Transcending ambivalence: Overcoming the ambiguity of theory and practices

Ambivalence is a deeply ambiguous concept. Contributions to the present book, viewed all together, exemplify that verdict, as they are situated in the intellectual space among theory, phenomena, research practices, and basic assumptions about the world..

Statistics of RNA–Seq data.

#<p>M: million reads.</p

Strand-specific RNA–Seq in five rhesus tissues reveals clear transcript structure for <i>de novo</i> genes.

<p>(A) An example of <i>de novo</i> gene <i>ENST00000315302</i> partially overlapped with a pre-existing gene <i>ODZ3</i>, transcribed by the other strand of the DNA. The ortholog of <i>ENST00000315302</i> in rhesus macaque was aligned according to genome-wide multiple alignments in UCSC. The junction reads generated by strand-specific RNA-Seq assays are highlighted by black bold lines, with fragments of junction reads crossing splicing junctions connected by thinner lines. The mapped reads well supported the transcription of the target <i>de novo</i> gene on the reverse strand, as most reads appeared in the track for ‘reads transcribed from the minus-strand’. Regions for all four splicing junctions are highlighted in dotted boxes and expanded in (B), including three in <i>ENST00000315302</i> transcribed from the minus strand and one from the other strand. All of these splicing junctions were well supported by the RNA-Seq reads mapped on the corresponding strand of the DNA. Vertical dotted lines in brown or blue highlight the exon boundaries in transcripts on the minus or plus strands, respectively. (C) Demo case for a discarded <i>de novo</i> gene in the manual curation process, in which the RNA-Seq data in rhesus macaque were not consistent with the putative splicing pattern predicted on the basis of human gene models. The common disabler is marked with a red star, and this was actually spliced out in rhesus macaque as indicated by the junction reads. Scale bar shown as benchmark for gene size.</p

Non-coding orthologs of human <i>de novo</i> protein-coding genes in rhesus macaque and chimpanzee show tissue expression profiles similar to human.

<p>(A) Hierarchical clustering chart of tissue expression proportions. For each gene in one species, tissue expression proportions were calculated by normalizing RPKM scores with the total expression level of the gene in that species. The scores were then clustered according to similarity using complete linkage hierarchical clustering. For each gene, cross-tissue correlation coefficients between human and chimpanzee (H–C), chimpanzee and rhesus macaque (C–R) and human and rhesus macaque (H–R) are shown. (B) Correlation coefficient scores for tissue expression profiles between human and rhesus macaque. Correlation coefficients for <i>de novo</i> genes (brown histograms) are illustrated with background simulated by 10,000 <i>Monte Carlo</i> simulations neglecting ortholog relationship for the tissue expression profile (blue histograms, mean scores are shown). (C) For each pair of tissues, Spearman correlation coefficients were computed separately and the extent of tissue-specific differences in <i>de novo</i> gene expressions are shown. Dotted lines highlight comparisons between pairs of corresponding tissues in different species. Grey boxes: missing data. ^*Correlation coefficient not available due to low tissue expressions in one or both species. ^#Gene reported in previous study as human-specific <i>de novo</i> protein-coding gene.</p

Orthologs of human <i>de novo</i> protein-coding genes encode structure-matched non-coding RNAs in rhesus macaque or chimpanzee.

<p>(A) Summed RPKM scores (log₂ transformed) of <i>de novo</i> genes in seven tissues from human and rhesus macaque. The human genes were ordered by decreasing expression level as a reference, and the rhesus genes were aligned accordingly. (B) For each <i>de novo</i> gene in Classes I and II, the base-level densities of RNA-Seq reads across the transcript (red), as well as the upstream/downstream regions (grey, 50% of the length of the transcript), are shown. The raw density scores computed from RNA-Seq reads coverage were normalized with the total reads across the region. (C) Splicing junctions with the sequence motifs near both the donor site and acceptor site, summarized by all splicing junctions in human <i>de novo</i> genes. (D) Venn diagram showing the numbers of human splicing junctions detected also in chimpanzee or rhesus macaque. Pie charts further illustrate the detailed status of human splicing junctions in chimpanzee and rhesus macaque.</p

Genome-wide identification of hominoid-specific <i>de novo</i> protein-coding genes.

<p>(A) On the basis of the gene locus and ORF age assignments, hominoid-specific <i>de novo</i> protein-coding genes were identified. Regions within dotted red lines indicate the repeating steps for each out-group species. We further filtered this list using stringent inclusion criteria and generated a smaller convincing list of 24 <i>de novo</i> genes. (B) Distribution of protein length for the 24 <i>de novo</i> genes, compared with the human genome as background. (C) Distribution of summed RPKM scores of the 24 <i>de novo</i> genes in seven human tissues, compared with the human genome as background. (D) Pie chart showing the distribution of the 24 <i>de novo</i> protein-coding genes in terms of the reuse of preexisting transcriptional context. Gene numbers in each category are marked. None: no evidence for the reuse of transcriptional context; bi: located downstream of bi-directional promoter; +: overlapping with preexisting genes on the same strand; −: overlapping with preexisting genes on the opposite strand. (E) Venn diagrams showing the contribution of <i>Alu</i> sequences to exons and splicing junctions in <i>de novo</i> protein-coding genes.</p

Emergence, Retention and Selection: A Trilogy of Origination for Functional <i>De Novo</i> Proteins from Ancestral LncRNAs in Primates

<div><p>While some human-specific protein-coding genes have been proposed to originate from ancestral lncRNAs, the transition process remains poorly understood. Here we identified 64 hominoid-specific <i>de novo</i> genes and report a mechanism for the origination of functional <i>de novo</i> proteins from ancestral lncRNAs with precise splicing structures and specific tissue expression profiles. Whole-genome sequencing of dozens of rhesus macaque animals revealed that these lncRNAs are generally not more selectively constrained than other lncRNA loci. The existence of these newly-originated <i>de novo</i> proteins is also not beyond anticipation under neutral expectation, as they generally have longer theoretical lifespan than their current age, due to their GC-rich sequence property enabling stable ORFs with lower chance of non-sense mutations. Interestingly, although the emergence and retention of these <i>de novo</i> genes are likely driven by neutral forces, population genetics study in 67 human individuals and 82 macaque animals revealed signatures of purifying selection on these genes specifically in human population, indicating a proportion of these newly-originated proteins are already functional in human. We thus propose a mechanism for creation of functional <i>de novo</i> proteins from ancestral lncRNAs during the primate evolution, which may contribute to human-specific genetic novelties by taking advantage of existed genomic contexts.</p></div

<i>De novo</i> protein-coding genes originating from lncRNAs.

<p>(<b>A</b>) Computational pipeline for <i>ab inito</i> identification and meta-analysis of <i>de novo</i> genes in the hominoid lineage. (<b>B</b>) Number of <i>de novo</i> genes on the phylogenetic tree, with the branch length proportional to the divergence time. (<b>C</b>) Stacked histogram showing the percentage of <i>de novo</i> gene orthologs that also show expression in chimpanzee or rhesus macaque. (<b>D</b>) Boxplot showing relative expression levels of the transcripts and their nearby regions corresponding to <i>de novo</i> genes (orthologs) in human (chimpanzee or macaque). The nearby regions are defined as upstream and downstream regions with equal length to the corresponding genes. For each region, the relative expression was calculated by normalizing the expression level of this region with the sum of the expression levels of the genic region and the nearby regions. (<b>E</b>) Percentage of splicing junctions with supporting RNA-Seq reads in human, chimpanzee and rhesus macaque. (<b>F</b>) For each pair of tissues, <i>Spearman</i> correlation coefficients were computed separately, and the extent of tissue-specific differences in <i>de novo</i> gene expressions are shown (based on the color scale). Dotted lines highlight parallel comparisons between two different species.</p

Profiling of polymorphisms in human and rhesus macaque.

<p><b>(A)</b> Comparison of human polymorphism sites profiled in this study with those in the 1000 Genomes Project. (<b>B</b>) The sequencing coverages of whole genome sequencing from one macaque animal and for the targeted re-sequencing of 82 macaque animals are summarized in green barplot and heatmaps inside the <i>Circos</i> map, respectively. The depths of the sequencing coverage are proportional to the color depth. Black rectangles outside the colored chromosome block represent the genomic locations of macaque orthologous regions of human <i>de novo</i> genes. The bottom panel illustrates the sequencing details of one region of interest. (<b>C</b>) Cumulative frequency of mean sequencing coverage on different genic regions of <i>de novo</i> genes is shown. Intergenic regions: 1-kb regions upstream and downstream of the gene. (<b>D, E</b>) Venn diagrams showing the distributions of macaque polymorphism sites identified by whole-genome sequencing and targeted re-sequencing, in terms of polymorphism sites (<b>D</b>) and genotypes (<b>E</b>).</p

<i>De novo</i> proteins originate from lncRNAs precursors irrespectively of their functional status at RNA level.

<p><b>(A)</b> Flow chart showing the computational pipeline of lncRNAome identification in rhesus macaque. (<b>B, C</b>) For lncRNAs identified in this study, the distribution of distances between 5’ end of lncRNAs and the nearest annotated transcript start site (TSS) (<b>B</b>) or CpG island (<b>C</b>) are shown. The numbers of TSS and CpG islands within 1-kb of the transcripts are shown in the inserted histograms. Annotated genes and randomly selected intergenic sites are also shown as positive and negative controls, respectively. (<b>D</b>) On the basis of population genetics data in rhesus macaque, the distribution of π for synonymous sites (<b><i>Syn Sites</i></b>), non-synonymous sites (<b><i>Nonsyn Sites</i></b>), all lncRNAs, lncRNA precursors and non-coding genes (<b><i>Functional</i></b>) are summarized in boxplots. <i>NS</i>: not significant, **<i>p</i>-value <0.01.</p