Structural basis of RNA polymerase inhibition by viral and host factors

The inhibition of RNA polymerases activity plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural basis underlying RNAP inhibition in archaea. The Acidianus two-tailed virus (ATV) encodes the RIP factor that binds to the inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus (STIV) induces the expression of the host factor TFS4, which binds in the RNAP secondary channel similarly to eukaryotic transcript cleavage factors. In contrast to RIP, TFS4 binding allosterically induces a widening of the DNA binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.

The Detection and Bioinformatic Analysis of Alternative 3′ UTR Isoforms as Potential Cancer Biomarkers

Alternative transcript cleavage and polyadenylation is linked to cancer cell transformation, proliferation and outcome. This has led researchers to develop methods to detect and bioinformatically analyse alternative polyadenylation as potential cancer biomarkers. If incorporated into standard prognostic measures such as gene expression and clinical parameters, these could advance cancer prognostic testing and possibly guide therapy. In this review, we focus on the existing methodologies, both experimental and computational, that have been applied to support the use of alternative polyadenylation as cancer biomarkers.

Conservation and turnover of miRNAs and their highly complementary targets in early branching animals

MicroRNAs (miRNAs) are crucial post-transcriptional regulators that have been extensively studied in Bilateria, a group comprising the majority of extant animals, where more than 30 conserved miRNA families have been identified. By contrast, bilaterian miRNA targets are largely not conserved. Cnidaria is the sister group to Bilateria and thus provides a unique opportunity for comparative studies. Strikingly, like their plant counterparts, cnidarian miRNAs have been shown to predominantly have highly complementary targets leading to transcript cleavage by Argonaute proteins. Here, we assess the conservation of miRNAs and their targets by small RNA sequencing followed by miRNA target prediction in eight species of Anthozoa (sea anemones and corals), the earliest-branching cnidarian class. We uncover dozens of novel miRNAs but only a few conserved ones. Further, given their high complementarity, we were able to computationally identify miRNA targets in each species. Besides evidence for conservation of specific miRNA target sites, which are maintained between sea anemones and stony corals across 500 Myr of evolution, we also find indications for convergent evolution of target regulation by different miRNAs. Our data indicate that cnidarians have only few conserved miRNAs and corresponding targets, despite their high complementarity, suggesting a high evolutionary turnover.

Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

ABSTRACTPromoter-proximal pausing regulates expression of many eukaryotic genes and serves as checkpoints for assembly of elongation/splicing machinery. Little is known how broadly the pausing is employed in transcriptional regulation in bacteria. We applied NET-seq combined with RNase I footprinting for genome-wide analysis of σ70-dependent transcription pauses in Escherichia coli. Many E. coli genes appear to contain clusters of strong backtracked pauses at 10-20-bp distance from the transcription start site caused by retention of σ70 subunit in RNA polymerase. The pauses in 10-15-bp register of the promoter are dictated by binding of σ70 to canonical −10 element, 6-7 nt spacer and “YR+1Y” motif centered at transcription start site all characteristic for strong E. coli promoters. The promoters for the pauses in 16-20-bp register contain an additional −10-like sequence positioned on the same face of the DNA duplex as the original −10 element suggesting that σ70 hopping was responsible for these pauses. Our in vitro analysis reveals that RNA polymerase backtracking and DNA scrunching are involved in these pauses that are relieved by Gre transcript cleavage factors. The genes coding for transcription factors are enriched in these pauses suggesting that σ70 and Gre proteins regulate transcription in response to changing environmental cues.

Computational Identification and Comparative Analysis of Conserved miRNAs and Their Putative Target Genes in the Juglans regia and J. microcarpa Genomes

MicroRNAs (miRNAs) are important factors for the post-transcriptional regulation of protein-coding genes in plants and animals. They are discovered either by sequencing small RNAs or computationally. We employed a sequence-homology-based computational approach to identify conserved miRNAs and their target genes in Persian (English) walnut, Juglans regia, and its North American wild relative, J. microcarpa. A total of 119 miRNA precursors (pre-miRNAs) were detected in the J. regia genome and 121 in the J. microcarpa genome and miRNA target genes were predicted and their functional annotations were performed in both genomes. In the J. regia genome, 325 different genes were targets; 87.08% were regulated by transcript cleavage and 12.92% by translation repression. In the J. microcarpa genome, 316 different genes were targets; 88.92% were regulated by transcript cleavage and 11.08% were regulated by translation repression. Totals of 1.3% and 2.0% of all resistance gene analogues (RGA) and 2.7% and 2.6% of all transcription factors (TFs) were regulated by miRNAs in the J. regia and J. microcarpa genomes, respectively. Juglans genomes evolved by a whole genome duplication (WGD) and consist of eight pairs of fractionated homoeologous chromosomes. Within each pair, the chromosome that has more genes with greater average transcription also harbors more pre-miRNAs and more target genes than its homoeologue. While only minor differences were detected in pre-miRNAs between the J. regia and J. microcarpa genomes, about one-third of the pre-miRNA loci were not conserved between homoeologous chromosome within each genome. Pre-miRNA and their corresponding target genes showed a tendency to be collocated within a subgenome.

Global analysis of SBP gene family in Brachypodium distachyon reveals its association with spike development

SQUAMOSA-promoter binding like proteins (SBPs/SPLs) are plant specific transcription factors targeted by miR156 and involved in various biological pathways, playing multi-faceted developmental roles. This gene family is not well characterized in Brachypodium. We identified a total of 18 SBP genes in B.distachyon genome. Phylogenetic analysis revealed that SBP gene family in Brachypodium expanded through large scale duplication. A total of 10 BdSBP genes were identified as targets of miR156. Transcript cleavage analysis of selected BdSBPs by miR156 confirmed their antagonistic connection. Alternative splicing was observed playing an important role in BdSBPs and miR156 interaction. Characterization of T-DNA Bdsbp9 mutant showed reduced plant growth and spike length, reflecting its involvement in the spike development. Expression of a majority of BdSBPs elevated during spikelet initiation. Specifically, BdSBP1 and BdSBP3 differentially expressed in response to vernalization. Differential transcript abundance of BdSBP1,BdSBP3,BdSBP8,BdSBP9,BdSBP14,BdSBP18 and BdSBP23 genes was observed during the spike development under high temperature. Co-expression network, protein–protein interaction and biological pathway analysis indicate that BdSBP genes mainly regulate transcription, hormone, RNA and transport pathways. Our work reveals the multi-layered control of SBP genes and demonstrates their association with spike development and temperature sensitivity in Brachypodium.

Insights into the global effect on Staphylococcus aureus growth arrest by induction of the endoribonuclease MazF toxin

A crucial bacterial strategy to avoid killing by antibiotics is to enter a growth arrested state, yet the molecular mechanisms behind this process remain elusive. The conditional overexpression of mazF, the endoribonuclease toxin of the MazEF toxin–antitoxin system in Staphylococcus aureus, is one approach to induce bacterial growth arrest, but its targets remain largely unknown. We used overexpression of mazF and high-throughput sequence analysis following the exact mapping of non-phosphorylated transcriptome ends (nEMOTE) technique to reveal in vivo toxin cleavage sites on a global scale. We obtained a catalogue of MazF cleavage sites and unearthed an extended MazF cleavage specificity that goes beyond the previously reported one. We correlated transcript cleavage and abundance in a global transcriptomic profiling during mazF overexpression. We observed that MazF affects RNA molecules involved in ribosome biogenesis, cell wall synthesis, cell division and RNA turnover and thus deliver a plausible explanation for how mazF overexpression induces stasis. We hypothesize that autoregulation of MazF occurs by directly modulating the MazEF operon, such as the rsbUVW genes that regulate the sigma factor SigB, including an observed cleavage site on the MazF mRNA that would ultimately play a role in entry and exit from bacterial stasis.

tasiR-ARFs Production and Target Regulation during In Vitro Maize Plant Regeneration

During in vitro maize plant regeneration somatic cells change their normal fate and undergo restructuring to generate pluripotent cells able to originate new plants. Auxins are essential to achieve such plasticity. Their physiological effects are mediated by auxin response factors (ARFs) that bind auxin responsive elements within gene promoters. Small trans-acting (ta)-siRNAs, originated from miR390-guided TAS3 primary transcript cleavage, target ARF3/4 class (tasiR-ARFs). Here we found that TAS3b precursor as well as derived tasiR-ARFbD5 and tasiR-ARFbD6 display significantly lower levels in non-embryogenic callus (NEC), while TAS3g, miR390 and tasiR-ARFg are more abundant in the same tissue. However, Argonaute (AGO7) and leafbladeless 1 (LBLl) required for tasiR-ARF biogenesis showed significantly higher transcript levels in EC suggesting limited tasiR-ARF biogenesis in NEC. The five maize ARFs targeted by tasiR-ARFs were also significantly enriched in EC and accompanied by higher auxin accumulation with punctuate patterns in this tissue. At hormone half-reduction and photoperiod implementation, plant regeneration initiated from EC with transient TAS3g, miR390 and tasiR-ARFg increase. Upon complete hormone depletion, TAS3b became abundant and derived tasiR-ARFs gradually increased at further regeneration stages. ZmARF transcripts targeted by tasiR-ARFs, as well as AGO7 and LBL1 showed significantly lower levels during regeneration than in EC. These results indicate a dynamic tasiR-ARF mediated regulation throughout maize in vitro plant regeneration.

Post-transcriptional regulation of several biological processes involved in latex production in Hevea brasiliensis

Background Small RNAs modulate plant gene expression at both the transcriptional and post-transcriptional level, mostly through the induction of either targeted DNA methylation or transcript cleavage, respectively. Small RNA networks are involved in specific plant developmental processes, in signaling pathways triggered by various abiotic stresses and in interactions between the plant and viral and non-viral pathogens. They are also involved in silencing maintenance of transposable elements and endogenous viral elements. Alteration in small RNA production in response to various environmental stresses can affect all the above-mentioned processes. In rubber trees, changes observed in small RNA populations in response to trees affected by tapping panel dryness, in comparison to healthy ones, suggest a shift from a transcriptional to a post-transcriptional regulatory pathway. This is the first attempt to characterise small RNAs involved in post-transcriptional silencing and their target transcripts in Hevea. Methods Genes producing microRNAs (MIR genes) and loci producing trans-activated small interfering RNA (ta-siRNA) were identified in the clone PB 260 re-sequenced genome. Degradome libraries were constructed with a pool of total RNA from six different Hevea tissues in stressed and non-stressed plants. The analysis of cleaved RNA data, associated with genomics and transcriptomics data, led to the identification of transcripts that are affected by 20–22 nt small RNA-mediated post-transcriptional regulation. A detailed analysis was carried out on gene families related to latex production and in response to growth regulators. Results Compared to other tissues, latex cells had a higher proportion of transcript cleavage activity mediated by miRNAs and ta-siRNAs. Post-transcriptional regulation was also observed at each step of the natural rubber biosynthesis pathway. Among the genes involved in the miRNA biogenesis pathway, our analyses showed that all of them are expressed in latex. Using phylogenetic analyses, we show that both the Argonaute and Dicer-like gene families recently underwent expansion. Overall, our study underlines the fact that important biological pathways, including hormonal signalling and rubber biosynthesis, are subject to post-transcriptional silencing in laticifers.