Evolution of a Human-Specific De Novo Open Reading Frame and Its Linked Transcriptional Silencer.

In the human genome, two short open reading frames (ORFs) separated by a transcriptional silencer and a small intervening sequence stem from the gene SMIM45. The two ORFs show different translational characteristics, and they also show divergent patterns of evolutionary development. The studies presented here describe the evolution of the components of SMIM45. One ORF consists of an ultra-conserved 68 amino acid (aa) sequence, whose origins can be traced beyond the evolutionary age of divergence of the elephant shark, ~462 MYA. The silencer also has ancient origins, but it has a complex and divergent pattern of evolutionary formation, as it overlaps both at the 68 aa ORF and the intervening sequence. The other ORF consists of 107 aa. It develops during primate evolution but is found to originate de novo from an ancestral non-coding genomic region with root origins within the Afrothere clade of placental mammals, whose evolutionary age of divergence is ~99 MYA. The formation of the complete 107 aa ORF during primate evolution is outlined, whereby sequence development is found to occur through biased mutations, with disruptive random mutations that also occur but lead to a dead-end. The 107 aa ORF is of particular significance, as there is evidence to suggest it is a protein that may function in human brain development. Its evolutionary formation presents a view of a human-specific ORF and its linked silencer that were predetermined in non-primate ancestral species. The genomic position of the silencer offers interesting possibilities for the regulation of transcription of the 107 aa ORF. A hypothesis is presented with respect to possible spatiotemporal expression of the 107 aa ORF in embryonic tissues.

From epigenotype to new genotypes: Relevance of epigenetic mechanisms in the emergence of genomic evolutionary novelty.

A fundamental question in evolutionary biology is how heritable variability originates. In Darwinian evolution a central focus was placed on the thinking that random variations are the material basis for the action of natural selection. Although randomness in Darwinian evolution can be interpreted from different angles, here I will focus on the assumption of equiprobability of mutations. I have reviewed the literature regarding epigenetic mechanisms causing biased genetic variability. Although it is interesting to find correlations between somatic epigenetic marks and evolution, causation between epigenetic changes and genomic evolutionary novelties can only be established when the epigenetic changes are interrogated in the germ line. Epigenetic changes are reported to influence the emergence of single nucleotide polymorphisms and copy number variations. On the other hand, epigenetic changes are known to be influenced by environmental exposures. This dual ability of epigenetic changes could mean that germ line epigenetically influenced mutations could have an important role in the emergence of genomic evolutionary novelties. The emergent knowledge on the relation of epigenetic changes and mutations will help to understand an underappreciated role of the environment in speciation and genomic divergence: that of influencer of genomic changes.

Somatic mutation patterns at Ig and Non-Ig Loci.

The reverse transcriptase (RT) model of immunoglobulin (Ig) somatic hypermutation (SHM) has received insufficient scientific attention. This is understandable given that DNA deamination mediated by activation-induced deaminase (AID), the initiating step of Ig SHM, has dominated experiments since 2002. We summarise some key history of the RT Ig SHM model dating to 1987. For example, it is now established that DNA polymerase η, the sole DNA repair polymerase involved in post-replication short-patch repair, is an efficient cellular RT. This implies that it is potentially able to initiate target site reverse transcription by RNA-directed DNA repair at AID-induced lesions. Recently, DNA polymerase θ has also been shown to be an efficient cellular RT. Since DNA polymerase θ plays no significant role in Ig SHM, it could serve a similar RNA-dependent DNA polymerase role as DNA polymerase η at non-Ig loci in the putative RNA-templated nucleotide excision repair of bulky adducts and other mutagenic lesions on the transcribed strand. A major yet still poorly recognised consequence of the proposed RT process in Ig SHM is the generation of significant and characteristic strand-biased mutation signatures at both deoxyadenosine/deoxythymidine and deoxyguanosine/deoxycytidine base pairs. In this historical perspective, we highlight how diagnostic strand-biased mutation signatures are detected in vivo during SHM at both Ig loci in germinal centre B lymphocytes and non-Ig loci in cancer genomes. These strand-biased signatures have been significantly obscured by technical issues created by improper use of the polymerase chain reaction technique. A heightened awareness of this fact should contribute to better data interpretation and somatic mutation pattern recognition both at Ig and non-Ig loci.

ADAR deaminase A-to-I editing of DNA and RNA moieties of RNA:DNA hybrids has implications for the mechanism of Ig somatic hypermutation.

The implications are discussed of recently published biochemical studies on ADAR-mediated A-to-I DNA and RNA deamination at RNA:DNA hybrids. The significance of these data are related to previous work on strand-biased and codon-context mutation signatures in B lymphocytes and cancer genomes. Those studies have established that there are two significant strand biases at A:T and G:C base pairs, A-site mutations exceed T-site mutations (A>>T) by 2.9 fold and G-site mutations exceed C-site mutations (G>>C) by 1.7 fold. Both these strand biases are inconsistent with alternative "DNA Deamination" mechanisms, yet are expected consequences of the RNA/RT-based "Reverse Transcriptase" mechanism of immunoglobulin (Ig) somatic hypermutation (SHM). The A-to-I DNA editing component at RNA:DNA hybrids that is likely to occur in Transcription Bubbles, while important, is of far lower A-to-I editing efficiency than in dsRNA substrates. The RNA moiety of RNA:DNA hybrids is also edited at similar lower frequencies relative to the editing rate at dsRNA substrates. Further, if the A-to-I DNA editing at RNA:DNA hybrids were the sole cause of A-to-I (read as A-to-G) mutation events for Ig SHM in vivo then the exact opposite strand biases at A:T base pairs (T>>A) of what is actually observed (A>>T) would be predicted. It is concluded that the strand-biased somatic mutation patterns at both A:T and G:C base pairs in vivo are best interpreted by the sequential steps of the RNA/RT-based mechanism. Further, the direct DNA A-to-I deamination at Transcription Bubbles is expected to contribute to the T-to-C component of the strand-biased Ig SHM spectrum.

Reverse Transcriptase Mechanism of Somatic Hypermutation: 60 Years of Clonal Selection Theory.

The evidence for the reverse transcriptase mechanism of somatic hypermutation is substantial and multifactorial. In this 60th anniversary year of the publication of Sir MacFarlane Burnet's Clonal Selection Theory, the evidence is briefly reviewed and updated.

Somatic hypermutation in immunity and cancer: Critical analysis of strand-biased and codon-context mutation signatures.

For 30 years two general mechanisms have competed to explain somatic hypermutation of immunoglobulin (Ig) genes. The first, the DNA-based model, is focused only on DNA substrates. The modern form is the Neuberger "DNA Deamination Model" based on activation-induced cytidine deaminase (AID) and short-patch error-prone DNA repair by DNA Polymerase-η operating around AID C-to-U lesions. The other is an RNA-based mechanism or the "Reverse Transcriptase Model" of SHM which produces strand-biased mutations at A:T and G:C base pairs. This involves error-prone cDNA synthesis via an RNA-dependent DNA polymerase copying the Ig pre-mRNA template and integrating the now error-filled cDNA copy back into the normal chromosomal site. The modern form of this mechanism depends on AID dC-to-dU lesions and long tract error-prone cDNA synthesis of the transcribed strand by DNA Polymerase-η acting as a reverse transcriptase. The evidence for and against each mechanism is critically evaluated. The conclusion is that all the SHM molecular data gathered since 1980 supports directly or indirectly the RNA/RT-based mechanism. All the data and critical analyses are systematically laid out so the reader can evaluate this conclusion for themselves. Recently we have investigated whether similar RNA/RT-based mutator mechanisms explain how de novo mutations arise in somatic tissues (cancer genomes). The data analyses indeed suggest that cancers arise via dysregulated "Ig-like SHM responses" involving rogue DNA and RNA deaminations coupled to genome-wide RT events. Further, Robyn Lindley has recently shown that the strand-biased mutations in cancer genome genes are also in "codon-context." This has been termed Targeted Somatic Mutation (TSM) to highlight that mutations are far more targeted than previously thought in somatic tissues associated with disease. The TSM process implies an "in-frame DNA reader" whereby DNA and RNA deaminases at transcribed regions are guided in their mutagenic action, by the codon reading frame of the DNA.