Search

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., (© 2022. The Author(s).)

Background: Comparative genomics approaches have facilitated the discovery of many novel non-coding and structured RNAs (ncRNAs). The increasing availability of related genomes now makes it possible to systematically search for compensatory base changes - and thus for conserved secondary structures - even in genomic regions that are poorly alignable in the primary sequence. The wealth of available transcriptome data can add valuable insight into expression and possible function for new ncRNA candidates. Earlier work identifying ncRNAs in Drosophila melanogaster made use of sequence-based alignments and employed a sliding window approach, inevitably biasing identification toward RNAs encoded in the more conserved parts of the genome., Results: To search for conserved RNA structures (CRSs) that may not be highly conserved in sequence and to assess the expression of CRSs, we conducted a genome-wide structural alignment screen of 27 insect genomes including D. melanogaster and integrated this with an extensive set of tiling array data. The structural alignment screen revealed ∼30,000 novel candidate CRSs at an estimated false discovery rate of less than 10%. With more than one quarter of all individual CRS motifs showing sequence identities below 60%, the predicted CRSs largely complement the findings of sliding window approaches applied previously. While a sixth of the CRSs were ubiquitously expressed, we found that most were expressed in specific developmental stages or cell lines. Notably, most statistically significant enrichment of CRSs were observed in pupae, mainly in exons of untranslated regions, promotors, enhancers, and long ncRNAs. Interestingly, cell lines were found to express a different set of CRSs than were found in vivo. Only a small fraction of intergenic CRSs were co-expressed with the adjacent protein coding genes, which suggests that most intergenic CRSs are independent genetic units., Conclusions: This study provides a more comprehensive view of the ncRNA transcriptome in fly as well as evidence for differential expression of CRSs during development and in cell lines.

Background: Structured non-coding RNAs play many different roles in the cells, but the annotation of these RNAs is lacking even within the human genome. The currently available computational tools are either too computationally heavy for use in full genomic screens or rely on pre-aligned sequences., Methods: Here we present a fast and efficient method, DotcodeR, for detecting structurally similar RNAs in genomic sequences by comparing their corresponding coarse-grained secondary structure dot plots at string level. This allows us to perform an all-against-all scan of all window pairs from two genomes without alignment., Results: Our computational experiments with simulated data and real chromosomes demonstrate that the presented method has good sensitivity., Conclusions: DotcodeR can be useful as a pre-filter in a genomic comparative scan for structured RNAs.

Background: Inflammatory bowel disease (IBD) is a complex multi-factorial inflammatory disease with Crohn's disease (CD) and ulcerative colitis (UC) being the two most common forms. A number of transcriptional profiling studies have provided compelling evidence that describe the role of protein-coding genes and microRNAs in modulating the immune responses in IBD., Methods: In the present study, we performed a genome-wide transcriptome profiling of lncRNAs and protein-coding genes in 96 colon pinch biopsies (inflamed and non-inflamed) extracted from multiple colonic locations from 45 patients (CD = 13, UC = 20, controls = 12) using an expression microarray platform., Results: In our study, we identified widespread dysregulation of lncRNAs and protein-coding genes in both inflamed and non-inflamed CD and UC compared to the healthy controls. In cases of inflamed CD and UC, we identified 438 and 745 differentially expressed lncRNAs, respectively, while in cases of the non-inflamed CD and UC, we identified 12 and 19 differentially expressed lncRNAs, respectively. We also observed significant enrichment (P-value <0.001, Pearson's Chi-squared test) for 96 differentially expressed lncRNAs and 154 protein-coding genes within the IBD susceptibility loci. Furthermore, we found strong positive expression correlations for the intersecting and cis-neighboring differentially expressed IBD loci-associated lncRNA-protein-coding gene pairs. The functional annotation analysis of differentially expressed genes revealed their involvement in the immune response, pro-inflammatory cytokine activity and MHC protein complex., Conclusions: The lncRNA expression profiling in both inflamed and non-inflamed CD and UC successfully stratified IBD patients from the healthy controls. Taken together, the identified lncRNA transcriptional signature along with clinically relevant parameters suggest their potential as biomarkers in IBD.

Background: Annotating mammalian genomes for noncoding RNAs (ncRNAs) is nontrivial since far from all ncRNAs are known and the computational models are resource demanding. Currently, the human genome holds the best mammalian ncRNA annotation, a result of numerous efforts by several groups. However, a more direct strategy is desired for the increasing number of sequenced mammalian genomes of which some, such as the pig, are relevant as disease models and production animals., Results: We present a comprehensive annotation of structured RNAs in the pig genome. Combining sequence and structure similarity search as well as class specific methods, we obtained a conservative set with a total of 3,391 structured RNA loci of which 1,011 and 2,314, respectively, hold strong sequence and structure similarity to structured RNAs in existing databases. The RNA loci cover 139 cis-regulatory element loci, 58 lncRNA loci, 11 conflicts of annotation, and 3,183 ncRNA genes. The ncRNA genes comprise 359 miRNAs, 8 ribozymes, 185 rRNAs, 638 snoRNAs, 1,030 snRNAs, 810 tRNAs and 153 ncRNA genes not belonging to the here fore mentioned classes. When running the pipeline on a local shuffled version of the genome, we obtained no matches at the highest confidence level. Additional analysis of RNA-seq data from a pooled library from 10 different pig tissues added another 165 miRNA loci, yielding an overall annotation of 3,556 structured RNA loci. This annotation represents our best effort at making an automated annotation. To further enhance the reliability, 571 of the 3,556 structured RNAs were manually curated by methods depending on the RNA class while 1,581 were declared as pseudogenes. We further created a multiple alignment of pig against 20 representative vertebrates, from which RNAz predicted 83,859 de novo RNA loci with conserved RNA structures. 528 of the RNAz predictions overlapped with the homology based annotation or novel miRNAs. We further present a substantial synteny analysis which includes 1,004 lineage specific de novo RNA loci and 4 ncRNA loci in the known annotation specific for Laurasiatheria (pig, cow, dolphin, horse, cat, dog, hedgehog)., Conclusions: We have obtained one of the most comprehensive annotations for structured ncRNAs of a mammalian genome, which is likely to play central roles in both health modelling and production. The core annotation is available in Ensembl 70 and the complete annotation is available at http://rth.dk/resources/rnannotator/susscr102/version1.02.

Background: MicroRNAs (miRNAs) are a class of non-protein-coding genes that play a crucial regulatory role in mammalian development and disease. Whereas a large number of miRNAs have been annotated at the structural level during the latest years, functional annotation is sparse. Actinobacillus pleuropneumoniae (APP) causes serious lung infections in pigs. Severe damage to the lungs, in many cases deadly, is caused by toxins released by the bacterium and to some degree by host mediated tissue damage. However, understanding of the role of microRNAs in the course of this infectious disease in porcine is still very limited., Results: In this study, the RNA extracted from visually unaffected and necrotic tissue from pigs infected with Actinobacillus pleuropneumoniae was subjected to small RNA deep sequencing. We identified 169 conserved and 11 candidate novel microRNAs in the pig. Of these, 17 were significantly up-regulated in the necrotic sample and 12 were down-regulated. The expression analysis of a number of candidates revealed microRNAs of potential importance in the innate immune response. MiR-155, a known key player in inflammation, was found expressed in both samples. Moreover, miR-664-5p, miR-451 and miR-15a appear as very promising candidates for microRNAs involved in response to pathogen infection., Conclusions: This is the first study revealing significant differences in composition and expression profiles of miRNAs in lungs infected with a bacterial pathogen. Our results extend annotation of microRNA in pig and provide insight into the role of a number of microRNAs in regulation of bacteria induced immune and inflammatory response in porcine lung.

Background: Transcription factors (TFs) have long been known to be principally activators of transcription in eukaryotes and prokaryotes. The growing awareness of the ubiquity of microRNAs (miRNAs) as suppressive regulators in eukaryotes, suggests the possibility of a mutual, preferential, self-regulatory connectivity between miRNAs and TFs. Here we investigate the connectivity from TFs and miRNAs to other genes and each other using text mining, TF promoter binding site and 6 different miRNA binding site prediction methods., Results: In the first approach text mining of PubMed abstracts reveal statistically significant associations between miRNAs and both TFs and signal transduction gene classes. Secondly, prediction of miRNA targets in human and mouse 3'UTRs show enrichment only for TFs but not consistently across prediction methods for signal transduction or other gene classes. Furthermore, a random sample of 986 TarBase entries was scored for experimental evidence by manual inspection of the original papers, and enrichment for TFs was observed to increase with score. Low-scoring TarBase entries, where experimental evidence is anticorrelated miRNA:mRNA expression with predicted miRNA targets, appear not to select for real miRNA targets to any degree. Our manually validated text-mining results also suggests that miRNAs may be activated by more TFs than other classes of genes, as 7% of miRNA:TF co-occurrences in the literature were TFs activating miRNAs. This was confirmed when thirdly, we found enrichment for predicted, conserved TF binding sites in miRNA and TF genes compared to other gene classes., Conclusions: We see enrichment of connections between miRNAs and TFs using several independent methods, suggestive of a network of mutual activating and suppressive regulation. We have also built regulatory networks (containing 2- and 3-loop motifs) for mouse and human using predicted miRNA and TF binding sites and we have developed a web server to search and display these loops, available for the community at http://rth.dk/resources/tfmirloop.

Background: Post-transcriptional control of gene expression is mostly conducted by specific elements in untranslated regions (UTRs) of mRNAs, in collaboration with specific binding proteins and RNAs. In several well characterized cases, these RNA elements are known to form stable secondary structures. RNA secondary structures also may have major functional implications for long noncoding RNAs (lncRNAs). Recent transcriptional data has indicated the importance of lncRNAs in brain development and function. However, no methodical efforts to investigate this have been undertaken. Here, we aim to systematically analyze the potential for RNA structure in brain-expressed transcripts., Results: By comprehensive spatial expression analysis of the adult mouse in situ hybridization data of the Allen Mouse Brain Atlas, we show that transcripts (coding as well as non-coding) associated with in silico predicted structured probes are highly and significantly enriched in almost all analyzed brain regions. Functional implications of these RNA structures and their role in the brain are discussed in detail along with specific examples. We observe that mRNAs with a structure prediction in their UTRs are enriched for binding, transport and localization gene ontology categories. In addition, after manual examination we observe agreement between RNA binding protein interaction sites near the 3' UTR structures and correlated expression patterns., Conclusions: Our results show a potential use for RNA structures in expressed coding as well as noncoding transcripts in the adult mouse brain, and describe the role of structured RNAs in the context of intracellular signaling pathways and regulatory networks. Based on this data we hypothesize that RNA structure is widely involved in transcriptional and translational regulatory mechanisms in the brain and ultimately plays a role in brain function.

Background: Fast seed-based alignment heuristics such as BLAST and BLAT have become indispensable tools in comparative genomics for all studies aiming at the evolutionary relations of proteins, genes, and non-coding RNAs. This is true in particular for the large mammalian genomes. The sensitivity and specificity of these tools, however, crucially depend on parameters such as seed sizes or maximum expectation values. In settings that require high sensitivity the amount of short local match fragments easily becomes intractable. Then, fragment chaining is a powerful leverage to quickly connect, score, and rank the fragments to improve the specificity., Results: Here we present a fast and flexible fragment chainer that for the first time also supports a sum-of-pair gap cost model. This model has proven to achieve a higher accuracy and sensitivity in its own field of application. Due to a highly time-efficient index structure our method outperforms the only existing tool for fragment chaining under the linear gap cost model. It can easily be applied to the output generated by alignment tools such as segemehl or BLAST. As an example we consider homology-based searches for human and mouse snoRNAs demonstrating that a highly sensitive BLAST search with subsequent chaining is an attractive option. The sum-of-pair gap costs provide a substantial advantage is this context., Conclusions: Chaining of short match fragments helps to quickly and accurately identify regions of homology that may not be found using local alignment heuristics alone. By providing both the linear and the sum-of-pair gap cost model, a wider range of application can be covered. The software clasp is available at http://www.bioinf.uni-leipzig.de/Software/clasp/.

Background: Many regulatory non-coding RNAs (ncRNAs) function through complementary binding with mRNAs or other ncRNAs, e.g., microRNAs, snoRNAs and bacterial sRNAs. Predicting these RNA interactions is essential for functional studies of putative ncRNAs or for the design of artificial RNAs. Many ncRNAs show clear signs of undergoing compensating base changes over evolutionary time. Here, we postulate that a non-negligible part of the existing RNA-RNA interactions contain preserved but covarying patterns of interactions., Methods: We present a novel method that takes compensating base changes across the binding sites into account. The algorithm works in two steps on two pre-generated multiple alignments. In the first step, individual base pairs with high reliability are found using the PETfold algorithm, which includes evolutionary and thermodynamic properties. In step two (where high reliability base pairs from step one are constrained as unpaired), the principle of cofolding is combined with hierarchical folding. The final prediction of intra- and inter-molecular base pairs consists of the reliabilities computed from the constrained expected accuracy scoring, which is an extended version of that used for individual multiple alignments., Results: We derived a rather extensive algorithm. One of the advantages of our approach (in contrast to other RNA-RNA interaction prediction methods) is the application of covariance detection and prediction of pseudoknots between intra- and inter-molecular base pairs. As a proof of concept, we show an example and discuss the strengths and weaknesses of the approach.

Background: Since at least half of the genes in mammalian genomes are subjected to alternative splicing, alternative pre-mRNA splicing plays an important contribution to the complexity of the mammalian proteome. Expressed sequence tags (ESTs) provide evidence of a great number of possible alternative isoforms. With the EST resource for the domestic pig now containing more than one million porcine ESTs, it is possible to identify alternative splice forms of the individual transcripts in this species from the EST data with some confidence., Results: The pig EST data generated by the Sino-Danish Pig Genome project has been assembled with publicly available ESTs and made available in the PigEST database. Using the Distiller package 2,515 EST clusters with candidate alternative isoforms were identified in the EST data with high confidence. In agreement with general observations in human and mouse, we find putative splice variants in about 30% of the contigs with more than 50 ESTs. Based on the criteria that a minimum of two EST sequences confirmed each splice event, a list of 100 genes with the most distinct tissue-specific alternative splice events was generated from the list of candidates. To confirm the tissue specificity of the splice events, 10 genes with functional annotation were randomly selected from which 16 individual splice events were chosen for experimental verification by quantitative PCR (qPCR). Six genes were shown to have tissue specific alternatively spliced transcripts with expression patterns matching those of the EST data. The remaining four genes had tissue-restricted expression of alternative spliced transcripts. Five out of the 16 splice events that were experimentally verified were found to be putative pig specific., Conclusions: In accordance with human and rodent studies we estimate that approximately 30% of the porcine genes undergo alternative splicing. We found a good correlation between EST predicted tissue-specificity and experimentally validated splice events in different porcine tissue. This study indicates that a cluster size of around 50 ESTs is optimal for in silico detection of alternative splicing. Although based on a limited number of splice events, the study supports the notion that alternative splicing could have an important impact on species differentiation since 31% of the splice events studied appears to be species specific.

Background: An essential driving component in the co-evolution of plants and insects is the ability to produce and handle bioactive compounds. Plants produce bioactive natural products for defense, but some insects detoxify and/or sequester the compounds, opening up for new niches with fewer competitors. To study the molecular mechanism behind the co-adaption in plant-insect interactions, we have investigated the interactions between Lotus corniculatus and Zygaena filipendulae. They both contain cyanogenic glucosides which liberate toxic hydrogen cyanide upon breakdown. Moths belonging to the Zygaena family are the only insects known, able to carry out both de novo biosynthesis and sequestration of the same cyanogenic glucosides as those from their feed plants. The biosynthetic pathway for cyanogenic glucoside biosynthesis in Z. filipendulae proceeds using the same intermediates as in the well known pathway from plants, but none of the enzymes responsible have been identified. A genomics strategy founded on 454 pyrosequencing of the Z. filipendulae transcriptome was undertaken to identify some of these enzymes in Z. filipendulae., Results: Comparisons of the Z. filipendulae transcriptome with the sequenced genomes of Bombyx mori, Drosophila melanogaster, Tribolium castaneum, Apis mellifera and Anopheles gambiae indicate a high coverage of the Z. filipendulae transcriptome. 11% of the Z. filipendulae transcriptome sequences were assigned to Gene Ontology categories. Candidate genes for enzymes functioning in the biosynthesis of cyanogenic glucosides (cytochrome P450 and family 1 glycosyltransferases) were identified based on sequence length, number of copies and presence/absence of close homologs in D. melanogaster, B. mori and the cyanogenic butterfly Heliconius. Examination of biased codon usage, GC content and selection on gene candidates support the notion of cyanogenesis as an "old" trait within Ditrysia, as well as its origins being convergent between plants and insects., Conclusion: Pyrosequencing is an attractive approach to gain access to genes in the biosynthesis of bio-active natural products from insects and other organisms, for which the genome sequence is not known. Based on analysis of the Z. filipendulae transcriptome, promising gene candidates for biosynthesis of cyanogenic glucosides was identified, and the suitability of Z. filipendulae as a model system for cyanogenesis in insects is evident.

Background: The mitochondria are involved in many basic functions in cells of vertebrates, and can be considered the power generator of the cell. Though the mitochondria have been extensively studied there appear to be only few expression studies of mitochondrial genes involving a large number of tissues and developmental stages. Here, we conduct an analysis using the PigEST resource 1 which contains expression information from 35 tissues distributed on one normalized and 97 non-normalized cDNA libraries of which 24 are from developmental stages. The mitochondrial PigEST resource contains 41,499 mitochondrial sequences., Results: The mitochondrial EST (Expressed Sequence Tag) sequences were assembled into contigs which covers more than 94 percent of the porcine mitochondrial genome, with an average of 976 EST sequences per nucleotide. This data was converted into expression values for the individual genes in each cDNA library revealing differential expression between genes expressed in cDNA libraries from developmental and adult stages. For the 13 protein coding genes (and several RNA genes), we find one set of six genes, containing all cytochrome oxidases, that are upregulated in developmental tissues, whereas the remaining set of seven genes, containing all ATPases, that are upregulated in adult muscle and brain tissues. Further, the COX I (Cytochrome oxidase subunit one) expression profile differs from that of the remaining genes, which could be explained by a tissue specific cleavage event or degradation pattern, and is especially pronounced in developmental tissues. Finally, as expected cDNA libraries from muscle tissues contain by far the largest amount (up to 20%) of expressed mitochondrial genes., Conclusion: Our results present novel insight into differences in mitochondrial gene expression, emphasizing differences between adult and developmental tissues. Our work indicates that there are presently unknown mechanisms which work to customize mitochondrial processes to the specific needs of the cell, illustrated by the different patterns between adult and developmental tissues. Furthermore, our results also provide novel insight into how in-depth sequencing can provide significant information about expression patterns.

Background: Non-coding RNAs (ncRNAs) are involved in a wide spectrum of regulatory functions. Within recent years, there have been increasing reports of observed polyadenylated ncRNAs and mRNA like ncRNAs in eukaryotes. To investigate this further, we examined the large data set in the Sino-Danish PigEST resource http://pigest.ku.dk which also contains expression information distributed on 97 non-normalized cDNA libraries., Results: We constructed a pipeline, EST2ncRNA, to search for known and novel ncRNAs. The pipeline utilises sequence similarity to ncRNA databases (blast), structure similarity to Rfam (RaveNnA) as well as multiple alignments to predict conserved novel putative RNA structures (RNAz). EST2ncRNA was fed with 48,000 contigs and 73,000 singletons available from the PigEST resource. Using the pipeline we identified known RNA structures in 137 contigs and single reads (conreads), and predicted high confidence RNA structures in non-protein coding regions of additional 1,262 conreads. Of these, structures in 270 conreads overlap with existing predictions in human. To sum up, the PigEST resource comprises trans-acting elements (ncRNAs) in 715 contigs and 340 singletons as well as cis-acting elements (inside UTRs) in 311 contigs and 51 singletons, of which 18 conreads contain both predictions of trans- and cis-acting elements. The predicted RNAz candidates were compared with the PigEST expression information and we identify 114 contigs with an RNAz prediction and expression in at least ten of the non-normalised cDNA libraries. We conclude that the contigs with RNAz and known predictions are in general expressed at a much lower level than protein coding transcripts. In addition, we also observe that our ncRNA candidates constitute about one to two percent of the genes expressed in the cDNA libraries. Intriguingly, the cDNA libraries from developmental (brain) tissues contain the highest amount of ncRNA candidates, about two percent. These observations are related to existing knowledge and hypotheses about the role of ncRNAs in higher organisms. Furthermore, about 80% porcine coding transcripts (of 18,600 identified) as well as less than one-third ORF-free transcripts are conserved at least in the closely related bovine genome. Approximately one percent of the coding and 10% of the remaining matches are unique between the PigEST data and cow genome. Based on the pig-cow alignments, we searched for similarities to 16 other organisms by UCSC available alignments, which resulted in a 87% coverage by the human genome for instance., Conclusion: Besides recovering several of the already annotated functional RNA structures, we predicted a large number of high confidence conserved secondary structures in polyadenylated porcine transcripts. Our observations of relatively low expression levels of predicted ncRNA candidates together with the observations of higher relative amount in cDNA libraries from developmental stages are in agreement with the current paradigm of ncRNA roles in higher organisms and supports the idea of polyadenylated ncRNAs.

Background: Comparative whole genome analysis of Mammalia can benefit from the addition of more species. The pig is an obvious choice due to its economic and medical importance as well as its evolutionary position in the artiodactyls., Results: We have generated approximately 3.84 million shotgun sequences (0.66X coverage) from the pig genome. The data are hereby released (NCBI Trace repository with center name "SDJVP", and project name "Sino-Danish Pig Genome Project") together with an initial evolutionary analysis. The non-repetitive fraction of the sequences was aligned to the UCSC human-mouse alignment and the resulting three-species alignments were annotated using the human genome annotation. Ultra-conserved elements and miRNAs were identified. The results show that for each of these types of orthologous data, pig is much closer to human than mouse is. Purifying selection has been more efficient in pig compared to human, but not as efficient as in mouse, and pig seems to have an isochore structure most similar to the structure in human., Conclusion: The addition of the pig to the set of species sequenced at low coverage adds to the understanding of selective pressures that have acted on the human genome by bisecting the evolutionary branch between human and mouse with the mouse branch being approximately 3 times as long as the human branch. Additionally, the joint alignment of the shot-gun sequences to the human-mouse alignment offers the investigator a rapid way to defining specific regions for analysis and resequencing.