Heat Stress Enhances the Accumulation of Polyadenylated Mitochondrial Transcripts in Arabidopsis thaliana

Background: Polyadenylation of RNA has a decisive influence on RNA stability. Depending on the organisms or subcellular compartment, it either enhances transcript stability or targets RNAs for degradation. In plant mitochondria, polyadenylation promotes RNA degradation, and polyadenylated mitochondrial transcripts are therefore widely considered to be rare and unstable. We followed up a surprising observation that a large number of mitochondrial transcripts are detectable in microarray experiments that used poly(A)-specific RNA probes, and that these transcript levels are significantly enhanced after heat treatment. Methodology/Principal Findings: As the Columbia genome contains a complete set of mitochondrial genes, we had to identify polymorphisms to differentiate between nuclear and mitochondrial copies of a mitochondrial transcript. We found that the affected transcripts were uncapped transcripts of mitochondrial origin, which were polyadenylated at multiple sites within their 39region. Heat-induced enhancement of these transcripts was quickly restored during a short recovery period. Conclusions/Significance: Our results show that polyadenylated transcripts of mitochondrial origin are more stable than previously suggested, and that their steady-state levels can even be significantly enhanced under certain conditions. As many microarrays contain mitochondrial probes, due to the frequent transfer of mitochondrial genes into the genome

Dynamic Interaction of cBid with Detergents, Liposomes and Mitochondria

The BH3-only protein Bid plays a key role in the induction of mitochondrial apoptosis, but its mechanism of action is still not completely understood. Here we studied the two main activation events of Bid: Caspase-8 cleavage and interaction with the membrane bilayer. We found a striking reversible behaviour of the dissociation-association events between the Bid fragments p15 and p7. Caspase-8 cleavage does not induce per se separation of the two Bid fragments, which remain in a stable complex resembling the full length Bid. Detergents trigger a complete dissociation, which can be fully reversed by detergent removal in a range of protein concentrations from 100 µM down to 500 nM. Incubation of cBid with cardiolipin-containing liposomes leads to partial dissociation of the complex. Only p15 (tBid) fragments are found at the membrane, while p7 shows no tendency to interact with the bilayer, but complete removal of p7 strongly increases the propensity of tBid to become membrane-associated. Despite the striking structural similarities of inactive Bid and Bax, Bid does not form oligomers and reacts differently in the presence of detergents and membranes, highlighting clear differences in the modes of action of the two proteins. The partial dissociation of cBid triggered by the membrane is suggested to depend on the strong and specific interaction between p15 and p7. The reversible disassembly and re-assembly of the cBid molecules at the membrane was as well proven by EPR using spin labeled cBid in the presence of isolated mitochondria. The observed dynamic dissociation of the two Bid fragments could allow the assistance to the pore-forming Bax to occur repeatedly and may explain the proposed “hit-and-run" mode of action of Bid at the bilayer

COMRADES determines in vivo RNA structures and interactions.

The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs.This work was supported by Cancer Research UK (C13474/A18583, C6946/A14492) and the Wellcome Trust (104640/Z/14/Z, 092096/Z/10/Z) to E.A.M. O.Z. was supported by the Human Frontier Science Program (HFSP, LT000558/2015), the European Molecular Biology Organization (EMBO, ALTF1622-2014), and the Blavatnik Family Foundation postdoctoral fellowship. G.K. and M.G. were supported by Wellcome Trust grant 207507 and UK Medical Research Council. A.T.L.L. and J.C.M. were supported by core funding from Cancer Research UK (award no. 17197 to JCM). J.C.M was also supported by core funding from EMBL. I.G. and L.W.M. were supported by the Wellcome Trust Senior Fellowship in Basic Biomedical Science to I.G. (207498/Z/17/Z). I.J.M., L.F.G. and J.S.-G. were supported by grants R01GM104475 and R01GM115649 from NIGMS. C.K.K was supported by City University of Hong Kong Projects 9610363 and 7200520, Croucher Foundation Project 9500030 and Hong Kong RGC Projects 9048103 and 9054020. C.-F.Q. was supported by the NSFC Excellent Young Scientist Fund 81522025 and the Newton Advanced Fellowship from the Academy of Medical Sciences, UK

Genomic fluidity: an integrative view of gene diversity within microbial populations

<p>Abstract</p> <p>Background</p> <p>The dual concepts of pan and core genomes have been widely adopted as means to assess the distribution of gene families within microbial species and genera. The core genome is the set of genes shared by a group of organisms; the pan genome is the set of all genes seen in any of these organisms. A variety of methods have provided drastically different estimates of the sizes of pan and core genomes from sequenced representatives of the same groups of bacteria.</p> <p>Results</p> <p>We use a combination of mathematical, statistical and computational methods to show that current predictions of pan and core genome sizes may have no correspondence to true values. Pan and core genome size estimates are problematic because they depend on the estimation of the occurrence of rare genes and genomes, respectively, which are difficult to estimate precisely because they are rare. Instead, we introduce and evaluate a robust metric - genomic fluidity - to categorize the gene-level similarity among groups of sequenced isolates. Genomic fluidity is a measure of the dissimilarity of genomes evaluated at the gene level.</p> <p>Conclusions</p> <p>The genomic fluidity of a population can be estimated accurately given a small number of sequenced genomes. Further, the genomic fluidity of groups of organisms can be compared robustly despite variation in algorithms used to identify genes and their homologs. As such, we recommend that genomic fluidity be used in place of pan and core genome size estimates when assessing gene diversity within genomes of a species or a group of closely related organisms.</p

The aged niche disrupts muscle stem cell quiescence

SUMMARY The niche is a conserved regulator of stem cell quiescence and function. During aging, stem cell function declines. To what extent and by which means age-related changes within the niche contribute to this phenomenon are unknown. We demonstrate that the aged muscle stem cell niche, the muscle fiber, expresses FGF2 under homeostatic conditions, driving a subset of satellite cells to break quiescence and lose self-renewing capacity. We show that relatively dormant aged satellite cells robustly express Sprouty1 (spry1), an inhibitor of FGF signalling. Increasing FGF signalling in aged satellite cells under homeostatic conditions by removing spry1, results in the loss of quiescence, satellite cell depletion and diminished regenerative capacity. Conversely, reducing niche-derived FGF activity through inhibition of FGFR1 signalling or overexpression of spry1 in satellite cells prevents their depletion. These experiments identify an age-dependent change in the stem cell niche that directly influences stem cell quiescence and function

Heterozygous Yeast Deletion Collection Screens Reveal Essential Targets of Hsp90

Hsp90 is an essential eukaryotic chaperone with a role in folding specific “client” proteins such as kinases and hormone receptors. Previously performed homozygous diploid yeast deletion collection screens uncovered broad requirements for Hsp90 in cellular transport and cell cycle progression. These screens also revealed that the requisite cellular functions of Hsp90 change with growth temperature. We present here for the first time the results of heterozygous deletion collection screens conducted at the hypothermic stress temperature of 15°C. Extensive bioinformatic analyses were performed on the resulting data in combination with data from homozygous and heterozygous screens previously conducted at normal (30°C) and hyperthermic stress (37°C) growth temperatures. Our resulting meta-analysis uncovered extensive connections between Hsp90 and (1) general transcription, (2) ribosome biogenesis and (3) GTP binding proteins. Predictions from bioinformatic analyses were tested experimentally, supporting a role for Hsp90 in ribosome stability. Importantly, the integrated analysis of the 15°C heterozygous deletion pool screen with previously conducted 30°C and 37°C screens allows for essential genetic targets of Hsp90 to emerge. Altogether, these novel contributions enable a more complete picture of essential Hsp90 functions

The N-Terminus of GalE Induces tmRNA Activity in Escherichia coli

BACKGROUND: The tmRNA quality control system recognizes stalled translation complexes and facilitates ribosome recycling in a process termed 'ribosome rescue'. During ribosome rescue, nascent chains are tagged with the tmRNA-encoded SsrA peptide, which targets tagged proteins for degradation. In Escherichia coli, tmRNA rescues ribosomes arrested on truncated messages, as well as ribosomes that are paused during elongation and termination. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a new translational pausing determinant that leads to SsrA peptide tagging of the E. coli GalE protein (UDP-galactose 4-epimerase). GalE chains are tagged at more than 150 sites, primarily within distinct clusters throughout the C-terminal domain. These tagging sites do not correspond to rare codon clusters and synonymous recoding of the galE gene had little effect on tagging. Moreover, tagging was largely unaffected by perturbations that either stabilize or destabilize the galE transcript. Examination of GalE-thioredoxin (TrxA) fusion proteins showed that the GalE C-terminal domain is no longer tagged when fused to an N-terminal TrxA domain. Conversely, the N-terminus of GalE induced tagging within the fused C-terminal TrxA domain. CONCLUSIONS/SIGNIFICANCE: These findings suggest that translation of the GalE N-terminus induces subsequent tagging of the C-terminal domain. We propose that co-translational maturation of the GalE N-terminal domain influences ribosome pausing and subsequent tmRNA activity

MicroRNA-sequence profiling reveals novel osmoregulatory microRNA expression patterns in catadromous eel anguilla marmorata

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that regulate gene expression by post-transcriptional repression of mRNAs. Recently, several miRNAs have been confirmed to execute directly or indirectly osmoregulatory functions in fish via translational control. In order to clarify whether miRNAs play relevant roles in the osmoregulation of Anguilla marmorata, three sRNA libraries of A. marmorata during adjusting to three various salinities were sequenced by Illumina sRNA deep sequencing methods. Totally 11,339,168, 11,958,406 and 12,568,964 clear reads were obtained from 3 different libraries, respectively. Meanwhile, 34 conserved miRNAs and 613 novel miRNAs were identified using the sequence data. MiR-10b-5p, miR-181a, miR-26a-5p, miR-30d and miR-99a-5p were dominantly expressed in eels at three salinities. Totally 29 mature miRNAs were significantly up-regulated, while 72 mature miRNAs were significantly down-regulated in brackish water (10‰ salinity) compared with fresh water (0‰ salinity); 24 mature miRNAs were significantly up-regulated, while 54 mature miRNAs were significantly down-regulated in sea water (25‰ salinity) compared with fresh water. Similarly, 24 mature miRNAs were significantly up-regulated, while 45 mature miRNAs were significantly down-regulated in sea water compared with brackish water. The expression patterns of 12 dominantly expressed miRNAs were analyzed at different time points when the eels transferred from fresh water to brackish water or to sea water. These miRNAs showed differential expression patterns in eels at distinct salinities. Interestingly, miR-122, miR-140-3p and miR-10b-5p demonstrated osmoregulatory effects in certain salinities. In addition, the identification and characterization of differentially expressed miRNAs at different salinities can clarify the osmoregulatory roles of miRNAs, which will shed lights for future studies on osmoregulation in fish

The great screen anomaly—a new frontier in product discovery through functional metagenomics

Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product

A novel μCT analysis reveals different responses of bioerosion and secondary accretion to environmental variability

Corals build reefs through accretion of calcium carbonate (CaCO3) skeletons, but net reef growth also depends on bioerosion by grazers and borers and on secondary calcification by crustose coralline algae and other calcifying invertebrates. However, traditional field methods for quantifying secondary accretion and bioerosion confound both processes, do not measure them on the same time-scale, or are restricted to 2D methods. In a prior study, we compared multiple environmental drivers of net erosion using pre- and post-deployment micro-computed tomography scans (μCT; calculated as the % change in volume of experimental CaCO3 blocks) and found a shift from net accretion to net erosion with increasing ocean acidity. Here, we present a novel μCT method and detail a procedure that aligns and digitally subtracts pre- and post-deployment μCT scans and measures the simultaneous response of secondary accretion and bioerosion on blocks exposed to the same environmental variation over the same time-scale. We tested our method on a dataset from a prior study and show that it can be used to uncover information previously unattainable using traditional methods. We demonstrated that secondary accretion and bioerosion are driven by different environmental parameters, bioerosion is more sensitive to ocean acidity than secondary accretion, and net erosion is driven more by changes in bioerosion than secondary accretion