YTHDC1 m6A-dependent and m6A-independent functions converge to preserve the DNA damage response.

Cells have evolved a robust and highly regulated DNA damage response to preserve their genomic integrity. Although increasing evidence highlights the relevance of RNA regulation, our understanding of its impact on a fully efficient DNA damage response remains limited. Here, through a targeted CRISPR-knockout screen, we identify RNA-binding proteins and modifiers that participate in the p53 response. Among the top hits, we find the m6A reader YTHDC1 as a master regulator of p53 expression. YTHDC1 binds to the transcription start sites of TP53 and other genes involved in the DNA damage response, promoting their transcriptional elongation. YTHDC1 deficiency also causes the retention of introns and therefore aberrant protein production of key DNA damage factors. While YTHDC1-mediated intron retention requires m6A, TP53 transcriptional pause-release is promoted by YTHDC1 independently of m6A. Depletion of YTHDC1 causes genomic instability and aberrant cancer cell proliferation mediated by genes regulated by YTHDC1. Our results uncover YTHDC1 as an orchestrator of the DNA damage response through distinct mechanisms of co-transcriptional mRNA regulation.

The Evolutionary History of Ephs and Ephrins: Toward Multicellular Organisms.

Eph receptor (Eph) and ephrin signaling regulate fundamental developmental processes through both forward and reverse signaling triggered upon cell-cell contact. In vertebrates, they are both classified into classes A and B, and some representatives have been identified in many metazoan groups, where their expression and functions have been well studied. We have extended previous phylogenetic analyses and examined the presence of Eph and ephrins in the tree of life to determine their origin and evolution. We have found that 1) premetazoan choanoflagellates may already have rudimental Eph/ephrin signaling as they have an Eph-/ephrin-like pair and homologs of downstream-signaling genes; 2) both forward- and reverse-downstream signaling might already occur in Porifera since sponges have most genes involved in these types of signaling; 3) the nonvertebrate metazoan Eph is a type-B receptor that can bind ephrins regardless of their membrane-anchoring structure, glycosylphosphatidylinositol, or transmembrane; 4) Eph/ephrin cross-class binding is specific to Gnathostomata; and 5) kinase-dead Eph receptors can be traced back to Gnathostomata. We conclude that Eph/ephrin signaling is of older origin than previously believed. We also examined the presence of protein domains associated with functional characteristics and the appearance and conservation of downstream-signaling pathways to understand the original and derived functions of Ephs and ephrins. We find that the evolutionary history of these gene families points to an ancestral function in cell-cell interactions that could contribute to the emergence of multicellularity and, in particular, to the required segregation of cell populations.

Emergence and evolutionary analysis of the human DDR network: implications in comparative genomics and downstream analyses.

The DNA damage response (DDR) is a crucial signaling network that preserves the integrity of the genome. This network is an ensemble of distinct but often overlapping subnetworks, where different components fulfill distinct functions in precise spatial and temporal scenarios. To understand how these elements have been assembled together in humans, we performed comparative genomic analyses in 47 selected species to trace back their emergence using systematic phylogenetic analyses and estimated gene ages. The emergence of the contribution of posttranslational modifications to the complex regulation of DDR was also investigated. This is the first time a systematic analysis has focused on the evolution of DDR subnetworks as a whole. Our results indicate that a DDR core, mostly constructed around metabolic activities, appeared soon after the emergence of eukaryotes, and that additional regulatory capacities appeared later through complex evolutionary process. Potential key posttranslational modifications were also in place then, with interacting pairs preferentially appearing at the same evolutionary time, although modifications often led to the subsequent acquisition of new targets afterwards. We also found extensive gene loss in essential modules of the regulatory network in fungi, plants, and arthropods, important for their validation as model organisms for DDR studies.

Intron detention tightly regulates the stemness/differentiation switch in the adult neurogenic niche.

The adult mammalian brain retains some capacity to replenish neurons and glia, holding promise for brain regeneration. Thus, understanding the mechanisms controlling adult neural stem cell (NSC) differentiation is crucial. Paradoxically, adult NSCs in the subependymal zone transcribe genes associated with both multipotency maintenance and neural differentiation, but the mechanism that prevents conflicts in fate decisions due to these opposing transcriptional programmes is unknown. Here we describe intron detention as such control mechanism. In NSCs, while multiple mRNAs from stemness genes are spliced and exported to the cytoplasm, transcripts from differentiation genes remain unspliced and detained in the nucleus, and the opposite is true under neural differentiation conditions. We also show that m6A methylation is the mechanism that releases intron detention and triggers nuclear export, enabling rapid and synchronized responses. m6A RNA methylation operates as an on/off switch for transcripts with antagonistic functions, tightly controlling the timing of NSCs commitment to differentiation.

Two distinct epithelial-to-mesenchymal transition programs control invasion and inflammation in segregated tumor cell populations.

Epithelial-to-mesenchymal transition (EMT) triggers cell plasticity in embryonic development, adult injured tissues and cancer. Combining the analysis of EMT in cell lines, embryonic neural crest and mouse models of renal fibrosis and breast cancer, we find that there is not a cancer-specific EMT program. Instead, cancer cells dedifferentiate and bifurcate into two distinct and segregated cellular trajectories after activating either embryonic-like or adult-like EMTs to drive dissemination or inflammation, respectively. We show that SNAIL1 acts as a pioneer factor in both EMT trajectories, and PRRX1 drives the progression of the embryonic-like invasive trajectory. We also find that the two trajectories are plastic and interdependent, as the abrogation of the EMT invasive trajectory by deleting Prrx1 not only prevents metastasis but also enhances inflammation, increasing the recruitment of antitumor macrophages. Our data unveil an additional role for EMT in orchestrating intratumor heterogeneity, driving the distribution of functions associated with either inflammation or metastatic dissemination.

Serine/threonine kinases and E2-ubiquitin conjugating enzymes in Planctomycetes: unexpected findings.

The regulation of signal transduction by phosphorylation and ubiquitination is essential to ensure the correct behavior of eukaryotic cells. We searched for protein families involved in such signaling in several eukaryotic species and in a limited set of prokaryotes, where two members of the Planctomycetes phylum were included as they exhibit eukaryote-like features (Gemmata obscuriglobus and Pirellula staleyi). We identified sequences homologous to eukaryotic serine/threonine kinases (STKs) and E2-ubiquitin conjugating enzymes in the two Planctomycetes species. To extend these analyses to the Planctomycetes/Verrucomicrobia/Chlamydia super-phylum, we performed comparative analyses using domains from kinases, phosphatases and GTPases that serve as signaling signatures, and we analyzed their distributions. We found substantial differences in kinome densities with regards to other prokaryote clades and among the groups in the Planctomycetes/Verrucomicrobia/Chlamydia super-phylum. In addition, we identified the presence of classic eukaryotic E2-conjugating ubiquitin proteins in prokaryotes, these having previously believed to exist only in eukaryotes. Our phylogenetic analyses of the STKs signature domains and E2-enzymes suggest the existence of horizontal gene transfer.

ORC1 binds to cis-transcribed RNAs for efficient activation of replication origins.

Cells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. Our results unveil a non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins.

Draft genome sequence of the electricigen Acidiphilium sp. strain PM (DSM 24941).

Acidiphilium sp. strain PM (DSM 24941) was isolated from Rio Tinto's acidic, heavy metal-rich waters. Voltammetry experiments revealed that this strain is capable of electricity production even under aerobic conditions. Here we report the draft genome sequence of Acidiphilium sp. PM and a preliminary genome analysis that reveals a versatile respiratory metabolism.

Environmental transcriptome analysis reveals physiological differences between biofilm and planktonic modes of life of the iron oxidizing bacteria Leptospirillum spp. in their natural microbial community.

BACKGROUND: Extreme acidic environments are characterized by their high metal content and lack of nutrients (oligotrophy). Macroscopic biofilms and filaments usually grow on the water-air interface or under the stream attached to solid substrates (streamers). In the Río Tinto (Spain), brown filaments develop under the water stream where the Gram-negative iron-oxidizing bacteria Leptospirillum spp. (L. ferrooxidans and L. ferriphilum) and Acidithiobacillus ferrooxidans are abundant. These microorganisms play a critical role in bioleaching processes for industrial (biominery) and environmental applications (acid mine drainage, bioremediation). The aim of this study was to investigate the physiological differences between the free living (planktonic) and the sessile (biofilm associated) lifestyles of Leptospirillum spp. as part of its natural extremely acidophilic community. RESULTS: Total RNA extracted from environmental samples was used to determine the composition of the metabolically active members of the microbial community and then to compare the biofilm and planktonic environmental transcriptomes by hybridizing to a genomic microarray of L. ferrooxidans. Genes up-regulated in the filamentous biofilm are involved in cellular functions related to biofilm formation and maintenance, such as: motility and quorum sensing (mqsR, cheAY, fliA, motAB), synthesis of cell wall structures (lnt, murA, murB), specific proteases (clpX/clpP), stress response chaperons (clpB, clpC, grpE-dnaKJ, groESL), etc. Additionally, genes involved in mixed acid fermentation (poxB, ackA) were up-regulated in the biofilm. This result, together with the presence of small organic acids like acetate and formate (1.36 mM and 0.06 mM respectively) in the acidic (pH 1.8) water stream, suggests that either L. ferrooxidans or other member of the microbial community are producing acetate in the acidophilic biofilm under microaerophilic conditions. CONCLUSIONS: Our results indicate that the acidophilic filaments are dynamic structures in which different mechanisms for biofilm formation/dispersion are operating. Specific transcriptomic fingerprints can be inferred for both planktonic and sessile cells, having the former a more active TCA cycle, while the mixed acid fermentation process dominate in the latter. The excretion of acetate may play a relevant ecological role as a source of electron donor for heterotrophic Fe3+ reducers like some Alphaproteobacteria, Acidobacterium spp. and Sulfobacillus spp., also present in the biofilm. Additionally, acetate may have a negative effect on bioleaching by inhibiting the growth of chemolithotrophic bacteria.

A gene regulatory network to control EMT programs in development and disease.

The Epithelial to Mesenchymal Transition (EMT) regulates cell plasticity during embryonic development and in disease. It is dynamically orchestrated by transcription factors (EMT-TFs), including Snail, Zeb, Twist and Prrx, all activated by TGF-ß among other signals. Here we find that Snail1 and Prrx1, which respectively associate with gain or loss of stem-like properties and with bad or good prognosis in cancer patients, are expressed in complementary patterns during vertebrate development and in cancer. We show that this complementarity is established through a feedback loop in which Snail1 directly represses Prrx1, and Prrx1, through direct activation of the miR-15 family, attenuates the expression of Snail1. We also describe how this gene regulatory network can establish a hierarchical temporal expression of Snail1 and Prrx1 during EMT and validate its existence in vitro and in vivo, providing a mechanism to switch and select different EMT programs with important implications in development and disease.

DDRprot: a database of DNA damage response-related proteins.

The DNA Damage Response (DDR) signalling network is an essential system that protects the genome's integrity. The DDRprot database presented here is a resource that integrates manually curated information on the human DDR network and its sub-pathways. For each particular DDR protein, we present detailed information about its function. If involved in post-translational modifications (PTMs) with each other, we depict the position of the modified residue/s in the three-dimensional structures, when resolved structures are available for the proteins. All this information is linked to the original publication from where it was obtained. Phylogenetic information is also shown, including time of emergence and conservation across 47 selected species, family trees and sequence alignments of homologues. The DDRprot database can be queried by different criteria: pathways, species, evolutionary age or involvement in (PTM). Sequence searches using hidden Markov models can be also used.Database URL: http://ddr.cbbio.es.

Whole-genome analysis of Azoarcus sp. strain CIB provides genetic insights to its different lifestyles and predicts novel metabolic features.

The genomic features of Azoarcus sp. CIB reflect its most distinguishing phenotypes as a diazotroph, facultative anaerobe, capable of degrading either aerobically and/or anaerobically a wide range of aromatic compounds, including some toxic hydrocarbons such as toluene and m-xylene, as well as its endophytic lifestyle. The analyses of its genome have expanded the catabolic potential of strain CIB toward common natural compounds, such as certain diterpenes, that were not anticipated as carbon sources. The high number of predicted solvent efflux pumps and heavy metal resistance gene clusters has provided the first evidence for two environmentally relevant features of this bacterium that remained unknown. Genome mining has revealed several gene clusters likely involved in the endophytic lifestyle of strain CIB, opening the door to the molecular characterization of some plant growth promoting traits. Horizontal gene transfer and mobile genetic elements appear to have played a major role as a mechanism of adaptation of this bacterium to different lifestyles. This work paves the way for a systems biology-based understanding of the abilities of Azoarcus sp. CIB to integrate aerobic and anaerobic metabolism of aromatic compounds, tolerate stress conditions, and interact with plants as an endophyte of great potential for phytostimulation and phytoremediation strategies. Comparative genomics provides an Azoarcus pan genome that confirms the global metabolic flexibility of this genus, and suggests that its phylogeny should be revisited.