The Single-Nucleotide Resolution Transcriptome of Pseudomonas aeruginosa Grown in Body Temperature

One of the hallmarks of opportunistic pathogens is their ability to adjust and respond to a wide range of environmental and host-associated conditions. The human pathogen Pseudomonas aeruginosa has an ability to thrive in a variety of hosts and cause a range of acute and chronic infections in individuals with impaired host defenses or cystic fibrosis. Here we report an in-depth transcriptional profiling of this organism when grown at host-related temperatures. Using RNA-seq of samples from P. aeruginosa grown at 28°C and 37°C we detected genes preferentially expressed at the body temperature of mammalian hosts, suggesting that they play a role during infection. These temperature-induced genes included the type III secretion system (T3SS) genes and effectors, as well as the genes responsible for phenazines biosynthesis. Using genome-wide transcription start site (TSS) mapping by RNA-seq we were able to accurately define the promoters and cis-acting RNA elements of many genes, and uncovered new genes and previously unrecognized non-coding RNAs directly controlled by the LasR quorum sensing regulator. Overall we identified 165 small RNAs and over 380 cis-antisense RNAs, some of which predicted to perform regulatory functions, and found that non-coding RNAs are preferentially localized in pathogenicity islands and horizontally transferred regions. Our work identifies regulatory features of P. aeruginosa genes whose products play a role in environmental adaption during infection and provides a reference transcriptional landscape for this pathogen

Comparative transcriptomics of pathogenic and non-pathogenic Listeria species

Comparative RNA-seq analysis of two related pathogenic and non-pathogenic bacterial strains reveals a hidden layer of divergence in the non-coding genome as well as conserved, widespread regulatory structures called ‘Excludons', which mediate regulation through long non-coding antisense RNAs

Comparative transcriptomics across the prokaryotic tree of life

Whole-transcriptome sequencing studies from recent years revealed an unexpected complexity in transcriptomes of bacteria and archaea, including abundant non-coding RNAs, cis-antisense transcription and regulatory untranslated regions (UTRs). Understanding the functional relevance of the plethora of non-coding RNAs in a given organism is challenging, especially since some of these RNAs were attributed to ‘transcriptional noise’. To allow the search for conserved transcriptomic elements we produced comparative transcriptome maps for multiple species across the microbial tree of life. These transcriptome maps are detailed in annotations, comparable by gene families, and BLAST-searchable by user provided sequences. Our transcriptome collection includes 18 model organisms spanning 10 phyla/subphyla of bacteria and archaea that were sequenced using standardized RNA-seq methods. The utility of the comparative approach, as implemented in our web server, is demonstrated by highlighting genes with exceptionally long 5′UTRs across species, which correspond to many known riboswitches and further suggest novel putative regulatory elements. Our study provides a standardized reference transcriptome to major clinically and environmentally important microbial phyla. The viewer is available at http://exploration.weizmann.ac.il/TCOL, setting a framework for comparative studies of the microbial non-coding genome

Transcriptome-wide discovery of circular RNAs in Archaea

Circular RNA forms had been described in all domains of life. Such RNAs were shown to have diverse biological functions, including roles in the life cycle of viral and viroid genomes, and in maturation of permuted tRNA genes. Despite their potentially important biological roles, discovery of circular RNAs has so far been mostly serendipitous. We have developed circRNA-seq, a combined experimental/computational approach that enriches for circular RNAs and allows profiling their prevalence in a whole-genome, unbiased manner. Application of this approach to the archaeon Sulfolobus solfataricus P2 revealed multiple circular transcripts, a subset of which was further validated independently. The identified circular RNAs included expected forms, such as excised tRNA introns and rRNA processing intermediates, but were also enriched with non-coding RNAs, including C/D box RNAs and RNase P, as well as circular RNAs of unknown function. Many of the identified circles were conserved in Sulfolobus acidocaldarius, further supporting their functional significance. Our results suggest that circular RNAs, and particularly circular non-coding RNAs, are more prevalent in archaea than previously recognized, and might have yet unidentified biological roles. Our study establishes a specific and sensitive approach for identification of circular RNAs using RNA-seq, and can readily be applied to other organisms

Transcriptome-Wide Mapping of 5-methylcytidine RNA Modifications in Bacteria, Archaea, and Yeast Reveals m⁵C within Archaeal mRNAs

<div><p>The presence of 5-methylcytidine (m⁵C) in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m⁵C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (<i>B. subtilis</i>) and gram negative (<i>E. coli</i>) bacteria, an archaeon (<i>S. solfataricus</i>) and a eukaryote (<i>S. cerevisiae</i>), followed by massively parallel sequencing. We were able to recover most previously documented m⁵C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m⁵C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m⁵C was absent were also discovered. Intriguingly, we detected m⁵C sites within archaeal mRNAs, and identified a consensus motif of AU<b><u>C</u></b>GANGU that directs methylation in <i>S. solfataricus</i>. Our results, which were validated using m⁵C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.</p></div

Transcription-mediated gene fusion in the human genome

Transcription of a gene usually ends at a regulated termination point, preventing the RNA-polymerase from reading through the next gene. However, sporadic reports suggest that chimeric transcripts, formed by transcription of two consecutive genes into one RNA, can occur in human. The splicing and translation of such RNAs can lead to a new, fused protein, having domains from both original proteins. Here, we systematically identified over 200 cases of intergenic splicing in the human genome (involving 421 genes), and experimentally demonstrated that at least half of these fusions exist in human tissues. We showed that unique splicing patterns dominate the functional and regulatory nature of the resulting transcripts, and found intergenic distance bias in fused compared with nonfused genes. We demonstrate that the hundreds of fused genes we identified are only a subset of the actual number of fused genes in human. We describe a novel evolutionary mechanism where transcription-induced chimerism followed by retroposition results in a new, active fused gene. Finally, we provide evidence that transcription-induced chimerism can be a mechanism contributing to the evolution of protein complexes

Sanger-based verification of two novel methylated positions in <i>S. solfataricus</i> rRNA identified using RNA-seq.

<p>Bisulfite-converted sequences were amplified using amplicon-specific primers and sequenced. (A) Position C1369 in the <i>S. solfataricus</i> 16S rRNA. (B) Position C2643 in the <i>S. solfataricus</i> 23S rRNA.</p

RNA immunoprecipitation with modification-specific antibodies.

<p>Shown is the coverage of Illumina-sequenced cDNA following RNA fragmentation, antibody pulldown, reverse transcription and sequencing. Black line, pulldown performed with an anti-5-methylcitosine (hm⁵C) antibody; green line, pulldown performed with an anti-5-hydroxy-methylcitosine antibody; red line, input RNA (no antibody applied). X-axis, position along the genome; Y-axis (right), read coverage of the sequenced anti-m⁵C library; Y-axis (left), fold enrichment of peaks related to median coverage along the gene. The coverage of the anti-hm⁵C and input libraries was normalized using the median of the anti-m⁵C library as a reference point. (A) The 23S gene of <i>S. solfataricus</i>. Peaks corresponding to positions 2121 and 2643 in the gene (875,473 and 875,995 relative to the <i>S. solfataricus</i> genome, respectively) are marked. Another peak, which did not come up in our bisulfite-based analysis, is observed around position 2760. (B) The 16S gene of <i>S. solfataricus</i>. A single peak corresponding to position 1369 in the gene (position 873,040 relative to the genome) is marked. (C–E) Antibody pulldown of m⁵C modifications in protein-coding genes from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003602#pgen-1003602-t003" target="_blank">Table 3</a>.</p