Direct RNA sequencing of Respiratory Syncytial Virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance

To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing on an Oxford nanopore device. This yielded over 150,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We were also able to examine frequencies with which polycistronic readthrough mRNAs were generated and to assess the length of the polyadenylated tails for each group of transcripts. We show that there is a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not consistent. We show that the polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host cells polyadenylation machinery and broadly declined in length for most transcripts as infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails is much less for N, SH and G transcripts compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates.

First finding of free-living representatives of Prokinetoplastina and their nuclear and mitochondrial genomes

Kinetoplastids are heterotrophic flagellated protists, including important parasites of humans and animals (trypanosomatids), and ecologically important free-living bacterial consumers (bodonids). Phylogenies have shown that the earliest-branching kinetoplastids are all parasites or obligate endosymbionts, whose highly-derived state makes reconstructing the ancestral state of the group challenging. We have isolated new strains of unusual free-living flagellates that molecular phylogeny shows to be most closely related to endosymbiotic and parasitic Perkinsela and Ichthyobodo species that, together with unidentified environmental sequences, form the clade at the base of kinetoplastids. These strains are therefore the first described free-living prokinetoplastids, and potentially very informative in understanding the evolution and ancestral states of morphological and molecular characteristics described in other kinetoplastids. Overall, we find that these organisms morphologically and ultrastructurally resemble some free-living bodonids and diplonemids, and possess nuclear genomes with few introns, polycistronic mRNA expression, high coding density, and derived traits shared with other kinetoplastids. Their genetic repertoires are more diverse than the best-studied free-living kinetoplastids, which is likely a reflection of their higher metabolic potential. Mitochondrial RNAs of these new species undergo the most extensive U insertion/deletion editing reported so far, and limited deaminative C-to-U and A-to-I editing, but we find no evidence for mitochondrial trans-splicing.

Molecular Characterization of the Burkholderia cenocepacia dcw Operon and FtsZ Interactors as New Targets for Novel Antimicrobial Design

The worldwide spread of antimicrobial resistance highlights the need of new druggable cellular targets. The increasing knowledge of bacterial cell division suggested the potentiality of this pathway as a pool of alternative drug targets, mainly based on the essentiality of these proteins, as well as on the divergence from their eukaryotic counterparts. People suffering from cystic fibrosis are particularly challenged by the lack of antibiotic alternatives. Among the opportunistic pathogens that colonize the lungs of these patients, Burkholderia cenocepacia is a well-known multi-drug resistant bacterium, particularly difficult to treat. Here we describe the organization of its division cell wall (dcw) cluster: we found that 15 genes of the dcw operon can be transcribed as a polycistronic mRNA from mraZ to ftsZ and that its transcription is under the control of a strong promoter regulated by MraZ. B. cenocepacia J2315 FtsZ was also shown to interact with the other components of the divisome machinery, with a few differences respect to other bacteria, such as the direct interaction with FtsQ. Using an in vitro sedimentation assay, we validated the role of SulA as FtsZ inhibitor, and the roles of FtsA and ZipA as tethers of FtsZ polymers. Together our results pave the way for future antimicrobial design based on the divisome as pool of antibiotic cellular targets.

uORF Shuffling Fine-Tunes Gene Expression at a Deep Level of the Process

Upstream open reading frames (uORFs) are present in the 5’ leader sequences (or 5’ untranslated regions) upstream of the protein-coding main ORFs (mORFs) in eukaryotic polycistronic mRNA. It is well known that a uORF negatively affects translation of the mORF. Emerging ribosome profiling approaches have revealed that uORFs themselves, as well as downstream mORFs, can be translated. However, it has also been revealed that plants can fine-tune gene expression by modulating uORF-mediated regulation in some situations. This article reviews several proposed mechanisms that enable genes to escape from uORF-mediated negative regulation and gives insight into the application of uORF-mediated regulation for precisely controlling gene expression.

Versatile transgenic multistage effector-gene combinations for Plasmodium falciparum suppression in Anopheles

The malaria parasite’s complex journey through the Anopheles mosquito vector provides multiple opportunities for targeting Plasmodium with recombinant effectors at different developmental stages and different host tissues. We have designed and expressed transgenes that efficiently suppress Plasmodium infection by targeting the parasite with multiple independent endogenous and exogenous effectors at multiple infection stages to potentiate suppression and minimize the probability for development of resistance to develop. We have also addressed the fitness impact of transgene expression on the mosquito. We show that highly potent suppression can be achieved by targeting both pre-oocyst stages by transgenically overexpressing either the endogenous immune deficiency immune pathway transcription factor Rel2 or a polycistronic mRNA encoding multiple antiparasitic effectors and simultaneously targeting the sporozoite stages with an anti-sporozoite single-chain antibody fused to the antiparasitic protein Scorpine. Expression of the selected endogenous effector systems appears to pose a lower fitness cost than does the use of foreign genes.

Identifying Core Operons in Metagenomic Data

An operon is a functional unit of DNA whose genes are co-transcribed on polycistronic mRNA, in a co-regulated fashion. Operons are a powerful mechanism of introducing functional complexity in bacteria, and are therefore of interest in microbial genetics, physiology, biochemistry, and evolution. Here we present a Pipeline for Operon Exploration in Metagenomes or POEM. At the heart of POEM lies the concept of a core operon, a functional unit enabled by a predicted operon in a metagenome. Using a series of benchmarks, we show the high accuracy of POEM, and demonstrate its use on a human gut metagenome sample. We conclude that POEM is a useful tool for analyzing metagenomes beyond the genomic level, and for identifying multi-gene functionalities and possible neofunctionalization in metagenomes. Availability: https://github.com/Rinoahu/POEM_py3k

Multiple roles of the polycistronic gene tarsaless/mille-pattes/polished-rice during embryogenesis of the kissing bug Rhodnius prolixus

Genes encoding small open-reading frames (smORFs) have been characterized as essential players of developmental processes. The smORF tarsaless/mille-pattes/polished-rice has been thoroughly investigated in holometabolous insects, such as the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum, while its function in hemimetabolous insects remains unknown. Thus, we analyzed the function of the tal/pri/mlpt ortholog in a hemimetabolous insect, the kissing bug Rhodnius prolixus (Rp). First, sequence analysis shows that Rp-tal/pri/mlpt polycistronic mRNA encodes two small peptides (11 to 14 amino acids) containing a LDPTG motif. Interestingly, a new hemipteran-specific conserved peptide of approximately 80 amino acids was also identified by in silico analysis. In silico docking analysis supports the high-affinity binding of the small LDPTG peptides to the transcription factor Shavenbaby. Rp-tal/pri/mlpt in situ hybridization and knockdown via RNA interference showed a conserved role of Rp-tal/pri/mlpt during embryogenesis, with a major role in the regulation of thoracic versus abdominal segmentation, leg development and head formation. Altogether, our study shows that tal/pri/mlpt segmentation role is conserved in the common ancestor of Paraneoptera and suggests that polycistronic genes might generate order specific smORFs.

A novel plasmid-transcribed regulatory sRNA, QfsR, controls chromosomal polycistronic mRNAs in Agrobacterium tumefaciens

Plasmids are mobile DNAs that adjust host cell functions for their own amplification and dissemination. We identified QfsR, a small RNA, transcribed from the Ti plasmid in the phytopathogen Agrobacterium fabrum. QfsR is widely conserved throughout RepABC plasmids carried by Rhizobiaceae. Target prediction, expression analysis and site-direct mutagenesis experiments show that QfsR directly pairs within polycistronic mRNAs transcribed from chromosomes (involved in flagella apparatus and succinoglycan biosynthesis) and Ti plasmid (involved in conjugative transfer). QfsR leads to a coordinated expression of whole polycistronic mRNA molecules. Whereas a lack of QfsR induces motility and reduces succinoglycan production, its overproduction increases the quorum sensing signal accumulation and the Ti plasmid conjugative transfer. Based on these observations, we propose QfsR as a hub connecting regulatory networks of motility, succinoglycan biosynthesis and plasmid conjugative transfer. To our knowledge, QfsR is the first example of a plasmid-encoded sRNA that controls chromosomal polycistronic mRNAs.SignificancePlasmids represent an important cost for the hosting cell although some are beneficial under certain circumstances. Agrobacterium tumefaciens harboring Tumor inducing plasmid (pTi) are able to infect plants and to use specific resources produced by the infected cells. We characterized QfsR, a novel small RNA (sRNA) from pTi, that directly regulates plasmid polycistronic mRNA but also chromosomal ones. QfsR contributes to a fine-tuned regulation of bacterial motility, exopolysaccharide biosynthesis and conjugative dissemination of pTi. Our results report the first plasmid-encoded sRNA able to modify and coordinate cellular behaviour probably for the benefit of the plasmid dissemination and tight crosstalk between plasmid and chromosome. This could be widespread since QfsR homologs were predicted in other plasmids of Rhizobiaceae symbionts and pathogens.

Tracing the ancestry of operons in bacteria

Motivation Complexity is a fundamental attribute of life. Complex systems are made of parts that together perform functions that a single component, or subsets of components, cannot. Examples of complex molecular systems include protein structures such as the F1Fo-ATPase, the ribosome, or the flagellar motor: each one of these structures requires most or all of its components to function properly. Given the ubiquity of complex systems in the biosphere, understanding the evolution of complexity is central to biology. At the molecular level, operons are classic examples of a complex system. An operon’s genes are co-transcribed under the control of a single promoter to a polycistronic mRNA molecule, and the operon’s gene products often form molecular complexes or metabolic pathways. With the large number of complete bacterial genomes available, we now have the opportunity to explore the evolution of these complex entities, by identifying possible intermediate states of operons. Results In this work, we developed a maximum parsimony algorithm to reconstruct ancestral operon states, and show a simple vertical evolution model of how operons may evolve from the individual component genes. We describe several ancestral states that are plausible functional intermediate forms leading to the full operon. We also offer Reconstruction of Ancestral Gene blocks Using Events or ROAGUE as a software tool for those interested in exploring gene block and operon evolution. Availability and implementation The software accompanying this paper is available under GPLv3 license on: https://github.com/nguyenngochuy91/Ancestral-Blocks-Reconstruction. Supplementary information Supplementary data are available at Bioinformatics online.

Noncontiguous operon is a genetic organization for coordinating bacterial gene expression

Bacterial genes are typically grouped into operons defined as clusters of adjacent genes encoding for proteins that fill related roles and are transcribed into a single polycistronic mRNA molecule. This simple organization provides an efficient mechanism to coordinate the expression of neighboring genes and is at the basis of gene regulation in bacteria. Here, we report the existence of a higher level of organization in operon structure that we named noncontiguous operon and consists in an operon containing a gene(s) that is transcribed in the opposite direction to the rest of the operon. This transcriptional architecture is exemplified by the genes menE-menC-MW1733-ytkD-MW1731 involved in menaquinone synthesis in the major human pathogen Staphylococcus aureus. We show that menE-menC-ytkD-MW1731 genes are transcribed as a single transcription unit, whereas the MW1733 gene, located between menC and ytkD, is transcribed in the opposite direction. This genomic organization generates overlapping transcripts whose expression is mutually regulated by transcriptional interference and RNase III processing at the overlapping region. In light of our results, the canonical view of operon structure should be revisited by including this operon arrangement in which cotranscription and overlapping transcription are combined to coordinate functionally related gene expression.