Separable roles for RNAi in regulation of transposable elements and viability in the fission yeast Schizosaccharomyces japonicus

RNA interference (RNAi) is a conserved mechanism of small RNA-mediated genome regulation commonly involved in suppression of transposable elements (TEs) through both post-transcriptional silencing, and transcriptional repression via heterochromatin assembly. The fission yeast Schizosaccharomyces pombe has been extensively utilised as a model for studying RNAi pathways. However, this species is somewhat atypical in that TEs are not major targets of RNAi, and instead small RNAs correspond primarily to non-coding pericentromeric repeat sequences, reflecting a specialised role for the pathway in promoting heterochromatin assembly in these regions. In contrast, in the related fission yeast Schizosaccharomyces japonicus, sequenced small RNAs correspond primarily to TEs. This suggests there may be fundamental differences in the operation of RNAi pathways in these two related species. To investigate these differences, we probed RNAi function in S. japonicus. Unexpectedly, and in contrast to S. pombe, we found that RNAi is essential in this species. Moreover, viability of RNAi mutants can be rescued by mutations implicated in enhanching RNAi-independent heterochromatin propagation. These rescued strains retain heterochromatic marks on TE sequences, but exhibit derepression of TEs at the post-transcriptional level. Our findings indicate that S. japonicus retains the ancestral role of RNAi in facilitating suppression of TEs via both post-transcriptional silencing and heterochromatin assembly, with specifically the heterochromatin pathway being essential for viability, likely due to a function in genome maintenance. The specialised role of RNAi in heterochromatin assembly in S. pombe appears to be a derived state that emerged after the divergence of S. japonicus.

Tutorial: Investigating SARS-CoV-2 evolution and phylogeny using MNHN-Tree-Tools

The Covid-19 pandemic has caused at more than 3 million deaths by Mai this year. It had a significant impact on the daily life and the global economy. The virus has since its first recorded outbreak in China mutated into new strains. The Nextstrain project has so far been monitoring the evolution of the virus. At the same time we were developing in our lab the MNHN-Tree-Tools toolkit, primarily for the investigation of DNA repeat sequences. We have further extended MNHN-Tree-Tools to guide phylogenetics. As such the toolkit has evolved into a high performance code, allowing for a fast investigation of millions of sequences. Given the context of the pandemic it became evident that we will use our versatile tool to investigate the evolution of SARS-CoV-2 sequences. Our efforts have cumulated in this tutorial that we share with the scientific community.

Measuring Proviral HIV-1 DNA: Hurdles and Improvements to an Assay Monitoring Integration Events Utilising Human Alu Repeat Sequences

Integrated HIV-1 DNA persists despite antiretroviral therapy and can fuel viral rebound following treatment interruption. Hence, methods to specifically measure the integrated HIV-1 DNA portion only are important to monitor the reservoir in eradication trials. Here, we provide an up-to-date overview of the literature on the different approaches used to measure integrated HIV-1 DNA. Further, we propose an implemented standard-curve free assay to quantify integrated HIV-1 DNA, so-called Alu-5LTR PCR, which utilises novel primer combinations. We tested the Alu-5LTR PCR in 20 individuals on suppressive ART for a median of nine years; the results were compared to those produced with the standard-free Alu-gag assay. The numbers of median integrated HIV-1 DNA copies were 5 (range: 1–12) and 14 (5–26) with the Alu-gag and Alu-5LTR, respectively. The ratios between Alu-gag vs Alu-5LTR results were distributed within the cohort as follows: most patients (12/20, 60%) provided ratios between 2–5, with 3/20 (15%) and 5/20 (25%) being below or above this range, respectively. Alu-5LTR assay sensitivity was also determined using an “integrated standard”; the data confirmed the increased sensitivity of the assay, i.e., equal to 0.25 proviruses in 10,000 genomes. This work represents an improvement in the field of measuring proviral HIV-1 DNA that could be employed in future HIV-1 persistence and eradication studies.

Comparative Chloroplast Genomics of Sophora Species: Evolution and Phylogenetic Relationships in the Early-Diverging Legume Subfamily Papilionoideae (Fabaceae)

The taxonomy and evolutionary history of Sophora L., a genus with high economic and medicinal value, remain uncertain due to the absence of genetic resource (especially in China) and low polymorphism of molecular markers. Our aim was to elucidate the molecular evolution and phylogenetic relationships in chloroplast genomes of Sophora species in the early-diverging legume subfamily Papilionoideae (Fabaceae). We reported nine Sophora chloroplast genome from China using Illumina sequencing. We performed a series of analyses with previously published genomes of Sophora species to investigate their genomic characteristics, identified simple sequence repeats, large repeat sequences, tandem repeats, and highly polymorphic loci. The genomes were 152,953–158,087 bp in length, and contained 111–113 unique genes, including 76–78 protein coding, 31 tRNA, and 4 rRNA. The expansion of inverted repeat boundary of Sophora resulted in rps12 entering into the LSC region and loss of trnT-CGU gene in some species. Also, we found an approximately 23 kb inversion between trnC-GCA and trnF-GAA within the genus. In addition, we identified seven highly polymorphic loci (pi (π) > 0.035) suitable for inferring the phylogeny of Sophora species. Among these, three regions also co-occurred with large repeat sequences and support use of repeats as a proxy for the identification of polymorphic loci. Based on whole chloroplast genome and protein-coding sequences data-set, a well-supported phylogenetic tree of Sophora and related taxa showed that this genus is monophyletic, but sect. Disamaea and sect. Sophora, are incongruent with traditional taxonomic classifications based on fruit morphology. Our finding provides significant genetic resources to support further investigation into the phylogenetic relationship and evolution of the genus Sophora.

Halovirus HF2 Intergenic Repeat Sequences Carry Promoters

Halovirus HF2 was the first member of the Haloferacalesvirus genus to have its genome fully sequenced, which revealed two classes of intergenic repeat (IR) sequences: class I repeats of 58 bp in length, and class II repeats of 29 bp in length. Both classes of repeat contain AT-rich motifs that were conjectured to represent promoters. In the present study, nine IRs were cloned upstream of the bgaH reporter gene, and all displayed promoter activity, providing experimental evidence for the previous conjecture. Comparative genomics showed that IR sequences and their relative genomic positions were strongly conserved among other members of the same virus genus. The transcription of HF2 was also examined by the reverse-transcriptase-PCR (RT-PCR) method, which demonstrated very long transcripts were produced that together covered most of the genome, and from both strands. The presence of long counter transcripts suggests a regulatory role or possibly unrecognized coding potential.

Roles for the 8-Oxoguanine DNA Repair System in Protecting Telomeres From Oxidative Stress

Telomeres are protective nucleoprotein structures that cap linear chromosome ends and safeguard genome stability. Progressive telomere shortening at each somatic cell division eventually leads to critically short and dysfunctional telomeres, which can contribute to either cellular senescence and aging, or tumorigenesis. Human reproductive cells, some stem cells, and most cancer cells, express the enzyme telomerase to restore telomeric DNA. Numerous studies have shown that oxidative stress caused by excess reactive oxygen species is associated with accelerated telomere shortening and dysfunction. Telomeric repeat sequences are remarkably susceptible to oxidative damage and are preferred sites for the production of the mutagenic base lesion 8-oxoguanine, which can alter telomere length homeostasis and integrity. Therefore, knowledge of the repair pathways involved in the processing of 8-oxoguanine at telomeres is important for advancing understanding of the pathogenesis of degenerative diseases and cancer associated with telomere instability. The highly conserved guanine oxidation (GO) system involves three specialized enzymes that initiate distinct pathways to specifically mitigate the adverse effects of 8-oxoguanine. Here we introduce the GO system and review the studies focused on investigating how telomeric 8-oxoguanine processing affects telomere integrity and overall genome stability. We also discuss newly developed technologies that target oxidative damage selectively to telomeres to investigate roles for the GO system in telomere stability.

Guide-directed DNA cleavage by a prokaryotic Argonaute protein induces chromosome recombination in Escherichia coli

Emerging evidence supports the argument that some prokaryotic argonautes (pAgos) serve as a defensive system against invasion of viruses and plasmids through guide DNAs (gDNAs) directed DNA cleavage. This DNA-guided DNA interference motivates research to induce genomic mutations via pAgo mediated cleavage. Here we demonstrate that CbAgo, a pAgo from Clostridium butyricum, is able to induce chromosomal recombination between direct repeat sequences via its gDNA-directed cleavage in Escherichia coli chromosome. We also show that CbAgo targeting can assist Lambda-Red recombineering in RecA-deficient strain. Our study reveals that cleavage by CbAgo in E. coli chromosome can be mutagenic and suggests its broader application in genetic manipulation.

Plastome Phylogenomics and Historical Biogeography of Aquatic Plant Genus Hydrocharis (Hydrocharitaceae)

Background Hydrocharis L. and Limnobium Rich. are small aquatic genera, including three and two species, respectively. The taxonomic status, phylogenetic relationships and biogeographical history of these genera have remained unclear, owing to the lack of Central African endemic H. chevalieri from all previous studies. We sequenced and assembled plastomes of all three Hydrocharis species and Limnobium laevigatum to explore the phylogenetic and biogeographical history of these aquatic plants. Results All four newly generated plastomes were conserved in genome structure, gene content, and gene order. However, they differed in size, the number of repeat sequences, and inverted repeat borders. Our phylogenomic analyses recovered non-monophyletic Hydrocharis. African species H. chevalieri was fully supported as sister to the rest of the species, and L. laevigatum was nested in Hydrocharis as a sister to H. dubia. Hydrocharis-Limnobium initially diverged from the remaining genera at ca. 53.3 Ma, then began to diversify at ca. 30.9 Ma. The biogeographic analysis suggested that Hydrocharis probably originated in Europe and Central Africa. Conclusion Based on the phylogenetic results, morphological similarity and small size of the genera, the most reasonable taxonomic solution to the non-monophyly of Hydrocharis is to treat Limnobium as its synonym. The African endemic H. chevalieri is fully supported as a sister to the remaining species. Hydrocharis mainly diversified in the Miocene, during which rapid climate change may have contributed to the speciation and extinctions. The American species of former Limnobium probably dispersed to America through the Bering Land Bridge during the Miocene.

In-silico evaluation of ‘Mirror Repeats’ In HIV Genome

The repetitive sequences played an important role in the characterization of both prokaryotic & eukaryotic organisms. Various different patterns of repetitive sequences have also been identified in organisms. Among all the repeat sequences. Mirror Repeats (MR`s) play an important role in various types of neurological disorders. These MR`s have also been reported for structure determination of genomes, triplex DNA formation & various other genome functions. We have followed a distinguished method referred to as FPCB (FASTA PARALLEL COMPLEMENT BLAST) for the identification of MR`s. The above said method used to identify MR’s in both types of HIV viruses (HIV-1 & HIV-2). Present investigation reported that MR’s are frequently distributed in all the regions of the genomes of both types. As a result, 232 & 248 total numbers of MR`s identified in both the HIV-1 & HIV-2 genome respectively. In addition, it was also revealed that the majority of the identified sequences are imperfect. The maximum length of MR`s in HIV-1 is of 47 nucleotides (NTD`s), however in case of HIV-2, it is of 49 nucleotides (NTD`s). Present investigation will be helpful for further development of a link between mirror repeats and host genome, which will be a new trend to block the viral integration as well as pathogenicity.