Hepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.

RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer3. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.

CRISPRi screen for enhancing heterologous α-amylase yield in Bacillus subtilis.

Yield improvements in cell factories can potentially be obtained by fine-tuning the regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two α-amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Surprisingly, we found among hundreds of non-coding and structured RNA candidates that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation. Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expression analysis, we selected 53 non-coding candidates. Although CRISPRi knockdowns target both the sense and the antisense strand, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3' UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.

Flagella disruption in Bacillus subtilis increases amylase production yield.

BACKGROUND: Bacillus subtilis is a Gram-positive bacterium used as a cell factory for protein production. Over the last decades, the continued optimization of production strains has increased yields of enzymes, such as amylases, and made commercial applications feasible. However, current yields are still significantly lower than the theoretically possible yield based on the available carbon sources. In its natural environment, B. subtilis can respond to unfavorable growth conditions by differentiating into motile cells that use flagella to swim towards available nutrients. RESULTS: In this study, we analyze existing transcriptome data from a B. subtilis α-amylase production strain at different time points during a 5-day fermentation. We observe that genes of the fla/che operon, essential for flagella assembly and motility, are differentially expressed over time. To investigate whether expression of the flagella operon affects yield, we performed CRISPR-dCas9 based knockdown of the fla/che operon with sgRNA target against the genes flgE, fliR, and flhG, respectively. The knockdown resulted in inhibition of mobility and a striking 2-threefold increase in α-amylase production yield. Moreover, replacing flgE (required for flagella hook assembly) with an erythromycin resistance gene followed by a transcription terminator increased α-amylase yield by about 30%. Transcript levels of the α-amylase were unaltered in the CRISPR-dCas9 knockdowns as well as the flgE deletion strain, but all manipulations disrupted the ability of cells to swim on agar. CONCLUSIONS: We demonstrate that the disruption of flagella in a B. subtilis α-amylase production strain, either by CRISPR-dCas9-based knockdown of the operon or by replacing flgE with an erythromycin resistance gene followed by a transcription terminator, increases the production of α-amylase in small-scale fermentation.

Detection of internal N7-methylguanosine (m7G) RNA modifications by mutational profiling sequencing.

Methylation of guanosine on position N7 (m7G) on internal RNA positions has been found in all domains of life and have been implicated in human disease. Here, we present m7G Mutational Profiling sequencing (m7G-MaP-seq), which allows high throughput detection of m7G modifications at nucleotide resolution. In our method, m7G modified positions are converted to abasic sites by reduction with sodium borohydride, directly recorded as cDNA mutations through reverse transcription and sequenced. We detect positions with increased mutation rates in the reduced and control samples taking the possibility of sequencing/alignment error into account and use replicates to calculate statistical significance based on log likelihood ratio tests. We show that m7G-MaP-seq efficiently detects known m7G modifications in rRNA with mutational rates up to 25% and we map a previously uncharacterised evolutionarily conserved rRNA modification at position 1581 in Arabidopsis thaliana SSU rRNA. Furthermore, we identify m7G modifications in budding yeast, human and arabidopsis tRNAs and demonstrate that m7G modification occurs before tRNA splicing. We do not find any evidence for internal m7G modifications being present in other small RNA, such as miRNA, snoRNA and sRNA, including human Let-7e. Likewise, high sequencing depth m7G-MaP-seq analysis of mRNA from E. coli or yeast cells did not identify any internal m7G modifications.

RNase H sequence preferences influence antisense oligonucleotide efficiency.

RNase H cleaves RNA in RNA-DNA duplexes. It is present in all domains of life as well as in multiple viruses and is essential for mammalian development and for human immunodeficiency virus replication. Here, we developed a sequencing-based method to measure the cleavage of thousands of different RNA-DNA duplexes and thereby comprehensively characterized the sequence preferences of HIV-1, human and Escherichia coli RNase H enzymes. We find that the catalytic domains of E. coli and human RNase H have nearly identical sequence preferences, which correlate with the efficiency of RNase H-recruiting antisense oligonucleotides. The sequences preferred by HIV-1 RNase H are distributed in the HIV genome in a way suggesting selection for efficient RNA cleavage during replication. Our findings can be used to improve the design of RNase H-recruiting antisense oligonucleotides and show that sequence preferences of HIV-1 RNase H may have shaped evolution of the viral genome and contributed to the use of tRNA-Lys3 as primer during viral replication.

SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data.

Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is an accurate method for probing of RNA secondary structure. In existing SHAPE methods, the SHAPE probing signal is normalized to a no-reagent control to correct for the background caused by premature termination of the reverse transcriptase. Here, we introduce a SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA), which retains the ability of SHAPE reagents to accurately probe RNA structure, but also allows covalent coupling between the SHAPES reagent and a biotin molecule. We demonstrate that SHAPES-based selection of cDNA-RNA hybrids on streptavidin beads effectively removes the large majority of background signal present in SHAPE probing data and that sequencing-based SHAPES data contain the same amount of RNA structure data as regular sequencing-based SHAPE data obtained through normalization to a no-reagent control. Moreover, the selection efficiently enriches for probed RNAs, suggesting that the SHAPES strategy will be useful for applications with high-background and low-probing signal such as in vivo RNA structure probing.

Dissecting the target specificity of RNase H recruiting oligonucleotides using massively parallel reporter analysis of short RNA motifs.

Processing and post-transcriptional regulation of RNA often depend on binding of regulatory molecules to short motifs in RNA. The effects of such interactions are difficult to study, because most regulatory molecules recognize partially degenerate RNA motifs, embedded in a sequence context specific for each RNA. Here, we describe Library Sequencing (LibSeq), an accurate massively parallel reporter method for completely characterizing the regulatory potential of thousands of short RNA sequences in a specific context. By sequencing cDNA derived from a plasmid library expressing identical reporter genes except for a degenerate 7mer subsequence in the 3'UTR, the regulatory effects of each 7mer can be determined. We show that LibSeq identifies regulatory motifs used by RNA-binding proteins and microRNAs. We furthermore apply the method to cells transfected with RNase H recruiting oligonucleotides to obtain quantitative information for >15000 potential target sequences in parallel. These comprehensive datasets provide insights into the specificity requirements of RNase H and allow a specificity measure to be calculated for each tested oligonucleotide. Moreover, we show that inclusion of chemical modifications in the central part of an RNase H recruiting oligonucleotide can increase its sequence-specificity.

Effect of chronic uremia on the transcriptional profile of the calcified aorta analyzed by RNA sequencing.

The development of vascular calcification (VC) in chronic uremia (CU) is a tightly regulated process controlled by factors promoting and inhibiting mineralization. Next-generation high-throughput RNA sequencing (RNA-seq) is a powerful and sensitive tool for quantitative gene expression profiling and the detection of differentially expressed genes. In the present study, we, for the first time, used RNA-seq to examine rat aorta transcriptomes from CU rats compared with control rats. Severe VC was induced in CU rats, which lead to extensive changes in the transcriptional profile. Among the 10,153 genes with an expression level of >1 reads/kilobase transcript/million mapped reads, 2,663 genes were differentially expressed with 47% upregulated genes and 53% downregulated genes in uremic rats. Significantly deregulated genes were enriched for ontologies related to the extracellular matrix, response to wounding, organic substance, and ossification. The individually affected genes were of relevance to osteogenic transformation, tissue calcification, and Wnt modulation. Downregulation of the Klotho gene in uremia is believed to be involved in the development of VC, but it is debated whether the effect is caused by circulating Klotho only or if Klotho is produced locally in the vasculature. We found that Klotho was neither expressed in the normal aorta nor calcified aorta by RNA-seq. In conclusion, we demonstrated extensive changes in the transcriptional profile of the uremic calcified aorta, which were consistent with a shift in phenotype from vascular tissue toward an osteochondrocytic transcriptome profile. Moreover, neither the normal vasculature nor calcified vasculature in CU expresses Klotho.

Pharmaco-miR: linking microRNAs and drug effects.

MicroRNAs (miRNAs) are short regulatory RNAs that down-regulate gene expression. They are essential for cell homeostasis and active in many disease states. A major discovery is the ability of miRNAs to determine the efficacy of drugs, which has given rise to the field of 'miRNA pharmacogenomics' through 'Pharmaco-miRs'. miRNAs play a significant role in pharmacogenomics by down-regulating genes that are important for drug function. These interactions can be described as triplet sets consisting of a miRNA, a target gene and a drug associated with the gene. We have developed a web server which links miRNA expression and drug function by combining data on miRNA targeting and protein-drug interactions. miRNA targeting information derive from both experimental data and computational predictions, and protein-drug interactions are annotated by the Pharmacogenomics Knowledge base (PharmGKB). Pharmaco-miR's input consists of miRNAs, genes and/or drug names and the output consists of miRNA pharmacogenomic sets or a list of unique associated miRNAs, genes and drugs. We have furthermore built a database, named Pharmaco-miR Verified Sets (VerSe), which contains miRNA pharmacogenomic data manually curated from the literature, can be searched and downloaded via Pharmaco-miR and informs on trends and generalities published in the field. Overall, we present examples of how Pharmaco-miR provides possible explanations for previously published observations, including how the cisplatin and 5-fluorouracil resistance induced by miR-148a may be caused by miR-148a targeting of the gene KIT. The information is available at www.Pharmaco-miR.org.

Massive parallel-sequencing-based hydroxyl radical probing of RNA accessibility.

Hydroxyl Radical Footprinting (HRF) is a tried-and-tested method for analysis of the tertiary structure of RNA and for identification of protein footprints on RNA. The hydroxyl radical reaction breaks accessible parts of the RNA backbone, thereby allowing ribose accessibility to be determined by detection of reverse transcriptase termination sites. Current methods for HRF rely on reverse transcription of a single primer and detection by fluorescent fragments by capillary electrophoresis. Here, we describe an accurate and efficient massive parallel-sequencing-based method for probing RNA accessibility with hydroxyl radicals, called HRF-Seq. Using random priming and a novel barcoding scheme, we show that HRF-Seq dramatically increases the throughput of HRF experiments and facilitates the parallel analysis of multiple RNAs or experimental conditions. Moreover, we demonstrate that HRF-Seq data for the Escherichia coli 16S rRNA correlates well with the ribose accessible surface area as determined by X-ray crystallography and have a resolution that readily allows the difference in accessibility caused by exposure of one side of RNA helices to be observed.

The impact of PrsA over-expression on the Bacillus subtilis transcriptome during fed-batch fermentation of alpha-amylase production.

The production of the alpha-amylase (AMY) enzyme in Bacillus subtilis at a high rate leads to the accumulation of unfolded AMY, which causes secretion stress. The over-expression of the PrsA chaperone aids enzyme folding and reduces stress. To identify affected pathways and potential mechanisms involved in the reduced growth, we analyzed the transcriptomic differences during fed-batch fermentation between a PrsA over-expressing strain and control in a time-series RNA-seq experiment. We observe transcription in 542 unannotated regions, of which 234 had significant changes in expression levels between the samples. Moreover, 1,791 protein-coding sequences, 80 non-coding genes, and 20 riboswitches overlapping UTR regions of coding genes had significant changes in expression. We identified putatively regulated biological processes via gene-set over-representation analysis of the differentially expressed genes; overall, the analysis suggests that the PrsA over-expression affects ATP biosynthesis activity, amino acid metabolism, and cell wall stability. The investigation of the protein interaction network points to a potential impact on cell motility signaling. We discuss the impact of these highlighted mechanisms for reducing secretion stress or detrimental aspects of PrsA over-expression during AMY production.

Mutation of miRNA target sequences during human evolution.

It has long-been hypothesized that changes in non-protein-coding genes and the regulatory sequences controlling expression could undergo positive selection. Here we identify 402 putative microRNA (miRNA) target sequences that have been mutated specifically in the human lineage and show that genes containing such deletions are more highly expressed than their mouse orthologs. Our findings indicate that some miRNA target mutations are fixed by positive selection and might have been involved in the evolution of human-specific traits.

Origin of introns by 'intronization' of exonic sequences.

The mechanisms of spliceosomal intron creation have proved elusive. Here we describe a new mechanism: the recruitment of internal exonic sequences ('intronization') in Caenorhabditis species. The numbers of intronization events and introns gained by other mechanisms are similar, suggesting that intronization significantly contributes to recent intron creation in nematodes. Intronization is more common than the reverse process, loss of splicing of retained introns. Finally, these findings link alternative splicing with modern intron creation.

Pharmacogenomics genes show varying perceptibility to microRNA regulation.

OBJECTIVE: The aim of pharmacogenomics is to identify individual differences in genome and transcriptome composition and their effect on drug efficacy. MicroRNAs (miRNAs) are short noncoding RNAs that negatively regulate expression of the majority of animal genes, including many genes involved in drug efficacy. Consequently, differences in the miRNA expression among individuals could be an important factor contributing to differential drug response. Pharmacogenomics genes can be divided into drug target genes termed as pharmacodynamics genes (PD) and genes involved in drug transport and metabolism termed as pharmacokinetics genes (PK). To clarify the regulatory potential of miRNAs in pharmacogenomics, we have examined the potential regulation by miRNAs of PK and PD genes. METHODS: We identified PK and PD genes as annotated by the Pharmacogenomics Knowledge Base and examined miRNA targeting of genes in the two groups according to several miRNA target prediction databases. We furthermore studied how differences between the two groups are reflected in the gene structure and across gene families. Lastly, we studied changes in expression levels of PK versus PD genes in cells depleted for miRNAs by shRNA-mediated knockdown of the miRNA-processing enzyme Dicer. RESULTS: Our analysis identify a striking difference in the level of miRNA regulation between PK and PD genes, with the former having less than half predicted conserved miRNA binding sites compared with the latter. Importantly, this finding is reflected in a highly significant difference in the shift in expression levels of PD versus PK genes after depletion of miRNAs. CONCLUSION: Our study emphasizes an intrinsic difference between PK and PD genes and helps clarify the role of miRNAs in pharmacogenomics.

Quantitative regulation of alternative splicing in evolution and development.

Alternative splicing (AS) is a widespread mechanism with an important role in increasing transcriptome and proteome diversity by generating multiple different products from the same gene. Evolutionary studies of AS have focused primarily on the conservation of alternatively spliced sequences or of the AS pattern of those sequences itself. Less is known about the evolution of the regulation of AS, but several studies, working from different perspectives, have recently made significant progress. Here, we categorize the different levels of AS evolution, and summarize the studies on evolution of AS regulation, which point to a high level of evolutionary conservation of the regulation of AS events conserved between related species. This suggests that the quantitative regulation of AS is an intrinsic part of AS function. We discuss the potential role of changes in developmental regulation of AS as an additional layer in complex gene regulatory networks and in the emergence of genetic novelties.

Light-Dependent Translation Change of Arabidopsis psbA Correlates with RNA Structure Alterations at the Translation Initiation Region.

mRNA secondary structure influences translation. Proteins that modulate the mRNA secondary structure around the translation initiation region may regulate translation in plastids. To test this hypothesis, we exposed Arabidopsis thaliana to high light, which induces translation of psbA mRNA encoding the D1 subunit of photosystem II. We assayed translation by ribosome profiling and applied two complementary methods to analyze in vivo RNA secondary structure: DMS-MaPseq and SHAPE-seq. We detected increased accessibility of the translation initiation region of psbA after high light treatment, likely contributing to the observed increase in translation by facilitating translation initiation. Furthermore, we identified the footprint of a putative regulatory protein in the 5' UTR of psbA at a position where occlusion of the nucleotide sequence would cause the structure of the translation initiation region to open up, thereby facilitating ribosome access. Moreover, we show that other plastid genes with weak Shine-Dalgarno sequences (SD) are likely to exhibit psbA-like regulation, while those with strong SDs do not. This supports the idea that changes in mRNA secondary structure might represent a general mechanism for translational regulation of psbA and other plastid genes.

BSGatlas: a unified Bacillus subtilis genome and transcriptome annotation atlas with enhanced information access.

A large part of our current understanding of gene regulation in Gram-positive bacteria is based on Bacillus subtilis, as it is one of the most well studied bacterial model systems. The rapid growth in data concerning its molecular and genomic biology is distributed across multiple annotation resources. Consequently, the interpretation of data from further B. subtilis experiments becomes increasingly challenging in both low- and large-scale analyses. Additionally, B. subtilis annotation of structured RNA and non-coding RNA (ncRNA), as well as the operon structure, is still lagging behind the annotation of the coding sequences. To address these challenges, we created the B. subtilis genome atlas, BSGatlas, which integrates and unifies multiple existing annotation resources. Compared to any of the individual resources, the BSGatlas contains twice as many ncRNAs, while improving the positional annotation for 70 % of the ncRNAs. Furthermore, we combined known transcription start and termination sites with lists of known co-transcribed gene sets to create a comprehensive transcript map. The combination with transcription start/termination site annotations resulted in 717 new sets of co-transcribed genes and 5335 untranslated regions (UTRs). In comparison to existing resources, the number of 5' and 3' UTRs increased nearly fivefold, and the number of internal UTRs doubled. The transcript map is organized in 2266 operons, which provides transcriptional annotation for 92 % of all genes in the genome compared to the at most 82 % by previous resources. We predicted an off-target-aware genome-wide library of CRISPR-Cas9 guide RNAs, which we also linked to polycistronic operons. We provide the BSGatlas in multiple forms: as a website (https://rth.dk/resources/bsgatlas/), an annotation hub for display in the UCSC genome browser, supplementary tables and standardized GFF3 format, which can be used in large scale -omics studies. By complementing existing resources, the BSGatlas supports analyses of the B. subtilis genome and its molecular biology with respect to not only non-coding genes but also genome-wide transcriptional relationships of all genes.