AGouTI-Flexible Annotation of Genomic and Transcriptomic Intervals.

The recent development of high-throughput workflows in genomics and transcriptomics revealed that efficient annotation of such results is essential for researchers to draw conclusions from obtained results. Although some tools are available, their functionality is limited. Here, we present AGouTI-a universal tool for flexible annotation of any genomic or transcriptomic coordinates using known genomic features deposited in different publicly available databases in the form of GTF or GFF files. In contrast to currently available tools, AGouTI is designed to provide a flexible selection of genomic features overlapping or adjacent to annotated intervals and can be used on custom column-based text files obtained from different data analysis pipelines. Although providing many unique options, AGouTI is straightforward in installation and usage, enabling effortless integration into existing data analysis workflows.

The Evaluation of SHAPE-MaP RNA Structure Probing Protocols Reveals a Novel Role of Mn2+ in the Detection of 2'-OH Adducts.

Chemical probing, for decades, has been one of the most popular tools for studying the secondary structure of RNA molecules. Recently, protocols for simultaneous analysis of multiple RNAs have been developed, enabling in vivo transcriptome-wide interrogation of the RNA structure dynamics. One of the most popular methods is the selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP). In this study, we describe the evaluation of this protocol by addressing the influence of the reverse transcription enzymes, buffer conditions, and chemical probes on the properties of the cDNA library and the quality of mutational profiling-derived structural signals. Our results reveal a SuperScript IV (SSIV) reverse transcriptase as a more efficient enzyme for mutational profiling of SHAPE adducts and shed new light on the role of Mn2+ cations in the modulation of SSIV readthrough efficiency.

An mRNA-derived noncoding RNA targets and regulates the ribosome.

The structural and functional repertoire of small non-protein-coding RNAs (ncRNAs) is central for establishing gene regulation networks in cells and organisms. Here, we show that an mRNA-derived 18-nucleotide-long ncRNA is capable of downregulating translation in Saccharomyces cerevisiae by targeting the ribosome. This 18-mer ncRNA binds to polysomes upon salt stress and is crucial for efficient growth under hyperosmotic conditions. Although the 18-mer RNA originates from the TRM10 locus, which encodes a tRNA methyltransferase, genetic analyses revealed the 18-mer RNA nucleotide sequence, rather than the mRNA-encoded enzyme, as the translation regulator. Our data reveal the ribosome as a target for a small regulatory ncRNA and demonstrate the existence of a yet unkown mechanism of translation regulation. Ribosome-targeted small ncRNAs are found in all domains of life and represent a prevalent but so far largely unexplored class of regulatory molecules.

The Role of RNA Secondary Structure in Regulation of Gene Expression in Bacteria.

Due to the high exposition to changing environmental conditions, bacteria have developed many mechanisms enabling immediate adjustments of gene expression. In many cases, the required speed and plasticity of the response are provided by RNA-dependent regulatory mechanisms. This is possible due to the very high dynamics and flexibility of an RNA structure, which provide the necessary sensitivity and specificity for efficient sensing and transduction of environmental signals. In this review, we will discuss the current knowledge about known bacterial regulatory mechanisms which rely on RNA structure. To better understand the structure-driven modulation of gene expression, we describe the basic theory on RNA structure folding and dynamics. Next, we present examples of multiple mechanisms employed by RNA regulators in the control of bacterial transcription and translation.

Efficiency of PacBio long read correction by 2nd generation Illumina sequencing.

Long sequencing reads offer unprecedented opportunities in analysis and reconstruction of complex genomic regions. However, the gain in sequence length is often traded for quality. Therefore, recently several approaches have been proposed (e.g. higher sequencing coverage, hybrid assembly or sequence correction) to enhance the quality of long sequencing reads. A simple and cost-effective approach includes use of the high quality 2nd generation sequencing data to improve the quality of long reads. We designed a dedicated testing procedure and selected universal programs for long read correction, which provide as the output sequences that can be used in further genomic and transcriptomic studies. Our results show that HALC is the best choice for correction of long PacBio reads, when both, read size and quality, are the main focus of the analysis. However, the tested tools show some unexpected behaviors, including read trimming and fragmentation.

Identification of RNA targets for the nuclear multidomain cyclophilin atCyp59 and their effect on PPIase activity.

AtCyp59 is a multidomain cyclophilin containing a peptidyl-prolyl cis/trans isomerase (PPIase) domain and an evolutionarily highly conserved RRM domain. Deregulation of this class of cyclophilins has been shown to affect transcription and to influence phosphorylation of the C-terminal repeat domain of the largest subunit of the RNA polymerase II. We used a genomic SELEX method for identifying RNA targets of AtCyp59. Analysis of the selected RNAs revealed an RNA-binding motif (G[U/C]N[G/A]CC[A/G]) and we show that it is evolutionarily conserved. Binding to this motif was verified by gel shift assays in vitro and by RNA immunopreciptation assays of AtCyp59 in vivo. Most importantly, we show that binding also occurs on unprocessed transcripts in vivo and that binding of specific RNAs inhibits the PPIase activity of AtCyp59 in vitro. Surprisingly, genome-wide analysis showed that the RNA motif is present in about 70% of the annotated transcripts preferentially in exons. Taken together, the available data suggest that these cyclophilins might have an important function in transcription regulation.

Revealing stable processing products from ribosome-associated small RNAs by deep-sequencing data analysis.

The exploration of the non-protein-coding RNA (ncRNA) transcriptome is currently focused on profiling of microRNA expression and detection of novel ncRNA transcription units. However, recent studies suggest that RNA processing can be a multi-layer process leading to the generation of ncRNAs of diverse functions from a single primary transcript. Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases. Thus the correct assessment of widespread RNA processing events is one of the major obstacles in transcriptome research. Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data. The major features include efficient handling of non-unique reads, detection of novel stable ncRNA transcripts and processing products and annotation of known transcripts based on multiple sources of information. To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae. By employing the APART pipeline, we were able to detect and confirm by independent experimental methods multiple novel stable RNA molecules differentially processed from well known ncRNAs, like rRNAs, tRNAs or snoRNAs, in a stress-dependent manner.

Identification of differentially expressed non-coding RNAs in embryonic stem cell neural differentiation.

Protein-coding genes, guiding differentiation of ES cells into neural cells, have extensively been studied in the past. However, for the class of ncRNAs only the involvement of some specific microRNAs (miRNAs) has been described. Thus, to characterize the entire small non-coding RNA (ncRNA) transcriptome, involved in the differentiation of mouse ES cells into neural cells, we have generated three specialized ribonucleo-protein particle (RNP)-derived cDNA libraries, i.e. from pluripotent ES cells, neural progenitors and differentiated neural cells, respectively. By high-throughput sequencing and transcriptional profiling we identified several novel miRNAs to be involved in ES cell differentiation, as well as seven small nucleolar RNAs. In addition, expression of 7SL, 7SK and vault-2 RNAs was significantly up-regulated during ES cell differentiation. About half of ncRNA sequences from the three cDNA libraries mapped to intergenic or intragenic regions, designated as interRNAs and intraRNAs, respectively. Thereby, novel ncRNA candidates exhibited a predominant size of 18-30 nt, thus resembling miRNA species, but, with few exceptions, lacking canonical miRNA features. Additionally, these novel intraRNAs and interRNAs were not only found to be differentially expressed in stem-cell derivatives, but also in primary cultures of hippocampal neurons and astrocytes, strengthening their potential function in neural ES cell differentiation.

Insight into metabolic sensors of nitrosative stress protection in Phytophthora infestans.

Phytophthora infestans, a representative of phytopathogenic oomycetes, have been proven to cope with redundant sources of internal and host-derived reactive nitrogen species (RNS). To gain insight into its nitrosative stress resistance mechanisms, metabolic sensors activated in response to nitrosative challenge during both in vitro growth and colonization of the host plant were investigated. The conducted analyses of gene expression, protein accumulation, and enzyme activity reveal for the first time that P. infestans (avirulent MP946 and virulent MP977 toward potato cv. Sarpo Mira) withstands nitrosative challenge and has an efficient system of RNS elimination. The obtained data indicate that the system protecting P. infestans against nitric oxide (NO) involved the expression of the nitric oxide dioxygenase (Pi-NOD1) gene belonging to the globin family. The maintenance of RNS homeostasis was also supported by an elevated S-nitrosoglutathione reductase activity and upregulation of peroxiredoxin 2 at the transcript and protein levels; however, the virulence pattern determined the expression abundance. Based on the experiments, it can be concluded that P. infestans possesses a multifarious system of metabolic sensors controlling RNS balance via detoxification, allowing the oomycete to exist in different micro-environments flexibly.

tRNA-derived fragments target the ribosome and function as regulatory non-coding RNA in Haloferax volcanii.

Nonprotein coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. These RNAs either originate from their individual transcription units or are processing products from longer precursor RNAs. For example, tRNA-derived fragments (tRFs) have been identified in all domains of life and represent a growing, yet functionally poorly understood, class of ncRNA candidates. Here we present evidence that tRFs from the halophilic archaeon Haloferax volcanii directly bind to ribosomes. In the presented genomic screen of the ribosome-associated RNome, a 26-residue-long fragment originating from the 5' part of valine tRNA was by far the most abundant tRF. The Val-tRF is processed in a stress-dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. As a consequence of ribosome binding, Val-tRF reduces protein synthesis by interfering with peptidyl transferase activity. Therefore this tRF functions as ribosome-bound small ncRNA capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine tuning the rate of protein production.

The role of the universally conserved A2450-C2063 base pair in the ribosomal peptidyl transferase center.

Despite the fact that all 23S rRNA nucleotides that build the ribosomal peptidyl transferase ribozyme are universally conserved, standard and atomic mutagenesis studies revealed the nucleobase identities being non-critical for catalysis. This indicates that these active site residues are highly conserved for functions distinct from catalysis. To gain insight into potential contributions, we have manipulated the nucleobases via an atomic mutagenesis approach and have utilized these chemically engineered ribosomes for in vitro translation reactions. We show that most of the active site nucleobases could be removed without significant effects on polypeptide production. Our data however highlight the functional importance of the universally conserved non-Watson-Crick base pair at position A2450-C2063. Modifications that disrupt this base pair markedly impair translation activities, while having little effects on peptide bond formation, tRNA drop-off and ribosome-dependent EF-G GTPase activity. Thus it seems that disruption of the A2450-C2063 pair inhibits a reaction following transpeptidation and EF-G action during the elongation cycle. Cumulatively our data are compatible with the hypothesis that the integrity of this A-C wobble base pair is essential for effective tRNA translocation through the peptidyl transferase center during protein synthesis.

RNPomics: defining the ncRNA transcriptome by cDNA library generation from ribonucleo-protein particles.

Up to 450,000 non-coding RNAs (ncRNAs) have been predicted to be transcribed from the human genome. However, it still has to be elucidated which of these transcripts represent functional ncRNAs. Since all functional ncRNAs in Eukarya form ribonucleo-protein particles (RNPs), we generated specialized cDNA libraries from size-fractionated RNPs and validated the presence of selected ncRNAs within RNPs by glycerol gradient centrifugation. As a proof of concept, we applied the RNP method to human Hela cells or total mouse brain, and subjected cDNA libraries, generated from the two model systems, to deep-sequencing. Bioinformatical analysis of cDNA sequences revealed several hundred ncRNP candidates. Thereby, ncRNAs candidates were mainly located in intergenic as well as intronic regions of the genome, with a significant overrepresentation of intron-derived ncRNA sequences. Additionally, a number of ncRNAs mapped to repetitive sequences. Thus, our RNP approach provides an efficient way to identify new functional small ncRNA candidates, involved in RNP formation.

Identification of Structural Variants in Two Novel Genomes of Maize Inbred Lines Possibly Related to Glyphosate Tolerance.

To study genetic variations between genomes of plants that are naturally tolerant and sensitive to glyphosate, we used two Zea mays L. lines traditionally bred in Poland. To overcome the complexity of the maize genome, two sequencing technologies were employed: Illumina and Single Molecule Real-Time (SMRT) PacBio. Eleven thousand structural variants, 4 million SNPs and approximately 800 thousand indels differentiating the two genomes were identified. Detailed analyses allowed to identify 20 variations within the EPSPS gene, but all of them were predicted to have moderate or unknown effects on gene expression. Other genes of the shikimate pathway encoding bifunctional 3-dehydroquinate dehydratase/shikimate dehydrogenase and chorismate synthase were altered by variants predicted to have a high impact on gene expression. Additionally, high-impact variants located within the genes involved in the active transport of glyphosate through the cell membrane encoding phosphate transporters as well as multidrug and toxic compound extrusion have been identified.

RNA polymerase II-binding aptamers in human ACRO1 satellites disrupt transcription in cis.

Transcription elongation is a highly regulated process affected by many proteins, RNAs and the underlying DNA. Here we show that the nascent RNA can interfere with transcription in human cells, extending our previous findings from bacteria and yeast. We identified a variety of Pol II-binding aptamers (RAPs), prominent in repeat elements such as ACRO1 satellites, LINE1 retrotransposons and CA simple repeats, and also in several protein-coding genes. ACRO1 repeat, when translated in silico, exhibits ~50% identity with the Pol II CTD sequence. Taken together with a recent proposal that proteins in general tend to interact with RNAs similar to their cognate mRNAs, this suggests a mechanism for RAP binding. Using a reporter construct, we show that ACRO1 potently inhibits Pol II elongation in cis. We propose a novel mode of transcriptional regulation in humans, in which the nascent RNA binds Pol II to silence its own expression.

Is Polymorphism in the Apoptosis and Inflammatory Pathway Genes Associated With a Primary Response to Anti-TNF Therapy in Crohn's Disease Patients?

Anti-tumor necrosis factor (TNF) therapy is used for the induction and maintenance of remission in Crohn's disease (CD) patients. However, primary nonresponders to initial treatment constitute 20%-40% of cases. The causes of this phenomenon are still unknown. In this study, we aimed to determine the genetic predictors of the variable reactions of CD patients to anti-TNF therapy. Using long-range PCR libraries and the next-generation sequencing (NGS) method, we performed broad pharmacogenetic studies including a panel of 23 genes (TNFRSF1A, TNFRSF1B, CASP9, FCGR3A, LTA, TNF, FAS, ADAM17, IL17A, IL6, MMP1, MMP3, S100A8, S100A9, S100A12, TLR2, TLR4, TLR9, CD14, IL23R, IL23, IL1R, and IL1B) in a group of 107 diagnosed and clinically characterized CD patients following anti-TNF therapy. In the studied group, we indicated, in total, 598 single nucleotide variants for all analyzed genomic targets. Twelve patients (11.2%) did not respond to the induction therapy, which was associated with alleles in 11 loci located in FCGR3A (rs7539036, rs6672453, rs373184583, and rs12128686), IL1R (rs2041747), TNFRSF1B (rs5746053), IL1B (rs1071676, rs1143639, rs1143637, and rs1143634), and FAS (rs7896789) genes. After multiple comparison corrections, the results were not statistically significant, however for nonresponders the alleles distribution for those loci presented large differences and specified scheme compared to responders and populations. These findings require further investigation in an independent larger cohort before introducing them for a clinical setting, however, we identified an interesting direction. Polymorphism of the FCGR3A, IL1R, TNFRSF1B, IL1B, and FAS genes could be a predictor of the primary response to anti-TNF therapy in CD patients.

A tRNA half modulates translation as stress response in Trypanosoma brucei.

In the absence of extensive transcription control mechanisms the pathogenic parasite Trypanosoma brucei crucially depends on translation regulation to orchestrate gene expression. However, molecular insight into regulating protein biosynthesis is sparse. Here we analyze the small non-coding RNA (ncRNA) interactome of ribosomes in T. brucei during different growth conditions and life stages. Ribosome-associated ncRNAs have recently been recognized as unprecedented regulators of ribosome functions. Our data show that the tRNAThr 3´half is produced during nutrient deprivation and becomes one of the most abundant tRNA-derived RNA fragments (tdRs). tRNAThr halves associate with ribosomes and polysomes and stimulate translation by facilitating mRNA loading during stress recovery once starvation conditions ceased. Blocking or depleting the endogenous tRNAThr halves mitigates this stimulatory effect both in vivo and in vitro. T. brucei and its close relatives lack the well-described mammalian enzymes for tRNA half processing, thus hinting at a unique tdR biogenesis in these parasites.

mRNA-specific translation regulation by a ribosome-associated ncRNA in Haloferax volcanii.

Regulation of gene expression at the translational level allows rapid adaptation of cellular proteomes to quickly changing environmental conditions and is thus central for prokaryotic organisms. Small non-coding RNAs (sRNAs) have been reported to effectively orchestrate translation control in bacteria and archaea mainly by targeting mRNAs by partial base complementarity. Here we report an unprecedented mechanism how sRNAs are capable of modulating protein biosynthesis in the halophilic archaeon Haloferax volcanii. By analyzing the ribosome-associated ncRNAs (rancRNAs) under different stress conditions we identified an intergenic sRNA, termed rancRNA_s194, that is primarily expressed during exponential growth under all tested conditions. By interaction with the ribosome rancRNA_s194 inhibits peptide bond formation and protein synthesis in vitro but appears to target a specific mRNA in vivo. The respective knock-out strain shows a reduced lag phase in media containing xylose as sole carbon source and outcompetes the wildtype cells under these conditions. Mass spectrometry, polysome profiling and mRNA binding competition experiments suggest that rancRNA_s194 prevents the cstA mRNA from being efficiently translated by H. volcanii ribosomes. These findings enlarge the regulatory repertoire of archaeal sRNAs in modulating post-transcriptional gene expression.

Maf1-mediated regulation of yeast RNA polymerase III is correlated with CCA addition at the 3' end of tRNA precursors.

In eukaryotic cells tRNA synthesis is negatively regulated by the protein Maf1, conserved from yeast to humans. Maf1 from yeast Saccharomyces cerevisiae mediates repression of trna transcription when cells are transferred from medium with glucose to medium with glycerol, a non-fermentable carbon source. The strain with deleted gene encoding Maf1 (maf1Δ) is viable but accumulates tRNA precursors. In this study tRNA precursors were analysed by RNA-Seq and Northern hybridization in wild type strain and maf1Δ mutant grown in glucose medium or upon shift to repressive conditions. A negative effect of maf1Δ mutant on the addition of the auxiliary CCA nucleotides to the 3' end of pre-tRNAs was observed in cells shifted to unfavourable growth conditions. This effect was reduced by overexpression of the yeast CCA1 gene encoding ATP(CTP):tRNA nucleotidyltransferase. The CCA sequence at the 3' end is important for export of tRNA precursors from the nucleus and essential for tRNA charging with amino acids. Data presented here indicate that CCA-addition to intron-containing end-processed tRNA precursors is a limiting step in tRNA maturation when there is no Maf1 mediated RNA polymerase III (Pol III) repression. The correlation between CCA synthesis and Pol III regulation by Maf1 could be important in coordination of tRNA transcription, processing and regulation of translation.

Evaluation of the dynamic structure of DsrA RNA from E. coli and its functional consequences.

DsrA RNA is an 87-nucleotide regulatory non-protein-coding RNA of Escherichia coli for which two secondary structure models (I and II) have been proposed. We have compared these models by the energy calculations, which revealed that the currently accepted model II should be rejected on the basis of thermodynamics. Here we provide new results of nuclease footprinting analysis and the application of RNA technologies that have not previously been used for DsrA RNA structural studies, such as hydrolysis with RNase H, DNAzyme, hydroxyl radicals and lead. These approaches together with bioinformatics calculations provided strong arguments for a new model III. This model clearly shows that the long U-rich region between hairpins 1 and 2 is double-stranded. These findings shed new light on DsrA RNA-Hfq interactions.

Emerging applications of riboswitches - from antibacterial targets to molecular tools.

The ability to precisely regulate gene expression is one of the most important features of the living cells as it enables the adaptation and survival in different environmental conditions. The majority of regulatory mechanisms involve protein action, however, multiple genes are controlled by nucleic acids. Among RNA-based regulators, the riboswitches present a large group of specific domains within messenger RNAs able to respond to small metabolites, tRNA, secondary messengers, ions, vitamins or amino acids. A simple, accurate, and efficient mechanism of action as well as easiness in handling and engineering make the riboswitches a potent practical tool in industry, medicine, pharmacy or environmental protection. Hereby, we summarize the current achievements and challenges in designing and practical employment of the riboswitch-based tools.