The long and short of microRNA.

MicroRNAs (miRNAs) are versatile regulators of gene expression in higher eukaryotes. In order to silence many different mRNAs in a precise manner, miRNA stability and efficacy is controlled by highly developed regulatory pathways and fine-tuning mechanisms both affecting miRNA processing and altering mature miRNA target specificity.

A DNA damage-induced phosphorylation circuit enhances Mec1ATR Ddc2ATRIP recruitment to Replication Protein A.

The cell cycle checkpoint kinase Mec1ATR and its integral partner Ddc2ATRIP are vital for the DNA damage and replication stress response. Mec1-Ddc2 "senses" single-stranded DNA (ssDNA) by being recruited to the ssDNA binding Replication Protein A (RPA) via Ddc2. In this study, we show that a DNA damage-induced phosphorylation circuit modulates checkpoint recruitment and function. We demonstrate that Ddc2-RPA interactions modulate the association between RPA and ssDNA and that Rfa1-phosphorylation aids in the further recruitment of Mec1-Ddc2. We also uncover an underappreciated role for Ddc2 phosphorylation that enhances its recruitment to RPA-ssDNA that is important for the DNA damage checkpoint in yeast. The crystal structure of a phosphorylated Ddc2 peptide in complex with its RPA interaction domain provides molecular details of how checkpoint recruitment is enhanced, which involves Zn2+. Using electron microscopy and structural modeling approaches, we propose that Mec1-Ddc2 complexes can form higher order assemblies with RPA when Ddc2 is phosphorylated. Together, our results provide insight into Mec1 recruitment and suggest that formation of supramolecular complexes of RPA and Mec1-Ddc2, modulated by phosphorylation, would allow for rapid clustering of damage foci to promote checkpoint signaling.

Dominant carnivore loss benefits native avian and invasive mammalian scavengers.

Scavenging by large carnivores is integral for ecosystem functioning by limiting the build-up of carrion and facilitating widespread energy flows. However, top carnivores have declined across the world, triggering trophic shifts within ecosystems. Here, we compare findings from previous work on predator decline against areas with recent native mammalian carnivore loss. Specifically, we investigate top-down control on utilization of experimentally placed carcasses by two mesoscavengers-the invasive feral cat and native forest raven. Ravens profited most from carnivore loss, scavenging for five times longer in the absence of native mammalian carnivores. Cats scavenged on half of all carcasses in the region without dominant native carnivores. This was eight times more than in areas where other carnivores were at high densities. All carcasses persisted longer than the three-week monitoring period in the absence of native mammalian carnivores, while in areas with high carnivore abundance, all carcasses were fully consumed. Our results reveal that top-carnivore loss amplifies impacts associated with carnivore decline-increased carcass persistence and carrion access for smaller scavengers. This suggests that even at low densities, native mammalian carnivores can fulfil their ecological functions, demonstrating the significance of global carnivore conservation and supporting management approaches, such as trophic rewilding.

Optimal translational termination requires C4 lysyl hydroxylation of eRF1.

Efficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis. We reveal that Jumonji domain-containing 4 (Jmjd4), a 2-oxoglutarate- and Fe(II)-dependent oxygenase, catalyzes carbon 4 (C4) lysyl hydroxylation of eRF1. This posttranslational modification takes place at an invariant lysine within the eRF1 NIKS motif and is required for optimal translational termination efficiency. These findings further highlight the role of 2-oxoglutarate/Fe(II) oxygenases in fundamental cellular processes and provide additional evidence that ensuring fidelity of protein translation is a major role of hydroxylation.

CLIC4/Arf6 Pathway.

RATIONALE: Increased expression of CLIC4 (chloride intracellular channel 4) is a feature of endothelial dysfunction in pulmonary arterial hypertension, but its role in disease pathology is not fully understood. OBJECTIVE: To identify CLIC4 effectors and evaluate strategies targeting CLIC4 signaling in pulmonary hypertension. METHODS AND RESULTS: Proteomic analysis of CLIC4-interacting proteins in human pulmonary artery endothelial cells identified regulators of endosomal trafficking, including Arf6 (ADP ribosylation factor 6) GTPase activating proteins and clathrin, while CLIC4 overexpression affected protein regulators of vesicular trafficking, lysosomal function, and inflammation. CLIC4 reduced BMPRII (bone morphogenetic protein receptor II) expression and signaling as a result of Arf6-mediated reduction in gyrating clathrin and increased lysosomal targeting of the receptor. BMPRII expression was restored by Arf6 siRNA, Arf inhibitor Sec7 inhibitor H3 (SecinH3), and inhibitors of clathrin-mediated endocytosis but was unaffected by chloride channel inhibitor, indanyloxyacetic acid 94 or Arf1 siRNA. The effects of CLIC4 on NF-κB (nuclear factor-kappa B), HIF (hypoxia-inducible factor), and angiogenic response were prevented by Arf6 siRNA and SecinH3. Sugen/hypoxia mice and monocrotaline rats showed elevated expression of CLIC4, activation of Arf6 and NF-κB, and reduced expression of BMPRII in the lung. These changes were established early during disease development. Lung endothelium-targeted delivery of CLIC4 siRNA or treatment with SecinH3 attenuated the disease, reduced CLIC4/Arf activation, and restored BMPRII expression in the lung. Endothelial colony-forming cells from idiopathic pulmonary hypertensive patients showed upregulation of CLIC4 expression and Arf6 activity, suggesting potential importance of this pathway in the human condition. CONCLUSIONS: Arf6 is a novel effector of CLIC4 and a new therapeutic target in pulmonary hypertension.

Roadkill islands: Carnivore extinction shifts seasonal use of roadside carrion by generalist avian scavenger.

Global road networks facilitate habitat modification and are integral to human expansion. Many animals, particularly scavengers, use roads as they provide a reliable source of food, such as carrion left after vehicle collisions. Tasmania is often cited as the 'roadkill capital of Australia', with the isolated offshore islands in the Bass Strait experiencing similar, if not higher, levels of roadkill. However, native mammalian predators on the islands are extirpated, meaning the remaining scavengers are likely to experience lower interference competition. In this study, we used a naturally occurring experiment to examine how the loss of mammalian carnivores within a community impacts roadside foraging behaviour by avian scavengers. We monitored the locations of roadkill and forest ravens Corvus tasmanicus, an abundant scavenger species, on eight road transects across the Tasmanian mainland (high scavenging competition) and the Bass Strait islands (low scavenging competition). We represented raven observations as one-dimensional point patterns, using hierarchical Bayesian models to investigate the dependence of raven spatial intensity on habitat, season, distance to roadkill and route location. We found that roadkill carcasses were a strong predictor of raven presence along road networks. The effect of roadkill was amplified on roads on the Bass Strait islands, where roadside carrion was a predictor of raven presence across the entire year. In contrast, ravens were more often associated with roadkill on Tasmanian mainland roads in the autumn, when other resources were low. This suggests that in the absence of competing mammalian scavengers, ravens choose to feed on roadside carrion throughout the year, even in seasons when other resources are available. This lack of competition could be disproportionately benefiting forest ravens, leading to augmented raven populations and changes to the vertebrate community structure. Our study provides evidence that scavengers modify their behaviour in response to reduced scavenger species diversity, potentially triggering trophic shifts and highlighting the importance of conserving or reintroducing carnivores within ecosystems.

Structural basis of homologous recombination.

Homologous recombination (HR) is a pathway to faithfully repair DNA double-strand breaks (DSBs). At the core of this pathway is a DNA recombinase, which, as a nucleoprotein filament on ssDNA, pairs with homologous DNA as a template to repair the damaged site. In eukaryotes Rad51 is the recombinase capable of carrying out essential steps including strand invasion, homology search on the sister chromatid and strand exchange. Importantly, a tightly regulated process involving many protein factors has evolved to ensure proper localisation of this DNA repair machinery and its correct timing within the cell cycle. Dysregulation of any of the proteins involved can result in unchecked DNA damage, leading to uncontrolled cell division and cancer. Indeed, many are tumour suppressors and are key targets in the development of new cancer therapies. Over the past 40 years, our structural and mechanistic understanding of homologous recombination has steadily increased with notable recent advancements due to the advances in single particle cryo electron microscopy. These have resulted in higher resolution structural models of the signalling proteins ATM (ataxia telangiectasia mutated), and ATR (ataxia telangiectasia and Rad3-related protein), along with various structures of Rad51. However, structural information of the other major players involved, such as BRCA1 (breast cancer type 1 susceptibility protein) and BRCA2 (breast cancer type 2 susceptibility protein), has been limited to crystal structures of isolated domains and low-resolution electron microscopy reconstructions of the full-length proteins. Here we summarise the current structural understanding of homologous recombination, focusing on key proteins in recruitment and signalling events as well as the mediators for the Rad51 recombinase.

Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain.

Kindlins co-activate integrins alongside talin. They possess, like talin, a FERM domain (4.1-erythrin-radixin-moiesin domain) comprising F0-F3 subdomains, but with a pleckstrin homology (PH) domain inserted in the F2 subdomain that enables membrane association. We present the crystal structure of murine kindlin-3 PH domain determined at a resolution of 2.23 Šand characterise its lipid binding using biophysical and computational approaches. Molecular dynamics simulations suggest flexibility in the PH domain loops connecting ß-strands forming the putative phosphatidylinositol phosphate (PtdInsP)-binding site. Simulations with PtdInsP-containing bilayers reveal that the PH domain associates with PtdInsP molecules mainly via the positively charged surface presented by the ß1-ß2 loop and that it binds with somewhat higher affinity to PtdIns(3,4,5)P3 compared with PtdIns(4,5)P2 Surface plasmon resonance (SPR) with lipid headgroups immobilised and the PH domain as an analyte indicate affinities of 300 µM for PtdIns(3,4,5)P3 and 1 mM for PtdIns(4,5)P2 In contrast, SPR studies with an immobilised PH domain and lipid nanodiscs as the analyte show affinities of 0.40 µM for PtdIns(3,4,5)P3 and no affinity for PtdIns(4,5)P2 when the inositol phosphate constitutes 5% of the total lipids (∼5 molecules per nanodisc). Reducing the PtdIns(3,4,5)P3 composition to 1% abolishes nanodisc binding to the PH domain, as does site-directed mutagenesis of two lysines within the ß1-ß2 loop. Binding of PtdIns(3,4,5)P3 by a canonical PH domain, Grp1, is not similarly influenced by SPR experimental design. These data suggest a role for PtdIns(3,4,5)P3 clustering in the binding of some PH domains and not others, highlighting the importance of lipid mobility and clustering for the biophysical assessment of protein-membrane interactions.

Structural plasticity of Cid1 provides a basis for its distributive RNA terminal uridylyl transferase activity.

Terminal uridylyl transferases (TUTs) are responsible for the post-transcriptional addition of uridyl residues to RNA 3' ends, leading in some cases to altered stability. The Schizosaccharomyces pombe TUT Cid1 is a model enzyme that has been characterized structurally at moderate resolution and provides insights into the larger and more complex mammalian TUTs, ZCCHC6 and ZCCHC11. Here, we report a higher resolution (1.74 Å) crystal structure of Cid1 that provides detailed evidence for uracil selection via the dynamic flipping of a single histidine residue. We also describe a novel closed conformation of the enzyme that may represent an intermediate stage in a proposed product ejection mechanism. The structural insights gained, combined with normal mode analysis and biochemical studies, demonstrate that the plasticity of Cid1, particularly about a hinge region (N164-N165), is essential for catalytic activity, and provide an explanation for its distributive uridylyl transferase activity. We propose a model clarifying observed differences between the in vitro apparently processive activity and in vivo distributive monouridylylation activity of Cid1. We suggest that modulating the flexibility of such enzymes-for example by the binding of protein co-factors-may allow them alternatively to add single or multiple uridyl residues to the 3' termini of RNA molecules.

Biophysical analysis of Kindlin-3 reveals an elongated conformation and maps integrin binding to the membrane-distal ß-subunit NPXY motif.

Kindlin-3, a 75-kDa protein, has been shown to be critical for hemostasis, immunity, and bone metabolism via its role in integrin activation. The Kindlin family is hallmarked by a FERM domain comprised of F1, F2, and F3 subdomains together with an N-terminal F0 domain and a pleckstrin homology domain inserted in the F2 domain. Recombinant Kindlin-3 was cloned, expressed, and purified, and its domain organization was studied by x-ray scattering and other techniques to reveal an extended conformation. This unusual elongated structure is similar to that found in the paralogue Talin head domain. Analytical ultracentrifugation experiments indicated that Kindlin-3 forms a ternary complex with the Talin and ß-integrin cytoplasmic tails. NMR showed that Kindlin-3 specifically recognizes the membrane-distal tail NPXY motif in both the ß(1A) and ß(1D) isoforms, although the interaction is stronger with ß(1A). An upstream Ser/Thr cluster in the tails also plays a critical role. Overall these data support current biological, clinical, and mutational data on Kindlin-3/ß-tail binding and provide novel insights into the overall conformation and interactions of Kindlin-3.

Structural and functional characterization of the kindlin-1 pleckstrin homology domain.

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin ß-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P(3); K(D) ∼100 µM) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P(3) as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P(2) on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P(3) is affected by the presence of PO(4) ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species.

Phosphoregulation of the checkpoint kinase Mec1ATR.

Yeast Mec1, and its mammalian ortholog, Ataxia-Telangiectasia and Rad3-related, are giant protein kinases central to replication stress and double strand DNA break repair. Mec1ATR, in complex with Ddc2ATRIP, is a 'sensor' of single stranded DNA, and phosphorylates numerous cell cycle and DNA repair factors to enforce cell cycle arrest and facilitate repair. Over the last several years, new techniques - particularly in structural biology - have provided molecular mechanisms for Mec1ATR function. It is becoming increasingly clear how post-translational modification of Mec1ATR and its interaction partners modulates the DNA damage checkpoint. In this review, we summarise the most recent work unravelling Mec1ATR function in the DNA damage checkpoint and provide a molecular context for its regulation by phosphorylation.

Spatial pattern analysis of line-segment data in ecology.

The spatial analysis of linear features (lines and curves) is a challenging and rarely attempted problem in ecology. Existing methods are typically expressed in abstract mathematical formalism, making it difficult to assess their relevance and transferability into an ecological setting. We introduce a set of concrete and accessible methods to analyze the spatial patterning of line-segment data. The methods include Monte Carlo techniques based on a new generalization of Ripley's K -function and a class of line-segment processes that can be used to specify parametric models: parameters are estimated using maximum likelihood and models compared using information-theoretic principles. We apply the new methods to fallen tree (dead log) data collected from two 1-ha Australian tall eucalypt forest plots. Our results show that the spatial pattern of the fallen logs is best explained by plot-level spatial heterogeneity in combination with a slope-dependent nonuniform distribution of fallen-log orientations. These methods are of a general nature and are applicable to any line-segment data. In the context of forest ecology, the integration of fallen logs as linear structural features in a landscape with the point locations of living trees, and a quantification of their interactions, can yield new insights into the functional and structural role of tree fall in forest communities and their enduring post-mortem ecological legacy as spatially distributed decomposing logs.

Regulation of DNA break repair by RNA.

Genomic stability is critical for cell survival and its effective repair when damaged is a vital process for preserving genetic information. Failure to correctly repair the genome can lead to the accumulation of mutations that ultimately drives carcinogenesis. Life has evolved sophisticated surveillance, repair pathways, and mechanisms to recognize and mend genomic lesions to preserve its integrity. Many of these pathways involve a cascade of protein effectors that act to identify the type of damage, such as double-strand (ds) DNA breaks, propagate the damage signal, and recruit an array of other protein factors to resolve the damage without loss of genetic information. It is now becoming increasingly clear that there are a number of RNA processing factors, such as the transcriptional machinery, and microRNA biogenesis components, as well as RNA itself, that facilitate the repair of DNA damage. Here, some of the recent work unravelling the role of RNA in the DNA Damage Response (DDR), in particular the dsDNA break repair pathway, will be reviewed.

Parsimonious model selection using information theory: a modified selection rule.

Information-theoretic approaches to model selection, such as Akaike's information criterion (AIC) and cross validation, provide a rigorous framework to select among candidate hypotheses in ecology, yet the persistent concern of overfitting undermines the interpretation of inferred processes. A common misconception is that overfitting is due to the choice of criterion or model score, despite research demonstrating that selection uncertainty associated with score estimation is the predominant influence. Here we introduce a novel selection rule that identifies a parsimonious model by directly accounting for estimation uncertainty, while still retaining an information-theoretic interpretation. The new rule, which is a modification of the existing one-standard-error rule, mitigates overfitting and reduces the likelihood that spurious effects will be included in the selected model, thereby improving its inferential properties. We present the rule and illustrative examples in the context of maximum-likelihood estimation and Kullback-Leibler discrepancy, although the rule is applicable in a more general setting, including Bayesian model selection and other types of discrepancy.

Mechanism of auto-inhibition and activation of Mec1ATR checkpoint kinase.

In response to DNA damage or replication fork stalling, the basal activity of Mec1ATR is stimulated in a cell-cycle-dependent manner, leading to cell-cycle arrest and the promotion of DNA repair. Mec1ATR dysfunction leads to cell death in yeast and causes chromosome instability and embryonic lethality in mammals. Thus, ATR is a major target for cancer therapies in homologous recombination-deficient cancers. Here we identify a single mutation in Mec1, conserved in ATR, that results in constitutive activity. Using cryo-electron microscopy, we determine the structures of this constitutively active form (Mec1(F2244L)-Ddc2) at 2.8 Å and the wild type at 3.8 Å, both in complex with Mg2+-AMP-PNP. These structures yield a near-complete atomic model for Mec1-Ddc2 and uncover the molecular basis for low basal activity and the conformational changes required for activation. Combined with biochemical and genetic data, we discover key regulatory regions and propose a Mec1 activation mechanism.

Structures and regulations of ATM and ATR, master kinases in genome integrity.

Homologous recombination (HR) is a faithful repair mechanism for double stranded DNA breaks. Two highly homologous master kinases, the tumour suppressors ATM and ATR (Tel1 and Mec1 in yeast), coordinate cell cycle progression with repair during HR. Despite their importance, our molecular understanding of these apical coordinators has been limited, in part due to their large sizes. With the recent development in cryo-electron microscopy, significant advances have been made in structural characterisation of these proteins in the last two years. These structures, combined with new biochemical studies, now provide a more detailed understanding of how a low basal activity is maintained and how activation may occur. In this review, we summarize recent advances in the structural and molecular understanding of these key components in HR, compare the common and distinct features of these kinases and suggest aspects of structural components that are likely to be involved in regulating its activity.

Cryo-EM Structure of Nucleotide-Bound Tel1ATM Unravels the Molecular Basis of Inhibition and Structural Rationale for Disease-Associated Mutations.

Yeast Tel1 and its highly conserved human ortholog ataxia-telangiectasia mutated (ATM) are large protein kinases central to the maintenance of genome integrity. Mutations in ATM are found in ataxia-telangiectasia (A-T) patients and ATM is one of the most frequently mutated genes in many cancers. Using cryoelectron microscopy, we present the structure of Tel1 in a nucleotide-bound state. Our structure reveals molecular details of key residues surrounding the nucleotide binding site and provides a structural and molecular basis for its intrinsically low basal activity. We show that the catalytic residues are in a productive conformation for catalysis, but the phosphatidylinositol 3-kinase-related kinase (PIKK) regulatory domain insert restricts peptide substrate access and the N-lobe is in an open conformation, thus explaining the requirement for Tel1 activation. Structural comparisons with other PIKKs suggest a conserved and common allosteric activation mechanism. Our work also provides a structural rationale for many mutations found in A-T and cancer.

A structural and dynamic model for the assembly of Replication Protein A on single-stranded DNA.

Replication Protein A (RPA), the major eukaryotic single stranded DNA-binding protein, binds to exposed ssDNA to protect it from nucleases, participates in a myriad of nucleic acid transactions and coordinates the recruitment of other important players. RPA is a heterotrimer and coats long stretches of single-stranded DNA (ssDNA). The precise molecular architecture of the RPA subunits and its DNA binding domains (DBDs) during assembly is poorly understood. Using cryo electron microscopy we obtained a 3D reconstruction of the RPA trimerisation core bound with ssDNA (∼55 kDa) at ∼4.7 Šresolution and a dimeric RPA assembly on ssDNA. FRET-based solution studies reveal dynamic rearrangements of DBDs during coordinated RPA binding and this activity is regulated by phosphorylation at S178 in RPA70. We present a structural model on how dynamic DBDs promote the cooperative assembly of multiple RPAs on long ssDNA.

Improved crystallization and diffraction of caffeine-induced death suppressor protein 1 (Cid1).

The post-transcriptional addition of uridines to the 3'-end of RNAs is an important regulatory process that is critical for coding and noncoding RNA stability. In fission yeast and metazoans this untemplated 3'-uridylylation is catalysed by a single family of terminal uridylyltransferases (TUTs) whose members are adapted to specific RNA targets. In Schizosaccharomyces pombe the TUT Cid1 is responsible for the uridylylation of polyadenylated mRNAs, targeting them for destruction. In metazoans, the Cid1 orthologues ZCCHC6 and ZCCHC11 uridylate histone mRNAs, targeting them for degradation, but also uridylate microRNAs, altering their maturation. Cid1 has been studied as a model TUT that has provided insights into the larger and more complex metazoan enzyme system. In this paper, two strategies are described that led to improvements both in the crystallogenesis of Cid1 and in the resolution of diffraction by ∼1.5 Å. These advances have allowed high-resolution crystallographic studies of this TUT system to be initiated.