RNA interaction format: a general data format for RNA interactions.

SUMMARY: RNA molecules play crucial roles in various biological processes. They mediate their function mainly by interacting with other RNAs or proteins. At present, information about these interactions is distributed over different resources, often providing the data in simple tab-delimited formats that differ between the databases. There is no standardized data format that can capture the nature of all these different interactions in detail. AVAILABILITY AND IMPLEMENTATION: Here, we propose the RNA interaction format (RIF) for the detailed representation of RNA-RNA and RNA-Protein interactions and provide reference implementations in C/C++, Python, and JavaScript. RIF is released under licence GNU General Public License version 3 (GNU GPLv3) and is available on https://github.com/RNABioInfo/rna-interaction-format.

RNAnue: efficient data analysis for RNA-RNA interactomics.

RNA-RNA inter- and intramolecular interactions are fundamental for numerous biological processes. While there are reasonable approaches to map RNA secondary structures genome-wide, understanding how different RNAs interact to carry out their regulatory functions requires mapping of intermolecular base pairs. Recently, different strategies to detect RNA-RNA duplexes in living cells, so called direct duplex detection (DDD) methods, have been developed. Common to all is the Psoralen-mediated in vivo RNA crosslinking followed by RNA Proximity Ligation to join the two interacting RNA strands. Sequencing of the RNA via classical RNA-seq and subsequent specialised bioinformatic analyses the result in the prediction of inter- and intramolecular RNA-RNA interactions. Existing approaches adapt standard RNA-seq analysis pipelines, but often neglect inherent features of RNA-RNA interactions that are useful for filtering and statistical assessment. Here we present RNAnue, a general pipeline for the inference of RNA-RNA interactions from DDD experiments that takes into account hybridisation potential and statistical significance to improve prediction accuracy. We applied RNAnue to data from different DDD studies and compared our results to those of the original methods. This showed that RNAnue performs better in terms of quantity and quality of predictions.

GLASSgo in Galaxy: high-throughput, reproducible and easy-to-integrate prediction of sRNA homologs.

MOTIVATION: The correct prediction of bacterial sRNA homologs is a prerequisite for many downstream analyses based on comparative genomics, but it is frequently challenging due to the short length and distinct heterogeneity of such homologs. GLobal Automatic Small RNA Search go (GLASSgo) is an efficient tool for the prediction of sRNA homologs from a single input query. To make the algorithm available to a broader community, we offer a Docker container along with a free-access web service. For non-computer scientists, the web service provides a user-friendly interface. However, capabilities were lacking so far for batch processing, version control and direct interaction with compatible software applications as a workflow management system can provide. RESULTS: Here, we present GLASSgo 1.5.2, an updated version that is fully incorporated into the workflow management system Galaxy. The improved version contains a new feature for extracting the upstream regions, allowing the search for conserved promoter elements. Additionally, it supports the use of accession numbers instead of the outdated GI numbers, which widens the applicability of the tool. AVAILABILITY AND IMPLEMENTATION: GLASSgo is available at https://github.com/lotts/GLASSgo/ under the MIT license and is accompanied by instruction and application data. Furthermore, it can be installed into any Galaxy instance using the Galaxy ToolShed.

CRISPR-Cas bioinformatics.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) are essential genetic elements in many archaeal and bacterial genomes, playing a key role in a prokaryote adaptive immune system against invasive foreign elements. In recent years, the CRISPR-Cas system has also been engineered to facilitate target gene editing in eukaryotic genomes. Bioinformatics played an essential role in the detection and analysis of CRISPR systems and here we review the bioinformatics-based efforts that pushed the field of CRISPR-Cas research further. We discuss the bioinformatics tools that have been published over the last few years and, finally, present the most popular tools for the design of CRISPR-Cas9 guides.

VisualGraphX: interactive graph visualization within Galaxy.

MOTIVATION: We developed VisualGraphX, a web-based, interactive visualization tool for large-scale graphs. Current graph visualization tools that follow the rich-internet paradigm lack an interactive and scalable visualization of graph-based data. VisualGraphX aims to provide a universal graph visualization tool that empowers the users to efficiently explore the data for themselves at a large scale. It is available as a visualization plugin for the Galaxy platform, such that VisualGraphX can be integrated into custom analysis pipelines. AVAILABILITY AND IMPLEMENTATION: VisualGraphX has been released as a visualization plugin for the Galaxy platform under AFL 3.0 and is available with instructions and application data at http://gitlab.com/comptrans/VisualGraphX/ CONTACT: bjoern.voss@ibvt.uni-stuttgart.de.

Carotenogenesis Is Regulated by 5'UTR-Mediated Translation of Phytoene Synthase Splice Variants.

Phytoene synthase (PSY) catalyzes the highly regulated, frequently rate-limiting synthesis of the first biosynthetically formed carotene. While PSY constitutes a small gene family in most plant taxa, the Brassicaceae, including Arabidopsis (Arabidopsis thaliana), predominantly possess a single PSY gene. This monogenic situation is compensated by the differential expression of two alternative splice variants (ASV), which differ in length and in the exon/intron retention of their 5'UTRs. ASV1 contains a long 5'UTR (untranslated region) and is involved in developmentally regulated carotenoid formation, such as during deetiolation. ASV2 contains a short 5'UTR and is preferentially induced when an immediate increase in the carotenoid pathway flux is required, such as under salt stress or upon sudden light intensity changes. We show that the long 5'UTR of ASV1 is capable of attenuating the translational activity in response to high carotenoid pathway fluxes. This function resides in a defined 5'UTR stretch with two predicted interconvertible RNA conformations, as known from riboswitches, which might act as a flux sensor. The translation-inhibitory structure is absent from the short 5'UTR of ASV2 allowing to bypass translational inhibition under conditions requiring rapidly increased pathway fluxes. The mechanism is not found in the rice (Oryza sativa) PSY1 5'UTR, consistent with the prevalence of transcriptional control mechanisms in taxa with multiple PSY genes. The translational control mechanism identified is interpreted in terms of flux adjustments needed in response to retrograde signals stemming from intermediates of the plastid-localized carotenoid biosynthesis pathway.

Analysing RNA-kinetics based on folding space abstraction.

BACKGROUND: RNA molecules, especially non-coding RNAs, play vital roles in the cell and their biological functions are mostly determined by structural properties. Often, these properties are related to dynamic changes in the structure, as in the case of riboswitches, and thus the analysis of RNA folding kinetics is crucial for their study. Exact approaches to kinetic folding are computationally expensive and, thus, limited to short sequences. In a previous study, we introduced a position-specific abstraction based on helices which we termed helix index shapes (hishapes) and a hishape-based algorithm for near-optimal folding pathway computation, called HiPath. The combination of these approaches provides an abstract view of the folding space that offers information about the global features. RESULTS: In this paper we present HiKinetics, an algorithm that can predict RNA folding kinetics for sequences up to several hundred nucleotides long. This algorithm is based on RNAHeliCes, which decomposes the folding space into abstract classes, namely hishapes, and an improved version of HiPath, namely HiPath2, which estimates plausible folding pathways that connect these classes. Furthermore, we analyse the relationship of hishapes to locally optimal structures, the results of which strengthen the use of the hishape abstraction for studying folding kinetics. Finally, we show the application of HiKinetics to the folding kinetics of two well-studied RNAs. CONCLUSIONS: HiKinetics can calculate kinetic folding based on a novel hishape decomposition. HiKinetics, together with HiPath2 and RNAHeliCes, is available for download at http://www.cyanolab.de/software/RNAHeliCes.htm.

Transcript mapping based on dRNA-seq data.

BACKGROUND: RNA-seq and its variant differential RNA-seq (dRNA-seq) are today routine methods for transcriptome analysis in bacteria. While expression profiling and transcriptional start site prediction are standard tasks today, the problem of identifying transcriptional units in a genome-wide fashion is still not solved for prokaryotic systems. RESULTS: We present RNAseg, an algorithm for the prediction of transcriptional units based on dRNA-seq data. A key feature of the algorithm is that, based on the data, it distinguishes between transcribed and un-transcribed genomic segments. Furthermore, the program provides many different predictions in a single run, which can be used to infer the significance of transcriptional units in a consensus procedure. We show the performance of our method based on a well-studied dRNA-seq data set for Helicobacter pylori. CONCLUSIONS: With our algorithm it is possible to identify operons and 5'- and 3'-UTRs in an automated fashion. This alleviates the need for labour intensive manual inspection and enables large-scale studies in the area of comparative transcriptomics.

The diversity of small non-coding RNAs in the diatom Phaeodactylum tricornutum.

BACKGROUND: Marine diatoms constitute a major component of eukaryotic phytoplankton and stand at the crossroads of several evolutionary lineages. These microalgae possess peculiar genomic features and novel combinations of genes acquired from bacterial, animal and plant ancestors. Furthermore, they display both DNA methylation and gene silencing activities. Yet, the biogenesis and regulatory function of small RNAs (sRNAs) remain ill defined in diatoms. RESULTS: Here we report the first comprehensive characterization of the sRNA landscape and its correlation with genomic and epigenomic information in Phaeodactylum tricornutum. The majority of sRNAs is 25 to 30 nt-long and maps to repetitive and silenced Transposable Elements marked by DNA methylation. A subset of this population also targets DNA methylated protein-coding genes, suggesting that gene body methylation might be sRNA-driven in diatoms. Remarkably, 25-30 nt sRNAs display a well-defined and unprecedented 180 nt-long periodic distribution at several highly methylated regions that awaits characterization. While canonical miRNAs are not detectable, other 21-25 nt sRNAs of unknown origin are highly expressed. Besides, non-coding RNAs with well-described function, namely tRNAs and U2 snRNA, constitute a major source of 21-25 nt sRNAs and likely play important roles under stressful environmental conditions. CONCLUSIONS: P. tricornutum has evolved diversified sRNA pathways, likely implicated in the regulation of largely still uncharacterized genetic and epigenetic processes. These results uncover an unexpected complexity of diatom sRNA population and previously unappreciated features, providing new insights into the diversification of sRNA-based processes in eukaryotes.

Abstract folding space analysis based on helices.

RNA has many pivotal functions especially in the regulation of gene expression by ncRNAs. Identification of their structure is an important requirement for understanding their function. Structure prediction alone is often insufficient for this task, due to algorithmic problems, parameter inaccuracies, and biological peculiarities. Among the latter, there are base modifications, cotranscriptional folding leading to folding traps, and conformational switching as in the case of riboswitches. All these require more in-depth analysis of the folding space. The major drawback, which all methods have to cope with, is the exponential growth of the folding space. Therefore, methods are often limited in the sequence length they can analyze, or they make use of heuristics, sampling, or abstraction. Our approach adopts the abstraction strategy and remedies some problems of existing methods. We introduce a position-specific abstraction based on helices that we term helix index shapes, or hishapes for short. Utilizing a dynamic programming framework, we have implemented this abstraction in the program RNAHeliCes. Furthermore, we developed two hishape-based methods, one for energy barrier estimation, called HiPath, and one for abstract structure comparison, termed HiTed. We demonstrate the superior performance of HiPath compared to other existing methods and the competitive accuracy of HiTed. RNAHeliCes, together with HiPath and HiTed, are available for download at http://www.cyanolab.de/software/RNAHeliCes.htm.

An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803.

There has been an increasing interest in cyanobacteria because these photosynthetic organisms convert solar energy into biomass and because of their potential for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires knowledge of their transcriptional organization. Using differential RNA sequencing, we have established a genome-wide map of 3,527 transcriptional start sites (TSS) of the model organism Synechocystis sp. PCC6803. One-third of all TSS were located upstream of an annotated gene; another third were on the reverse complementary strand of 866 genes, suggesting massive antisense transcription. Orphan TSS located in intergenic regions led us to predict 314 noncoding RNAs (ncRNAs). Complementary microarray-based RNA profiling verified a high number of noncoding transcripts and identified strong ncRNA regulations. Thus, ∼64% of all TSS give rise to antisense or ncRNAs in a genome that is to 87% protein coding. Our data enhance the information on promoters by a factor of 40, suggest the existence of additional small peptide-encoding mRNAs, and provide corrected 5' annotations for many genes of this cyanobacterium. The global TSS map will facilitate the use of Synechocystis sp. PCC6803 as a model organism for further research on photosynthesis and energy research.

Classified Dynamic Programming in RNA Structure Analysis.

Analysis of the folding space of RNA generally suffers from its exponential size. With classified Dynamic Programming algorithms, it is possible to alleviate this burden and to analyse the folding space of RNA in great depth. Key to classified DP is that the search space is partitioned into classes based on an on-the-fly computed feature. A class-wise evaluation is then used to compute class-wide properties, such as the lowest free energy structure for each class, or aggregate properties, such as the class' probability. In this paper we describe the well-known shape and hishape abstraction of RNA structures, their power to help better understand RNA function and related methods that are based on these abstractions.

Adaptation and modification of three CRISPR loci in two closely related cyanobacteria.

An RNA-based screen was performed to reveal a possible evolutionary scenario for the CRISPR-Cas systems in two cyanobacterial model strains. Following the analysis of a draft genome sequence of Synechocystis sp PCC6714, three different CRISPR-Cas systems were characterized that have different degrees of relatedness to another three CRISPR-Cas systems in Synechocystis sp PCC6803. A subtype III-B system was identified that is extremely conserved between both strains. Strong signals in northern hybridizations and the presence of different spacers (but identical repeats) indicated this system to be active, despite the absence of a known endonuclease candidate gene involved in the maturation of its crRNAs in the two strains. The other two systems were found to differ significantly from each other, with different sets of repeat-spacer arrays and different Cas genes. In view of the otherwise very close relatedness of the two analyzed strains, this is suggestive of an unknown mechanism involved in the replacement of CRISPR-Cas cassettes as a whole. Further RNA analyses revealed the accumulation of crRNAs to be impacted by environmental conditions critical for photoautotropic growth. All six systems are associated with a gene for a possible transcriptional repressor. Indeed, we identified one of these genes, sll7009, as encoding a negative regulator specific for the CRISPR1 subtype I-D system in Synechocystis sp PCC6803.

Small RNA-mediated control of the Agrobacterium tumefaciens GABA binding protein.

Wounded plants activate a complex defence programme in response to Agrobacterium tumefaciens. They synthesize the non-proteinogenic amino acid γ-aminobutyric acid (GABA), which stimulates degradation of the quorum sensing signal N-(3-oxo-octanoyl) homoserine lactone. GABA is transported into A. tumefaciens via an ABC transporter dependent on the periplasmic binding protein Atu2422. We demonstrate that expression of atu2422 and two other ABC transporter genes is downregulated by the conserved small RNA (sRNA) AbcR1 (for ABC regulator). AbcR1 is encoded in tandem with another sRNA, which is similar in sequence and structure. Both sRNAs accumulate during stationary phase but only the absence of AbcR1 resulted in significant accumulation of Atu2422 and increased GABA import. AbcR1 inhibits initiation of atu2422 translation by masking its Shine-Dalgarno sequence and thereby reduces stability of the atu2422 transcript. It is the first described bacterial sRNA that controls uptake of a plant-generated signalling molecule. Given that similar sRNAs and ABC transporter genes are present in various Rhizobiaceae and in Brucella, it is likely that such sRNA-mediated control impacts a number of host-microbe interactions.

SIMA: simultaneous multiple alignment of LC/MS peak lists.

MOTIVATION: Alignment of multiple liquid chromatography/mass spectrometry (LC/MS) experiments is a necessity today, which arises from the need for biological and technical repeats. Due to limits in sampling frequency and poor reproducibility of retention times, current LC systems suffer from missing observations and non-linear distortions of the retention times across runs. Existing approaches for peak correspondence estimation focus almost exclusively on solving the pairwise alignment problem, yielding straightforward but suboptimal results for multiple alignment problems. RESULTS: We propose SIMA, a novel automated procedure for alignment of peak lists from multiple LC/MS runs. SIMA combines hierarchical pairwise correspondence estimation with simultaneous alignment and global retention time correction. It employs a tailored multidimensional kernel function and a procedure based on maximum likelihood estimation to find the retention time distortion function that best fits the observed data. SIMA does not require a dedicated reference spectrum, is robust with regard to outliers, needs only two intuitive parameters and naturally incorporates incomplete correspondence information. In a comparison with seven alternative methods on four different datasets, we show that SIMA yields competitive and superior performance on real-world data. AVAILABILITY: A C++ implementation of the SIMA algorithm is available from http://hci.iwr.uni-heidelberg.de/MIP/Software.

Hemin and magnesium-protoporphyrin IX induce global changes in gene expression in Chlamydomonas reinhardtii.

Retrograde signaling is a pathway of communication from mitochondria and plastids to the nucleus in the context of cell differentiation, development, and stress response. In Chlamydomonas reinhardtii, the tetrapyrroles magnesium-protoporphyrin IX and heme are only synthesized within the chloroplast, and they have been implicated in the retrograde control of nuclear gene expression in this unicellular green alga. Feeding the two tetrapyrroles to Chlamydomonas cultures was previously shown to transiently induce five nuclear genes, three of which encode the heat shock proteins HSP70A, HSP70B, and HSP70E. In contrast, controversial results exist on the possible role of magnesium-protoporphyrin IX in the repression of genes for light-harvesting proteins in higher plants, raising the question of how important this mode of regulation is. Here, we used genome-wide transcriptional profiling to measure the global impact of these tetrapyrroles on gene regulation and the scope of the response. We identified almost 1,000 genes whose expression level changed transiently but significantly. Among them were only a few genes for photosynthetic proteins but several encoding enzymes of the tricarboxylic acid cycle, heme-binding proteins, stress-response proteins, as well as proteins involved in protein folding and degradation. More than 50% of the latter class of genes was also regulated by heat shock. The observed drastic fold changes at the RNA level did not correlate with similar changes in protein concentrations under the tested experimental conditions. Phylogenetic profiling revealed that genes of putative endosymbiontic origin are not overrepresented among the responding genes. This and the transient nature of changes in gene expression suggest a signaling role of both tetrapyrroles as secondary messengers for adaptive responses affecting the entire cell and not only organellar proteins.

Small RNAs of the Bradyrhizobium/Rhodopseudomonas lineage and their analysis.

Small RNAs (sRNAs) play a pivotal role in bacterial gene regulation. However, the sRNAs of the vast majority of bacteria with sequenced genomes still remain unknown since sRNA genes are usually difficult to recognize and thus not annotated. Here, expression of seven sRNAs (BjrC2a, BjrC2b, BjrC2c, BjrC68, BjrC80, BjrC174 and BjrC1505) predicted by genome comparison of Bradyrhizobium and Rhodopseudomonas members, was verified by RNA gel blot hybridization, microarray and deep sequencing analyses of RNA from the soybean symbiont Bradyrhizobium japonicum USDA 110. BjrC2a, BjrC2b and BjrC2c belong to the RNA family RF00519, while the other sRNAs are novel. For some of the sRNAs we observed expression differences between free-living bacteria and bacteroids in root nodules. The amount of BjrC1505 was decreased in nodules. By contrast, the amount of BjrC2a, BjrC68, BjrC80, BjrC174 and the previously described 6S RNA was increased in nodules, and accumulation of truncated forms of these sRNAs was observed. Comparative genomics and deep sequencing suggest that BjrC2a is an antisense RNA regulating the expression of inositol-monophosphatase. The analyzed sRNAs show a different degree of conservation in Rhizobiales, and expression of homologs of BjrC2, BjrC68, BjrC1505, and 6S RNA was confirmed in the free-living purple bacterium Rhodopseudomonas palustris 5D.

Bioinformatic prediction and experimental verification of sRNAs in the haloarchaeon Haloferax volcanii.

Recently a small-scale RNomics study led to the experimental identification of 21 intergenic and 18 antisense sRNA genes in the haloarchaeon Haloferax volcanii. To broaden the knowledge about sRNAs in haloarchaea, two bioinformatic approaches were used to predict sRNA genes in the genome of H. volcanii. More than 120 putative intergenic sRNA genes were identified by these comparative genomic approaches. The expression of 61 of the predicted genes was analyzed using DNA microarrays, and 37 were found to be expressed under at least one of three conditions tested. Using the results of Northern blot analyses and of a high throughput sequencing study the number of expressed genes was raised to 54 and the small size was verified for 26 predicted sRNAs. An analysis of the coding capacity revealed that the set of predicted sRNAs most likely does not encode proteins or peptides. In two cases it turned out that the predictions had not identified bona fide sRNAs but conserved regions in UTRs of large protein-encoding transcripts. Taken together, the combination of bioinformatic prediction and experimental verification has more than tripled the number of known haloarchaeal sRNAs, underscoring the importance of regulatory RNAs in the third domain of life, the archaea. Further analyses of the biological functions of selected sRNAs, including the construction of deletion mutants, are currently under way.

Simulation of Folding Kinetics for Aligned RNAs.

Studying the folding kinetics of an RNA can provide insight into its function and is thus a valuable method for RNA analyses. Computational approaches to the simulation of folding kinetics suffer from the exponentially large folding space that needs to be evaluated. Here, we present a new approach that combines structure abstraction with evolutionary conservation to restrict the analysis to common parts of folding spaces of related RNAs. The resulting algorithm can recapitulate the folding kinetics known for single RNAs and is able to analyse even long RNAs in reasonable time. Our program RNAliHiKinetics is the first algorithm for the simulation of consensus folding kinetics and addresses a long-standing problem in a new and unique way.

A Small RNA Is Linking CRISPR-Cas and Zinc Transport.

The function and mode of action of small regulatory RNAs is currently still understudied in archaea. In the halophilic archaeon Haloferax volcanii, a plethora of sRNAs have been identified; however, in-depth functional analysis is missing for most of them. We selected a small RNA (s479) from Haloferax volcanii for detailed characterization. The sRNA gene is encoded between a CRISPR RNA locus and the Cas protein gene cluster, and the s479 deletion strain is viable and was characterized in detail. Transcriptome studies of wild-type Haloferax cells and the deletion mutant revealed upregulation of six genes in the deletion strain, showing that this sRNA has a clearly defined function. Three of the six upregulated genes encode potential zinc transporter proteins (ZnuA1, ZnuB1, and ZnuC1) suggesting the involvement of s479 in the regulation of zinc transport. Upregulation of these genes in the deletion strain was confirmed by northern blot and proteome analyses. Furthermore, electrophoretic mobility shift assays demonstrate a direct interaction of s479 with the target znuC1 mRNA. Proteome comparison of wild-type and deletion strains further expanded the regulon of s479 deeply rooting this sRNA within the metabolism of H. volcanii especially the regulation of transporter abundance. Interestingly, s479 is not only encoded next to CRISPR-cas genes, but the mature s479 contains a crRNA-like 5' handle, and experiments with Cas protein deletion strains indicate maturation by Cas6 and interaction with Cas proteins. Together, this might suggest that the CRISPR-Cas system is involved in s479 function.