CtIP-dependent nascent RNA expression flanking DNA breaks guides the choice of DNA repair pathway

The RNA world is changing our views about sensing and resolution of DNA damage. Here, we developed single-molecule DNA/RNA analysis approaches to visualize how nascent RNA facilitates the repair of DNA double-strand breaks (DSBs). RNA polymerase II (RNAPII) is crucial for DSB resolution in human cells. DSB-flanking, RNAPII-generated nascent RNA forms RNA:DNA hybrids, guiding the upstream DNA repair steps towards favouring the error-free Homologous Recombination (HR) pathway over Non-Homologous End Joining. Specific RNAPII inhibitor, THZ1, impairs recruitment of essential HR proteins to DSBs, implicating nascent RNA in DNA end resection, initiation and execution of HR repair. We further propose that resection factor CtIP interacts with and re-activates RNAPII when paused by the RNA:DNA hybrids, collectively promoting faithful repair of chromosome breaks to maintain genomic integrity.

Viroids and Viroid-like Circular RNAs: Do They Descend from Primordial Replicators?

Viroids are a unique class of plant pathogens that consist of small circular RNA molecules, between 220 and 450 nucleotides in size. Viroids encode no proteins and are the smallest known infectious agents. Viroids replicate via the rolling circle mechanism, producing multimeric intermediates which are cleaved to unit length either by ribozymes formed from both polarities of the viroid genomic RNA or by coopted host RNAses. Many viroid-like small circular RNAs are satellites of plant RNA viruses. Ribozyviruses, represented by human hepatitis delta virus, are larger viroid-like circular RNAs that additionally encode the viral nucleocapsid protein. It has been proposed that viroids are direct descendants of primordial RNA replicons that were present in the hypothetical RNA world. We argue, however, that much later origin of viroids, possibly, from recently discovered mobile genetic elements known as retrozymes, is a far more parsimonious evolutionary scenario. Nevertheless, viroids and viroid-like circular RNAs are minimal replicators that are likely to be close to the theoretical lower limit of replicator size and arguably comprise the paradigm for replicator emergence. Thus, although viroid-like replicators are unlikely to be direct descendants of primordial RNA replicators, the study of the diversity and evolution of these ultimate genetic parasites can yield insights into the earliest stages of the evolution of life.

A small RNA that cooperatively senses two stacked metabolites in one pocket for gene control

Riboswitches are structured non-coding RNAs often located upstream of essential genes in bacterial messenger RNAs. Such RNAs regulate expression of downstream genes by recognizing a specific cellular effector. Although nearly 50 riboswitch classes are known, only a handful recognize multiple effectors. Here, we report the 2.60-Å resolution co-crystal structure of a class I type I preQ1-sensing riboswitch that reveals two effectors stacked atop one another in a single binding pocket. These effectors bind with positive cooperativity in vitro and both molecules are necessary for gene regulation in bacterial cells. Stacked effector recognition appears to be a hallmark of the largest subgroup of preQ1 riboswitches, including those from pathogens such as Neisseria gonorrhoeae. We postulate that binding to stacked effectors arose in the RNA World to closely position two substrates for RNA-mediated catalysis. These findings expand known effector recognition capabilities of riboswitches and have implications for antimicrobial development.

The Perennial Riddle of Life’s Origin

The origin of life is the most consequential problem in biology, possibly in all of science, and it remains unsolved. This chapter summarizes what has been learned and highlights questions that remain open, including How, Where, When, and especially Why. LUCA, some four billion years ago, already featured the basic capacities of contemporary cells. These must have evolved still earlier, at a nebulous proto-cellular stage. There is good reason to believe that enzymes, DNA, ribosomes, electron-transport chains, and the rotary ATP synthase all predate LUCA and were shaped by the standard process of variation and natural selection, but we know next to nothing about how cells ever got started. I favor the proposal that it began with a purely chemical dynamic network capable of reproducing itself, that may have originated by chance. Natural selection would have favored the incorporation of any ancillary factors that promoted its kinetic stability, especially ones that improved reproduction or gave access to energy. All the specifics are in dispute, including the role of a prebiotic broth of organic chemicals, the nature and origin of enclosure, the RNA world, and a venue in submarine hydrothermal vents. My sense is that critical pieces of the puzzle remain to be discovered.

High-Resolution, Multidimensional Phylogenetic Metrics Identify Class I Aminoacyl-tRNA Synthetase Evolutionary Mosaicity and Inter-modular Coupling

The provenance of the aminoacyl-tRNA synthetases (aaRS) poses challenging questions because of their role in the emergence and evolution of genetic coding. We investigate evidence about their ancestry from curated structure-based multiple sequence alignments of a structurally invariant “scaffold” shared by all 10 canonical Class I aaRS. Three uncorrelated phylogenetic metrics—residue-by-residue conservation, its variance, and row-by-row cladistic congruence—imply that the Class I scaffold is a mosaic assembled from distinct, successive genetic sources. These data are especially significant in light of: (i) experimental fragmentations of the Class I scaffold into three partitions that retain catalytic activities in proportion to their length; and (ii) evidence that two of these partitions arose from an ancestral Class I aaRS gene encoding a Class II ancestor in frame on the opposite strand. Phylogenetic metrics of different modules vary in accordance with their presumed functionality. A 46-residue Class I “protozyme” roots the Class I molecular tree prior to the adaptive radiation of the Rossmann dinucleotide binding fold that refined substrate discrimination. Such rooting is consistent with near simultaneous emergence of genetic coding and the origin of the proteome, resolving a conundrum posed by previous inferences that Class I aaRS evolved long after the genetic code had been implemented in an RNA world. Further, pinpointing discontinuous enhancements of aaRS fidelity establishes a timeline for the growth of coding from a binary amino acid alphabet.

A Prebiotic Ribosylation of Pyrimidine Nucleobases Enabled by Metal Cations and Clay Minerals

We report a prebiotically relevant solution to the N1-ribosylation of pyrimidine nucleobases, a well-known challenge to the RNA world hypothesis. We found that the presence of metal cations and clay minerals enable the previously unachievable direct ribosylation of uracil. Spectroscopy and chromatography analyses confirmed the formation of ribosylated uracil. The method can be extended to the ribosylation of 2-pyrimidinone. These findings are also compatible with the metal-doped-clay model, developed by our lab for the unified route of the selection of ribose and subsequent syntheses of nucleotide and RNA.

Toward a Taxonomy of Virus-ncRNAs Interactions in an RNA World for Disentangling Some Tiny Secrets of Dengue Virus

In recent years, the role of non-coding RNAs (ncRNAs) in regulating cell physiology has begun to be better understood. Recent discoveries in viral molecular biology have revealed that such cellular functions are disturbed during viral infections mainly due to host cell ncRNAs, cellular factors, and virus-derived ncRNAs. Apart from the interplay between those molecules, other interactions derive from the specific folding of RNA virus genomes. These fulfill canonical regulation functions such as replication, translation, and viral packaging. In some cases, folds serve as precursors of small viral RNAs whose biogenesis is not yet clearly understood. Since ncRNAs and RNA viral genomes modulate complex molecular and cellular processes in viral infections, a new taxonomy is being proposed here overarching three main categories, considering the current information about ncRNA interactions in some well-known viral infections. The first category shows examples of host ncRNAs associated with the trigger of the immune response under viral infections. The second category describes interactions between the virus and host ncRNAs. The last category shows how the shape of the RNA viral genome is essential in processing RNAs derived from viruses. Finally, we introduce evidence of how these three categories can also work as a framework in order to organize known interactions of ncRNAs and cellular factors under DENV infection. This new taxonomy of interactions provides a comprehensive framework for organizing the ncRNA regulatory roles in the context of viral interactions and an RNA world.

A Prebiotic Ribosylation of Pyrimidine Nucleobases Promoted by Metal Cations and Clay Minerals

We report a prebiotically relevant solution to the N1-ribosylation of pyrimidine nucleobases, a well-known challenge in the RNA World hypothesis. It is found that the presence of metal cations and clay mineral enables the previously unachievable direct ribosylation of uracil, providing by far the highest yield. Spectroscopy and chromatography analyses confirmed the formation of ribosylated uracil. The method can also be extended to the ribosylation of 2-pyrimidinone. These findings are also compatible with the metal-doped-clay model developed by our lab for the unified route of the selection of ribose and subsequent syntheses of nucleotide and RNA.

Evolution towards increasing complexity through functional diversification in a protocell model of the RNA world

The encapsulation of genetic material inside compartments together with the creation and sustenance of functionally diverse internal components are likely to have been key steps in the formation of ‘live’, replicating protocells in an RNA world. Several experiments have shown that RNA encapsulated inside lipid vesicles can lead to vesicular growth and division through physical processes alone. Replication of RNA inside such vesicles can produce a large number of RNA strands. Yet, the impact of such replication processes on the emergence of the first ribozymes inside such protocells and on the subsequent evolution of the protocell population remains an open question. In this paper, we present a model for the evolution of protocells with functionally diverse ribozymes. Distinct ribozymes can be created with small probabilities during the error-prone RNA replication process via the rolling circle mechanism. We identify the conditions that can synergistically enhance the number of different ribozymes inside a protocell and allow functionally diverse protocells containing multiple ribozymes to dominate the population. Our work demonstrates the existence of an effective pathway towards increasing complexity of protocells that might have eventually led to the origin of life in an RNA world.

Direct RNA nanopore sequencing of Pseudomonas aeruginosa clone C transcriptomes

The transcriptomes of Pseudomonas aeruginosa clone C isolates NN2 and SG17M during the mid-exponential and early stationary phase of planktonic growth were evaluated by direct RNA sequencing on the nanopore platform and compared with established short-read cDNA sequencing on the Illumina platform. Fifty to ninety percent of the sense RNAs turned out to be rRNA molecules followed by similar proportions of mRNA transcripts and non-coding RNAs. Both platforms detected similar proportions of uncharged tRNAs and 29 yet undescribed antisense tRNAs. For example, the rarest arginine codon was paired with the most abundant tRNA Arg , and the tRNA Arg gene is missing for the most frequent arginine codon. More than 90% of the antisense RNA molecules were complementary to a coding sequence. The antisense RNAs were evenly distributed in the genomes. Direct RNA sequencing identified more than 4,000 distinct non-overlapping antisense RNAs during exponential and stationary growth. Besides highly expressed small antisense RNAs less than 200 bases in size, a population of longer antisense RNAs was sequenced that covered a broad range of a few hundred to thousands of bases and could be complementary to a contig of several genes. In summary, direct RNA sequencing identified yet undescribed RNA molecules and an unexpected composition of the pools of tRNAs, sense and antisense RNAs. IMPORTANCE Genome-wide gene expression of bacteria is commonly studied by high-throughput sequencing of size-selected cDNA fragment libraries of reverse-transcribed RNA preparations. However, the depletion of ribosomal RNAs, enzymatic reverse transcription and the fragmentation, size selection and amplification during library preparation lead to inevitable losses of information about the initial composition of the RNA pool. We demonstrate that direct RNA sequencing on the nanopore platform can overcome these limitations. Nanopore sequencing of total RNA yielded novel insights into the Pseudomonas aeruginosa transcriptome that – if replicated in other species – will change our view of the bacterial RNA world. The discovery of sense – antisense pairs of tmRNA, tRNAs and mRNAs indicates a further and unknown level of gene regulation in bacteria.