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Ribosome profiling (or Ribo-seq) is currently the most popular methodology for studying translation; it has been employed in recent years to decipher various fundamental gene expression regulation aspects. The main promise of the approach is its ability to detect ribosome densities over an entire transcriptome in high resolution of single codons. Indeed, dozens of ribo-seq studies have included results related to local ribosome densities in different parts of the transcript; nevertheless, the performance of Ribo-seq has yet to be quantitatively evaluated and reported in a large-scale multi-organismal and multi-protocol study of currently available datasets. Here we provide the first objective evaluation of Ribo-seq at the resolution of a single nucleotide(s) using clear, interpretable measures, based on the analysis of 15 experiments, 6 organisms, and a total of 612, 961 transcripts. Our major conclusion is that the ability to infer signals of ribosomal densities at nucleotide scale is considerably lower than previously thought, as signals at this level are not reproduced well in experimental replicates. In addition, we provide various quantitative measures that connect the expected error rate with Ribo-seq analysis resolution. The analysis of Ribo-seq data at the resolution of codons and nucleotides provides a challenging task, calls for task-specific statistical methods and further protocol improvements. We believe that our results are important for every researcher studying translation and specifically for researchers analyzing data generated by the Ribo-seq approach. This article was reviewed by Dmitrij Frishman, Eugene Koonin and Frank Eisenhaber.

Rhynchophylline (RHY) is an alkaloid component of Uncaria, which are plants extensively used in traditional Asian medicines. Uncaria treatments increase sleep time and quality in humans, and RHY induces sleep in rats. However, like many traditional natural treatments, the mechanisms of action of RHY and Uncaria remain evasive. Moreover, it is unknown whether RHY modifies key brain oscillations during sleep. We thus aimed at defining the effects of RHY on sleep architecture and oscillations throughout a 24-h cycle, as well as identifying the underlying molecular mechanisms. Mice received systemic RHY injections at two times of the day (beginning and end of the light period), and vigilance states were studied by electrocorticographic recordings. RHY enhanced slow wave sleep (SWS) after both injections, suppressed paradoxical sleep (PS) in the light but enhanced PS in the dark period. Furthermore, RHY modified brain oscillations during both wakefulness and SWS (including delta activity dynamics) in a time-dependent manner. Interestingly, most effects were larger in females. A brain spatial transcriptomic analysis showed that RHY modifies the expression of genes linked to cell movement, apoptosis/necrosis, and transcription/translation in a brain region-independent manner, and changes those linked to sleep regulation (e.g., Hcrt, Pmch) in a brain region-specific manner (e.g., in the hypothalamus). The findings provide support to the sleep-inducing effect of RHY, expose the relevance to shape wake/sleep oscillations, and highlight its effects on the transcriptome with a high spatial resolution. The exposed molecular mechanisms underlying the effect of a natural compound should benefit sleep- and brain-related medicine.

Genome sequencing has revolutionized our view of the relationships among genomes, particularly in revealing the confounding effects of lateral genetic transfer (LGT). Phylogenomic techniques have been used to construct purported trees of microbial life. Although such trees are easily interpreted and allow the use of a subset of genomes as "proxies" for the full set, LGT and other phenomena impact the positioning of different groups in genome trees, confounding and potentially invalidating attempts to construct a phylogeny-based taxonomy of microorganisms. Network and graph approaches can reveal complex sets of relationships, but applying these techniques to large data sets is a significant challenge. Notwithstanding the question of what exactly it might represent, generating and interpreting a Tree or Network of All Genomes will only be feasible if current algorithms can be improved upon. Complex relationships among even the most-similar genomes demonstrate that proxy-based approaches to simplifying large sets of genomes are not alone sufficient to solve the analysis problem. A phylogenomic analysis of 1173 sequenced bacterial and archaeal genomes generated phylogenetic trees for 159,905 distinct homologous gene sets. The relationships inferred from this set can be heavily dependent on the inclusion of other taxa: for example, phyla such as Spirochaetes, Proteobacteria and Firmicutes are recovered as cohesive groups or split depending on the presence of other specific lineages. Furthermore, named groups such as Acidithiobacillus, Coprothermobacter and Brachyspira show a multitude of affiliations that are more consistent with their ecology than with small subunit ribosomal DNA-based taxonomy. Network and graph representations can illustrate the multitude of conflicting affinities, but all methods impose constraints on the input data and create challenges of construction and interpretation. These complex relationships highlight the need for an inclusive approach to genomic data, and current methods with minor alterations will likely scale to allow the analysis of data sets with 10,000 or more genomes. The main challenges lie in the visualization and interpretation of genomic relationships, and the redefinition of microbial taxonomy when subsets of genomic data are so evidently in conflict with one another, and with the "canonical" molecular taxonomy. The manuscript was reviewed by William Martin, W. Ford Doolittle, Joel Velasco and Eugene Koonin.

Abstract Background TATA Binding Protein (TBP) is required for transcription initiation by all three eukaryotic RNA polymerases. It participates in transcriptional initiation at the majority of eukaryotic gene promoters, either by direct association to the TATA box upstream of the transcription start site or by indirectly localizing to the promoter through other proteins. TBP exists in solution in a dimeric form but binds to DNA as a monomer. Here, we present the first mathematical model for auto-catalytic TBP expression and use it to study the role of dimerization in maintaining the steady state TBP level. Results We show that the autogenous regulation of TBP results in a system that is capable of exhibiting three steady states: an unstable low TBP state, one stable state corresponding to a physiological TBP concentration, and another stable steady state corresponding to unviable cells where no TBP is expressed. Our model predicts that a basal level of TBP is required to establish the transcription of the TBP gene, and hence for cell viability. It also predicts that, for the condition corresponding to a typical mammalian cell, the high-TBP state and cell viability is sensitive to variation in DNA binding strength. We use the model to explore the effect of the dimer in buffering the response to changes in TBP levels, and show that for some physiological conditions the dimer is not important in buffering against perturbations. Conclusions Results on the necessity of a minimum basal TBP level support the in vivo observations that TBP is maternally inherited, providing the small amount of TBP required to establish its ubiquitous expression. The model shows that the system is sensitive to variations in parameters indicating that it is vulnerable to mutations in TBP. A reduction in TBP-DNA binding constant can lead the system to a regime where the unviable state is the only steady state. Contrary to the current hypotheses, we show that under some physiological conditions the dimer is not very important in restoring the system to steady state. This model demonstrates the use of mathematical modelling to investigate system behaviour and generate hypotheses governing the dynamics of such nonlinear biological systems. Reviewers This article was reviewed by Tomasz Lipniacki, James Faeder and Anna Marciniak-Czochra.

Maelstrom (MAEL) plays a crucial role in a recently-discovered piRNA pathway; however its specific function remains unknown. Here a novel MAEL-specific domain characterized by a set of conserved residues (Glu-His-His-Cys-His-Cys, EHHCHC) was identified in a broad range of species including vertebrates, sea squirts, insects, nematodes, and protists. It exhibits ancient lineage-specific expansions in several species, however, appears to be lost in all examined teleost fish species. Functional involvement of MAEL domains in DNA- and RNA-related processes was further revealed by its association with HMG, SR-25-like and HDAC_interact domains. A distant similarity to the DnaQ-H 3'–5' exonuclease family with the RNase H fold was discovered based on the evidence that all MAEL domains adopt the canonical RNase H fold; and several protist MAEL domains contain the conserved 3'–5' exonuclease active site residues (Asp-Glu-Asp-His-Asp, DEDHD). This evolutionary link together with structural examinations leads to a hypothesis that MAEL domains may have a potential nuclease activity or RNA-binding ability that may be implicated in piRNA biogenesis. The observed transition of two sets of characteristic residues between the ancestral DnaQ-H and the descendent MAEL domains may suggest a new mode for protein function evolution called "active site switch", in which the protist MAEL homologues are the likely evolutionary intermediates due to harboring the specific characteristics of both 3'–5' exonuclease and MAEL domains. This article was reviewed by L Aravind, Wing-Cheong Wong and Frank Eisenhaber. For the full reviews, please go to the Reviewers' Comments section.

The CRISPR-Cas systems of adaptive antivirus immunity are present in most archaea and many bacteria, and provide resistance to specific viruses or plasmids by inserting fragments of foreign DNA into the host genome and then utilizing transcripts of these spacers to inactivate the cognate foreign genome. The recent development of powerful genome engineering tools on the basis of CRISPR-Cas has sharply increased the interest in the diversity and evolution of these systems. Comparative genomic data indicate that during evolution of prokaryotes CRISPR-Cas loci are lost and acquired via horizontal gene transfer at high rates. Mathematical modeling and initial experimental studies of CRISPR-carrying microbes and viruses reveal complex coevolutionary dynamics. We performed a bifurcation analysis of models of coevolution of viruses and microbial host that possess CRISPR-Cas hereditary adaptive immunity systems. The analyzed Malthusian and logistic models display complex, and in particular, quasi-chaotic oscillation regimes that have not been previously observed experimentally or in agent-based models of the CRISPR-mediated immunity. The key factors for the appearance of the quasi-chaotic oscillations are the non-linear dependence of the host immunity on the virus load and the partitioning of the hosts into the immune and susceptible populations, so that the system consists of three components. Bifurcation analysis of CRISPR-host coevolution model predicts complex regimes including quasi-chaotic oscillations. The quasi-chaotic regimes of virus-host coevolution are likely to be biologically relevant given the evolutionary instability of the CRISPR-Cas loci revealed by comparative genomics. The results of this analysis might have implications beyond the CRISPR-Cas systems, i.e. could describe the behavior of any adaptive immunity system with a heritable component, be it genetic or epigenetic. These predictions are experimentally testable. This manuscript was reviewed by Sandor Pongor, Sergei Maslov and Marek Kimmel. For the complete reports, go to the Reviewers’ Reports section.

Transcription of the antisense strand of RTL1 produces a sense mRNA that is targeted for degradation by antisense microRNAs transcribed from the sense strand. Translation of the mRNA produces a retrotransposon-derived protein that is implicated in placental development. The sense and antisense transcripts are oppositely imprinted: sense mRNAs are expressed from the paternally-derived chromosome, antisense microRNAs from the maternally-derived chromosome. Two microRNAs at the RTL1 locus, miR-431 and the rodent-specific miR-434, are derived from within tandem repeats. We present an evolutionary model for the establishment of a new self-targeting microRNA derived from within a tandem repeat that inhibits production of RTL1 protein when maternally-derived in heterozygotes but not when paternally-derived. The interaction of sense and antisense transcripts can be interpreted as a form of communication between maternally-derived and paternally-derived RTL1 alleles that possesses many of the features of a greenbeard effect. This interaction is evolutionary stable, unlike a typical greenbeard effect, because of the necessary complementarity between microRNAs and mRNA transcribed from opposite strands of the same double helix. We conjecture that microRNAs and mRNA cooperate to reduce demands on mothers when an allele is paired with itself in homozygous offspring. This article was reviewed by Eugene Berezikov and Bernard Crespi.

“Which came first, the Chicken or the Egg?” We suggest this question is not a paradox. The Modern Synthesis envisions speciation through genetic changes in germ cells via random mutations, an “Egg first” scenario, but perhaps epigenetic inheritance mechanisms can transmit adaptive changes initiated in the soma (“Chicken first”). The article was reviewed by Dr. Eugene Koonin, Dr. Itai Yanai, Dr. Laura Landweber.