACE, (2) - intergenic RACEfrags, (3) - internal RACEfrags (i.e., RACEfrags detected within the RACE-primed gene). <b>B- RACEfrag descriptive analysis.</b> The top bar plot represents proportions of genomic domains covered by RACEfrags, and the bottom bar plot represents proportions of RACEfrags in different genomic domains (refinement of part A). As RACE is carried out in the two possible directions, 5′ and 3′, each bar plot is thus sub-divided into two sub-bar plots: proportions relative to 5′ RACEfrags in gray, and proportions relative to 3′ RACEfrags in blue. As expected: (1) RACEfrag coming from a given gene covers this gene more than any other gene; (2) for a given RACE-interrogated gene, internal exons and introns are equally covered by 5′ and 3′ RACEfrags, whereas 5′ most exons are more covered by 5′ RACEfrags and 3′ most exons by 3′ RACEfrags. The bottom bar plot also shows that most RACEfrags are exonic, then intronic and finally intergenic, and that exonic RACEfrags are first found in internal exons, then in most 3′ exons and finally in most 5′ exons.</p

RACEarray experiment flowchart.

<p>The successive steps of the RACEarray experiments are represented as a flowchart for both chromosomes 21 and 22. It should be read from left to right and from top to bottom.</p

Reciprocal gene/gene connections.

<p><b>A - General definition of reciprocal gene/gene connections</b>. Top panel: graphical illustration of reciprocity. Exons are symbolized by light blue boxes, introns by solid black lines. Dashed arrows, directed from the index exon to the RACEfrag, correspond to chimeric connections in distinct cell types, which are rendered in different colors. Two reciprocal gene/gene connections can be observed in this example, between genes A and B, and B and C. The (A–B) reciprocal pair is said to be (i), <i>unique to cell type 2</i>, and (ii), <i>pure</i> (<i>i.e.</i>, its reciprocity is observed at least once in the same condition, cell type <i>2</i> in this example), whereas (B–C) is <i>composite</i> (<i>i.e.</i>, its reciprocity can only be deduced from connections observed in different cell types). The counts of each connection type in this example are summarized in the tables in the bottom panel. <b>B - Observed numbers of reciprocal gene/gene connections across 10 different cell types</b>. This table is based on the template used in part A.</p

RNAse protection assays to validate predicted fusion/chimeric transcripts.

<p>Each panel on top shows the autoradiographs of the probe that covers the predicted chimeric RNA fragment against human RNA from tissue pools detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028213#pone.0028213.s023" target="_blank">table S2</a>. In each panel, “+” and “−” indicate the tracks with presence and absence of input RNA, respectively. The RNA size ladder is shown on the left of the figure (unit: nucleotides). The bottom of the figure schematically shows the two components of each predicted chimeric transcript. For example, clone 7556-D12 contains 97 bases of an exon of gene TOP3D (designated A) joined to a 153-base exonic fragment of gene PIK4A (designated B); both fragments are in the same genome strand orientation (shown by arrows). In the top first panel corresponding to clone 7556-D12, the protected chimeric fragment in the RNA“+” lane is labeled A+B. The protected (non-chimeric) exons B and A are also shown. The other 2 validated chimeric fragments are shown in the second and third top panels. The fourth panel contains a control exon-exon junction of gene HIRA. The fifth panel contains a control actin gene fragment. A total of 3 out of 15 predicted and tested chimeric fragments were validated using this method. For more details on these experiments see Material and Methods, and for the genomic coordinates of the fragments see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028213#pone.0028213.s023" target="_blank">table S2</a>.</p

Characteristics of hub genes. A- Expression of hub genes.

<p>The distribution of expression of the 74 hubs and of the 362 non hubs is plotted in blue and orange respectively. The expression of a gene is computed based on tiling array experiments performed on the same 16 cell lines and tissues as the RACE experiments (see details in the text). As we can see hubs tend to have higher expression values than non hubs. <b>B- </b><b>Phylogenetic conservation of hub genes.</b> In each of the three gene network categories (<i>i.e.</i>, hubs, non-hubs, and all RACEd genes), the proportion of genes having a detected ortholog in each eukaryotic species represented on the X axis (ordered by decreasing phylogenetic distance from human) is reported on the Y axis. Instances where the proportion of orthologs found in the <i>hub</i> category is significantly higher than for <i>non-hubs</i> (<i>p</i><0.01, Fisher test) are marked with an asterisk.</p

Gene expression coordination within cliques as a function of clique size.

<p>The width of the box plots is proportional to the number of connections involved in cliques of a given size. The number of observed cliques of sizes 2 to 7 is reported, as well as the numbers in randomized networks (mean, +/− standard deviation), in the form of a table at the bottom. Randomized networks are generated so that they have the same degree distribution as the original network (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028213#pone.0028213.s001" target="_blank">Materials S1</a>).</p

Distribution of genomic distances between RACEfrags and their respective index RACE primers.

<p>The raw histogram is shown in purple, the corresponding curve fit in red <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028213#pone.0028213-Khanin1" target="_blank">[25]</a>.</p

Reciprocal gene/gene connections supported by 5C on chromosome 21.

<p>The 638 reciprocal gene/gene connections on chromosome 21 are represented as blue inner links if they are supported by 5C, and as yellow inner links otherwise.</p