Laurence D. Hurst

While it is tempting to suppose that everything that happens inside our cells has a function, a recent study in PLOS Biology adds to the growing consensus that, for large-bodied species, the high diversity of transcripts is down to the fact that accidents happen.

Background: Understanding the causes of the exceptional rate of evolution of the mammalian placenta is likely to aid the understanding of placental development and the etiology of the human-specific pregnancy disorder pre-eclampsia (PE). As retroelements are often lineage-specific and known to be co-opted for placental function, here we consider the binding of the transcription factors GATA3 and DLX5 to retroelements. These factors are dysregulated in pre-eclampsia, as are their downstream consequences. Results: We identify retrovirus-derived LTR8B as a placentally-relevant cis-regulatory element (CRE), not least within the PSG array, a primate-specific genomic region that exhibits high intraspecies variability. LTR8B at PSG9 is particularly influential affecting other PSG family members. Moreover, unique among PSGs, PSG9 produces both secreted and membrane-anchored isoforms. The retroelement MER65-int provides alternative polyA signals that enable the evolution of secreted PSG variants by truncating the ancestral CEACAM protein’s transmembrane domain. Functional characterization finds that LTR8B/PSG9 regulates the differentiation of multinucleated trophoblasts (syncytialization) and, like chorionic gonadotropin and syncytin1, determines the identity of syncytiotrophoblasts. Notably, PSG9 is the most upregulated PSG in PE, with levels correlated with GATA3 and DLX5 levels. Conclusions: Retroelements contribute to the structural and expression evolution of PSG genes, facilitating lineage-specific placental evolution. The LTR8B/PSG9 regulatory network plays a central role in syncytiotrophoblast differentiation. Given the association between DLX5/GATA3 dysregulation and elevated PSG9 levels, along with PSG9’s expression in the first trimester, PSG9 shows potential as a predictive biomarker for preeclampsia.

Despite early assumptions of neutrality, numerous mechanisms are now thought to cause selection on synonymous mutations, commonly supported by a low evolutionary rate at synonymous sites ( K _s ). This has been best evidenced in the first ~10 codons of genes in E. coli, where K _s is less than around half that of the gene body. Diverse lines of evidence support the hypothesis that these first ~10 codons are under selection for high AT content which causes low mRNA stability that in turn enables ribosomal initiation. There remains one enigmatic discrepancy, however, namely that the low K _s domain extends far beyond the first 10 codons. Here we ask why this is. As we see no evidence that the zone influencing protein levels has been misestimated, we consider three further hypotheses: that reduced K _s is a) owing to overlapping genes, b) reflects an extended slow translational “ramp,” and c) is mutational. We reject the first two as in both E. coli and Bacillus sp. the extended low K _s domain persists on analysis of non-overlapping genes and in Bacillus , where fast optimal codons tend to be A/T-ending, a fast-to-slow codon trend is seen. We fail to falsify the third hypothesis. Employing mutation accumulation data for E. coli we show that the 5′ end has a lower mutation rate, with the first 10 codons having a rate around half that of the gene body, this then steadily increasing following the trend seen for K _s . Compositional variation is likely to explain some of the difference, the 5′ end lacking GC-rich runs while these are most mutagenic. We conclude that even a highly reduced K _s is not always adequate to substantiate selection on synonymous mutations. This result has broad implications for inference of the causes of evolutionary rate variation.

Background: Owing to their transcription factor binding sites, endogenous retroviruses (ERVs) can act as cis-regulatory-elements (CREs). By invading genomes in waves, ERVs offer a substrate for lineage-specific adaptations but also, when dysregulated, for lineage-specific disorders. Their role as CREs in rapid placental evolution, and in the human-specific placental disorder preeclampsia, may thus provide a paradigmatic exemplar. Here then we systematically identify ERV-derived CREs controlling human placental gene expression with dysregulation in preeclampsia. Results: We identify 87 ERV-derived CREs located upstream of genes expressed in the placenta. A subset of nine, all belonging to the ERV3-MLT1/2 families and dating to the mouse–human common ancestor, are consistently dysregulated in trophoblasts from preeclampsia samples. Of the nine ERV3-MLT1-linked genes dysregulated in preeclampsia, five are novel candidates, while four were previously associated with preeclampsia, though their ERV-based regulation was not recognized. Focusing on EPS8L1, we predict enhancer activity of upstream MLT1(G1) and validate using reporter assay and genome editing. This vertebrate-specific gene is expressed in progenitor cytotrophoblasts and syncytiotrophoblasts and is overexpressed in preeclampsia, correlating with preeclampsia biomarkers and is not elevated in related pregnancy disorders. A soluble form of EPS8L1 is detectable in maternal plasma as early as between 24 weeks of gestation. EPS8L1 knockout in trophoblast in vitro is lethal, and its overexpression alters trophoblast behaviors characteristic of preeclampsia. Conclusions: We conclude that ERV3-MLT1functions as a trophoblast-specific CRE for several human genes and may be dysregulated in preeclampsia. As EPS8L1 has a form in maternal circulation, it may have utility in diagnostics.

What accounts for the variation between proteins in their rate of evolution per synonymous substitution (i.e. dN/dS, alias ω)? Previous analyses suggested that cell location is predictive, with intracellular proteins evolving slower than membrane proteins, a result considered supportive of the extended complexity hypothesis. However, as they occur in 3D space, cytoplasmic proteins are expected to be more abundant. As the level of gene expression is the strongest predictor of ω, and many predictors of protein rate variation are explained by covariance with it, here we ask whether the cell compartment effect is explained by covariates. We employ two single-celled species for which there exist exceptional data, the bacterium (Escherichia coli) and the eukaryote (Saccharomyces cerevisiae). For both, we establish informative species trios to determine branch-specific ω values. In both species, in the absence of covariate control, cytoplasmic proteins evolve relatively slowly, while membrane proteins evolve fast, as originally claimed. After controlling for protein abundance, however, membrane proteins have the lowest rates, this inversion being resilient to multiple alternative abundance control methods. The effect size of the cell compartment as a predictor is of a comparable magnitude to the essentiality effect and remains when allowing for essentiality. We conclude that the effects of the cell compartments are real, but their direction is dependent on the presence or absence of abundance control. These results question any model, such as the extended complexity hypothesis, that claims support from a slower evolution of cytoplasmic proteins and underscore the importance of covariate control.

In bacteria, high A and low G content of the 5′ end of the coding sequence (CDS) promotes low RNA stability, facilitating ribosomal initiation and subsequently a high protein to transcript ratio. Additionally, 5′ NGG codons are suppressive owing to peptidyl-tRNA drop off. It was, therefore, surprising that the first large-scale transgene experiment to interrogate the 5′ effect by codon randomization found the NGG, G-rich codon AGG to be the most associated with high transgene output. Why is this? We show that this is not replicated in other large transgene datasets, where AGG and NGG are associated with low efficiency. More generally, there is limited agreement between the first experiment and others. This we find to be a consequence of non-random construct design. In constructs of the first experiment, AGG disproportionately occurs with non-AGG codons associated with low stability and high protein output, making AGG’s association with high output an artefact. While translationally non-optimal codons like AGG are conjectured to slow ribosomes for orderly initiation, we find that in the less biased constructs high, not low, translational adaptation in the first 10 codons is (weakly) predictive of higher translational efficiency. These results have implications for both transgene and experimental design.

To maintain genome integrity, repeat sequences are subject to heterochromatin inactivation and, in Neurospora, repeat-induced point mutation (RIP). The initiating factors behind both are poorly understood. We resolve the paradoxical observation that newly introduced Repeat-Linker-Repeat (R-L-R) constructs require RID alone for RIP, while genomic repeats are RIPed in the absence of RID, showing that eu- and hetero- chromatic repeats are handled differently, the latter additionally requiring DIM-2. The differences between mechanisms associated with older and newer duplicates caution against extrapolation from mechanisms inferred from model experimental systems. Additionally, while chromatin status affects RIP, we also show that RID, when tethered with LexA, acts as a nucleation center for the transition from euchromatin to heterochromatin in an HDA-1 dependent fashion. Constitutive heterochromatin by contrast is largely HDA1 independent and depends on HDA-1 paralogs. RID is thus a dual function initiator of both RIP and the transition to heterochromatin.

Although translational selection to favour codons that match the most abundant tRNAs is not readily observed in humans, there is nonetheless selection in humans on synonymous mutations. We hypothesize that much of this synonymous site selection can be explained in terms of protection against unwanted RNAs — spurious transcripts, mis-spliced forms or RNAs derived from transposable elements or viruses. We propose not only that selection on synonymous sites functions to reduce the rate of creation of unwanted transcripts (for example, through selection on exonic splice enhancers and cryptic splice sites) but also that high-GC content (but low-CpG content), together with intron presence and position, is both particular to functional native mRNAs and used to recognize transcripts as native. In support of this hypothesis, transcription, nuclear export, liquid phase condensation and RNA degradation have all recently been shown to promote GC-rich transcripts and suppress AU/CpG-rich ones. With such ‘traps’ being set against AU/CpG-rich transcripts, the codon usage of native genes has, in turn, evolved to avoid such suppression. That parallel filters against AU/CpG-rich transcripts also affect the endosomal import of RNAs further supports the unwanted transcript hypothesis of synonymous site selection and explains the similar design rules that have enabled the successful use of transgenes and RNA vaccines.

In many species highly expressed genes (HEGs) over-employ the synonymous codons that match the more abundant iso-acceptor tRNAs. Bacterial transgene codon randomization experiments report, however, that enrichment with such “translationally optimal” codons has little to no effect on the resultant protein level. By contrast, consistent with the view that ribosomal initiation is rate limiting, synonymous codon usage following the 5’ ATG greatly influences protein levels, at least in part by modifying RNA stability. For the design of bacterial transgenes, for simple codon based in silico inference of protein levels and for understanding selection on synonymous mutations, it would be valuable to computationally determine initiation optimality (IO) scores for codons for any given species. One attractive approach is to characterize the 5’ codon enrichment of HEGs compared with the most lowly expressed genes, just as translational optimality scores of codons have been similarly defined employing the full gene body. Here we determine the viability of this approach employing a unique opportunity: for Escherichia coli there is both the most extensive protein abundance data for native genes and a unique large-scale transgene codon randomization experiment enabling objective definition of the 5’ codons that cause, rather than just correlate with, high protein abundance (that we equate with initiation optimality, broadly defined). Surprisingly, the 5’ ends of native genes that specify highly abundant proteins avoid such initiation optimal codons. We find that this is probably owing to conflicting selection pressures particular to native HEGs, including selection favouring low initiation rates, this potentially enabling high efficiency of ribosomal usage and low noise. While the classical HEG enrichment approach does not work, rendering simple prediction of native protein abundance from 5’ codon content futile, we report evidence that initiation optimality scores derived from the transgene experiment may hold relevance for in silico transgene design for a broad spectrum of bacteria.