Max Perutz Labs

Large phylogenomics data sets require fast tree inference methods, especially for maximum-likelihood (ML) phylogenies. Fast programs exist, but due to inherent heuristics to find optimal trees, it is not clear whether the best tree is found. Thus, there is need for additional approaches that employ different search strategies to find ML trees and that are at the same time as fast as currently available ML programs. We show that a combination of hill-climbing approaches and a stochastic perturbation method can be time-efficiently implemented. If we allow the same CPU time as RAxML and PhyML, then our software IQ-TREE found higher likelihoods between 62.2% and 87.1% of the studied alignments, thus efficiently exploring the tree-space. If we use the IQ-TREE stopping rule, RAxML and PhyML are faster in 75.7% and 47.1% of the DNA alignments and 42.2% and 100% of the protein alignments, respectively. However, the range of obtaining higher likelihoods with IQ-TREE improves to 73.3-97.1%. IQ-TREE is freely available at http://www.cibiv.at/software/iqtree.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

IQ-TREE (http://www.iqtree.org, last accessed February 6, 2020) is a user-friendly and widely used software package for phylogenetic inference using maximum likelihood. Since the release of version 1 in 2014, we have continuously expanded IQ-TREE to integrate a plethora of new models of sequence evolution and efficient computational approaches of phylogenetic inference to deal with genomic data. Here, we describe notable features of IQ-TREE version 2 and highlight the key advantages over other software.

Several reactive oxygen species (ROS) are continuously produced in plants as byproducts of aerobic metabolism. Depending on the nature of the ROS species, some are highly toxic and rapidly detoxified by various cellular enzymatic and nonenzymatic mechanisms. Whereas plants are surfeited with mechanisms to combat increased ROS levels during abiotic stress conditions, in other circumstances plants appear to purposefully generate ROS as signaling molecules to control various processes including pathogen defense, programmed cell death, and stomatal behavior. This review describes the mechanisms of ROS generation and removal in plants during development and under biotic and abiotic stress conditions. New insights into the complexity and roles that ROS play in plants have come from genetic analyses of ROS detoxifying and signaling mutants. Considering recent ROS-induced genome-wide expression analyses, the possible functions and mechanisms for ROS sensing and signaling in plants are compared with those in animals and yeast.

The standard bootstrap (SBS), despite being computationally intensive, is widely used in maximum likelihood phylogenetic analyses. We recently proposed the ultrafast bootstrap approximation (UFBoot) to reduce computing time while achieving more unbiased branch supports than SBS under mild model violations. UFBoot has been steadily adopted as an efficient alternative to SBS and other bootstrap approaches. Here, we present UFBoot2, which substantially accelerates UFBoot and reduces the risk of overestimating branch supports due to polytomies or severe model violations. Additionally, UFBoot2 provides suitable bootstrap resampling strategies for phylogenomic data. UFBoot2 is 778 times (median) faster than SBS and 8.4 times (median) faster than RAxML rapid bootstrap on tested data sets. UFBoot2 is implemented in the IQ-TREE software package version 1.6 and freely available at http://www.iqtree.org.

In 2008 we published the fi rst set of guidelines for standardiz- ing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the fi eld. Our knowledge base and relevant new tech- nologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in differ- ent organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between meas urements that monitor the num- bers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the com- plete process including the amoun t and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accu mulation must be differentiated from stimuli that increas e autophagic activity, de fi ned as increased autophagy induction coupled with in creased delivery to, and degra- dation within, lysosomes (in most higher eukaryotes and some pro- tists such as Dictyostelium ) or the vacuole (in plants and fungi). In otherwords,itisespeciallyimportantthatinvestigatorsnewtothe fi eld understand that the appearance of more autophagosomes does not necessarily equate with mo re autophagy. In fact, in many cases, autophagosomes accumu late because of a block in traf fi cking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may re fl ect a reductionindegradativeactivity .Itisworthemphasizingherethat lysosomal digestion is a stage of au tophagy and evaluating its com- petence is a crucial part of the evaluation of autophagic fl ux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appro- priate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no indi- vidual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy- related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a speci fi c marker for an autophagic process. In these guidelines, we con- sider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the fi eld

This article presents W-IQ-TREE, an intuitive and user-friendly web interface and server for IQ-TREE, an efficient phylogenetic software for maximum likelihood analysis. W-IQ-TREE supports multiple sequence types (DNA, protein, codon, binary and morphology) in common alignment formats and a wide range of evolutionary models including mixture and partition models. W-IQ-TREE performs fast model selection, partition scheme finding, efficient tree reconstruction, ultrafast bootstrapping, branch tests, and tree topology tests. All computations are conducted on a dedicated computer cluster and the users receive the results via URL or email. W-IQ-TREE is available at http://iqtree.cibiv.univie.ac.at It is free and open to all users and there is no login requirement.

Nonparametric bootstrap has been a widely used tool in phylogenetic analysis to assess the clade support of phylogenetic trees. However, with the rapidly growing amount of data, this task remains a computational bottleneck. Recently, approximation methods such as the RAxML rapid bootstrap (RBS) and the Shimodaira-Hasegawa-like approximate likelihood ratio test have been introduced to speed up the bootstrap. Here, we suggest an ultrafast bootstrap approximation approach (UFBoot) to compute the support of phylogenetic groups in maximum likelihood (ML) based trees. To achieve this, we combine the resampling estimated log-likelihood method with a simple but effective collection scheme of candidate trees. We also propose a stopping rule that assesses the convergence of branch support values to automatically determine when to stop collecting candidate trees. UFBoot achieves a median speed up of 3.1 (range: 0.66-33.3) to 10.2 (range: 1.32-41.4) compared with RAxML RBS for real DNA and amino acid alignments, respectively. Moreover, our extensive simulations show that UFBoot is robust against moderate model violations and the support values obtained appear to be relatively unbiased compared with the conservative standard bootstrap. This provides a more direct interpretation of the bootstrap support. We offer an efficient and easy-to-use software (available at http://www.cibiv.at/software/iqtree) to perform the UFBoot analysis with ML tree inference.

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

In phylogenomics the analysis of concatenated gene alignments, the so-called supermatrix, is commonly accompanied by the assumption of partition models. Under such models each gene, or more generally partition, is allowed to evolve under its own evolutionary model. Although partition models provide a more comprehensive analysis of supermatrices, missing data may hamper the tree search algorithms due to the existence of phylogenetic (partial) terraces. Here, we introduce the phylogenetic terrace aware (PTA) data structure for the efficient analysis under partition models. In the presence of missing data PTA exploits (partial) terraces and induced partition trees to save computation time. We show that an implementation of PTA in IQ-TREE leads to a substantial speedup of up to 4.5 and 8 times compared with the standard IQ-TREE and RAxML implementations, respectively. PTA is generally applicable to all types of partition models and common topological rearrangements thus can be employed by all phylogenomic inference software.

50 years ago Isaacs and Lindenmann (1Isaacs A. Lindenmann J. Proc. R. Soc. Lond. B. Biol. Sci. 1957; 147: 258-267Crossref PubMed Google Scholar) first described interferons (IFNs) 2The abbreviations used are: IFN, interferon; JAK, Janus kinase; STAT, signal transducers and activators of transcription; JH, JAK homology; γC, γ-chain; SH2, Src homology 2; IL, interleukin; IFNAR, IFN-α receptor chain; ISGF, IFN-stimulated gene factor; ISRE, IFN-stimulated response element; TAD, transcriptional activation domain; GAS, γ-IFN-activated site; aa, amino acid(s); GM-CSF, granulocyte/macrophage-colony-stimulating factor; SOCS, suppressors of cytokine signaling; CBP, CREB-binding factor; pol II, polymerase II; HDAC, histone deacetylase; LIF, leukemia inhibitory factor. as founding members of the cytokine family. Over the next 25 years, these and several other four-helix bundle cytokines were characterized. The subsequent 25 years witnessed an exponential growth in number of four-helix bundle cytokines and their corresponding receptors. The early availability of recombinant IFNs afforded an opportunity to investigate how cytokines induce gene expression, culminating in the identification of the JAK-STAT signaling paradigm (see Fig. 1). Subsequent studies identified 7 STATs and 4 JAKs, providing important insight into how the ∼50 members of the four-helix bundle cytokine family transduce their potent biological responses. This review will briefly summarize this signaling paradigm (reviewed in Refs. 2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 3Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google Scholar, 4Decker T. Muller M. Stockinger S. Nat. Rev. Immunol. 2005; 5: 675-687Crossref PubMed Scopus (381) Google Scholar, 5O'Shea J.J. Gadina M. Schreiber R.D. Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar) and then focus on STAT-dependent transcription. Members of the JAK family, Jak1, Jak2, Jak3, and Tyk2, were initially identified as orphan tyrosine kinases (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 3Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google Scholar, 5O'Shea J.J. Gadina M. Schreiber R.D. Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 6Firmbach-Kraft I. Byers M. Shows T. Dalla-Favera R. Krolewski J.J. Oncogene. 1990; 5: 1329-1336PubMed Google Scholar, 7Wilks A.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1603-1607Crossref PubMed Scopus (365) Google Scholar). All exhibited broad patterns of expression, except Jak3, in which expression was restricted to leukocytes. Genetic studies linking Tyk2 to the biological response to type I IFNs (IFN-I; also IFN-α/β) inspired studies associating these kinases with cytokine signaling (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 5O'Shea J.J. Gadina M. Schreiber R.D. Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 8Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (709) Google Scholar). Specifically, these studies determined that ligand binding stimulated the rapid activation of receptor-associated JAKs, initiating JAK-STAT signaling (see Fig. 1). JAKs range in size from 120 to 140 kDa and feature seven conserved JAK homology (JH) domains. The two carboxyl-terminal JH regions represent the kinase (JH1/Ki) and pseudo kinase (JH2/ΨKi) domains (see Fig. 2). As with other tyrosine kinases, activation is driven by phosphorylation of critical tyrosines in the “inactivation loop.” The four amino-terminal JH domains (JH7–5 and half of JH4) constitute a FERM (four point one, ezrin, radixin, moesin) domain that mediates association with receptors. Specifically, JAKs associate with the proline-rich, membrane-proximal box1/box2 domain on cytokine receptors. An SH2-related domain (SH2; JH3 and half of JH4), of unknown function, lies between the pseudokinase and FERM domains. Tyk2—Tyk2 associates with receptors for IFN-I, IL-6, IL-10 and IL-12/23 cytokine families (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 9Watford W.T. O'Shea J.J. Immunity. 2006; 25: 695-697Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In Tyk2-deficient humans, the combined defects in the response to IFN-I, IL-6, IL-10, IL-12, and IL-23 are associated with enhanced allergic and impaired antimicrobial responses (9Watford W.T. O'Shea J.J. Immunity. 2006; 25: 695-697Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). By comparison, Tyk2 knock-out mice exhibit a less severe defect, indicating that murine Tyk2 is more of a response amplifier and not absolutely required (9Watford W.T. O'Shea J.J. Immunity. 2006; 25: 695-697Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Like humans, however, Tyk2-deficient mice exhibit a proclivity toward type 2 immune response (9Watford W.T. O'Shea J.J. Immunity. 2006; 25: 695-697Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In addition, Tyk2 contributes to the lethal effects of endotoxin through an ill defined and largely Stat1-independent pathway (10Karaghiosoff M. Steinborn R. Kovarik P. Kriegshauser G. Baccarini M. Donabauer B. Reichart U. Kolbe T. Bogdan C. Leanderson T. Levy D. Decker T. Muller M. Nat. Immunol. 2003; 4: 471-477Crossref PubMed Scopus (307) Google Scholar). Jak1—Initially identified in a screen for novel kinases (7Wilks A.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1603-1607Crossref PubMed Scopus (365) Google Scholar), biochemical and genetic studies have revealed a functional and physical association with the type I (IFN-α/β), type II (IFN-γ), IL-2, and IL-6 receptors (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 3Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google Scholar, 5O'Shea J.J. Gadina M. Schreiber R.D. Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). Evidence that the two IFN-α receptor chains, IFNAR1 and IFNAR2, associated with Tyk2 and Jak1, respectively, led to the notion that JAKs activate each other through transphosphorylation. Importantly, Jak1 knockout mice die perinatally, reflecting a defect in LIF (an IL-6 family member) receptor signaling (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 5O'Shea J.J. Gadina M. Schreiber R.D. Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). Characterization of Jak1 knock-out tissues, however, confirmed a critical role for this kinase in the response to IFN, IL-10, IL-2/IL-4 and IL-6 cytokine families. Jak2—Initial biochemical studies implicated Jak2 in the response to receptors from the single-chain (i.e. Epo-R, GH-R, Prl-R) and IL-3 (IL-3R, IL-5R, and GM-CSFR) cytokine families, as well as the IFN-γ receptor (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 3Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google Scholar, 5O'Shea J.J. Gadina M. Schreiber R.D. Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). Consistent with a critical role in definitive erythropoiesis, Jak2 knock-out mice died of anemia at E12.5 (5O'Shea J.J. Gadina M. Schreiber R.D. Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). Analysis of Jak2–/– tissues confirmed an important role in directing the responses to members of the single-chain, IL-3, and IFN-γ receptor families. Intriguingly, humans with Jak2 mutations exhibit myeloproliferative disorders (11Delhommeau F. Pisani D.F. James C. Casadevall N. Constantinescu S. Vainchenker W. Cell Mol. Life Sci. 2006; 63: 2939-2953Crossref PubMed Scopus (61) Google Scholar). Finally, elegant biochemical studies with chimeric erythropoietin receptors provide compelling evidence that ligand binding drives two receptor associated Jak2s into close proximity, enabling them to activate each other by transphosphorylation (12Remy I. Wilson I.A. Michnick S.W. Science. 1999; 283: 990-993Crossref PubMed Scopus (538) Google Scholar). Jak3—Leukocyte-specific Jak3 exclusively associates with the IL-2 receptor γ-chain (γC). This chain also serves as a component for the receptors of several lymphotrophic cytokines, including IL-4, IL-7, IL-9, IL-15, and IL-21. Underscoring the critical roles that γC and Jak3 play in lymphoid activity, mutations in either molecule are associated with severe combined immunodeficiency disease (5O'Shea J.J. Gadina M. Schreiber R.D. Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). Intriguingly, Jak3 knock-out mice develop a similar, but less severe, immunodeficiency syndrome (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 3Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google Scholar, 5O'Shea J.J. Gadina M. Schreiber R.D. Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). Because of the unique role Jak3 plays in regulating lymphocytes, it has become an important pharmaceutical target. The seven members of the mammalian STAT family (STATs 1, 2, 3, 4, 5a, 5b, and 6) range in size from 750 to 900 amino acids and feature several conserved domains, notably including an SH2 domain (see Fig. 2). In resting cells, STATs reside largely in the cytoplasm as inactive homodimers (13Mertens C. Zhong M. Krishnaraj R. Zou W. Chen X. Darnell Jr., J.E. Genes Dev. 2006; 20: 3372-3381Crossref PubMed Scopus (110) Google Scholar). However, upon ligand binding, receptor-associated JAKs become activated (see above), leading to the phosphorylation of specific receptor tyrosine residues (see Fig. 1). These receptor phosphotyrosyl residues direct the SH2-dependent recruitment of specific STATs, which in turn become JAK substrates. Activated STATs are released from the receptor as they reorient into an antiparallel dimer, where the SH2 domain of one STAT binds the phosphotyrosine of the other STAT. Activated STAT dimers translocate to the nucleus and bind to specific enhancer elements. STAT homodimers bind to members of the GAS family of enhancers (a palindrome, TTTCCNGGAAA; Fig. 1). In contrast, IFN-Is promote the formation of Stat1-Stat2 heterodimers, which associate with IRF-9 (IFN regulatory factor) to form ISGF-3 and bind to the ISRE enhancer family (a direct repeat, AGTTTN3TTTCC; Fig. 1). STAT Structure—Biochemical, genetic, and structural studies have identified seven conserved STAT domains, including the amino-terminal (NH2), coiled-coil, DNA-binding (DBD), linker (Lk), SH2, tyrosine activation (Y), and transcriptional activation domains (TAD) (Fig. 2; Ref. 14Becker S. Groner B. Müller C.W. Nature. 1998; 394: 145-151Crossref PubMed Scopus (670) Google Scholar, 15Chen X. Vinkemeier U. Zhao Y. Jeruzalmi D. Darnell Jr., J.E. Kuriyan J. Cell. 1998; 93: 827-839Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar). The NH2 domain (∼125 residues) is a structurally independent moiety and appears to direct homotypic dimerization of inactive STATs (13Mertens C. Zhong M. Krishnaraj R. Zou W. Chen X. Darnell Jr., J.E. Genes Dev. 2006; 20: 3372-3381Crossref PubMed Scopus (110) Google Scholar). This domain has also been implicated in cooperative DNA binding to tandem GAS elements, as well as in nuclear import and export (16McBride K.M. Reich N.C. Sci. STKE 2003. 2003; : RE13Google Scholar, 17Vinkemeier U. J. Cell Biol. PubMed Scopus Google Scholar). The domain of a bundle that from the This domain a and binds The DNA-binding domain of a that binding to the GAS family of enhancers with The corresponding of the Stat1-Stat2 has not been The linker domain an between the DNA-binding and dimerization domains. important role in receptor recruitment and the SH2 domain is the conserved The tyrosine activation domain is to the SH2 (i.e. The carboxyl-terminal which STAT family constitute the This an opportunity to associate with transcriptional (see founding STAT was initially identified as a component of the (Fig. Ref. C. Darnell J.E. Science. 1992; PubMed Scopus Google Scholar). Subsequent studies determined that the of homodimers Schindler C. Darnell J.E. Science. 1992; PubMed Scopus Google Scholar). studies confirmed the role that plays in the biological response to type I and type II IFNs D. R. M. Schreiber R.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, J.E. R. Levy D.E. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Consistent with humans exhibit to and A. S. G. J. A. A. C. C. J. C. L. Schreiber R.D. C. A. J. 2006; PubMed Scopus Google Scholar). Intriguingly, to promote and This the and associated with (see the of several cytokines to activate and members of the and IL-6 an to a more was also initially identified as a component of and genetic studies have revealed that plays a role in the biological response to type I a critical role for in regulating the T. Muller M. Stockinger S. Nat. Rev. Immunol. 2005; 5: 675-687Crossref PubMed Scopus (381) Google Scholar, C. S. Schindler C. Immunity. Full Text Full Text PDF PubMed Scopus Google Scholar). the of this family. In to the STAT in in with a TAD, is evidence that homodimers form bind with Finally, the by which is to was initially identified as an that gene expression S. Y. M. S. T. T. M. T. Cell. Full Text PDF PubMed Scopus Google Scholar). is to transduce for the IL-6 LIF, and IL-10 families, as well as and (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 3Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google Scholar). studies in have that is activated by several growth and gene has a role for (i.e. die at (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 3Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google In contrast, specific have an important role for (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 3Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google Scholar). important of is association with has been identified in and and other Consistent with the expression of and (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 3Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google Scholar). expression of a G. Zhao Y. C. Darnell J.E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). and genetic have implicated in (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 3Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google Scholar). However, the potent of is to to these responses. Finally, several studies that growth through in the of tyrosine phosphorylation DNA binding J. M. Levy D.E. Stark G.R. 2005; Google Scholar). gene for identified through homology to was also to to the and genetic studies have the important role plays in directing the biological response to and which receptor (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, Nat. Rev. Immunol. 2005; 5: PubMed Scopus Google Scholar). the of into potent (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 3Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google Scholar, 5O'Shea J.J. Gadina M. Schreiber R.D. Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). plays an role in studies have implicated tyrosine and phosphorylation in these immune (see has been to important in the of and an associated Nat. Rev. Immunol. 2005; 5: PubMed Scopus Google Scholar). tandem and with their these STATs exhibit the of homology to STATs Y. G. J. L. T. L. PubMed Scopus Google Scholar). Consistent with this they are and genetic studies have the important role that and play in directing a biological response to the IL-3 and single-chain and and γC (i.e. the IL-2, IL-7, IL-9, IL-15, and receptor families. between and their functional the responses to and and Finally, gene studies have revealed an important role for in and Y. W.T. D. A. L. O'Shea J.J. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar). for and which receptor (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, 3Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google Scholar, 5O'Shea J.J. Gadina M. Schreiber R.D. Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). Like is one of the more also a which with transcriptional (see and Ref. D. G. J. A. Rev. 2006; PubMed Scopus Google Scholar). Intriguingly, homodimers a GAS that an studies have confirmed a critical role for in the of into as well as in These studies have also an important role for in function, including and and feature of JAK-STAT signaling is rapid and subsequent As activated STATs in the a of however, the signal and the STATs are to the cytoplasm for the next of This of receptors and JAKs, as well as STAT transcriptional well of STAT signal nuclear and of cytokine However, a number of have been including and Oncogene. PubMed Scopus Google Scholar, T. Immunity. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). play an important role in regulating signaling Genetic and biochemical have implicated several in the of cytokine receptors and JAKs, including and (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, T. T. N. Nat. Rev. Immunol. 2005; 5: PubMed Scopus Google Scholar). have a role for and in STAT (2Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (907) Google Scholar, T. T. N. Nat. Rev. Immunol. 2005; 5: PubMed Scopus Google Scholar, M. T. Immunity. Full Text Full Text PDF PubMed Scopus Google Scholar). However, two of these and have been implicated in nuclear STAT which appears to critical for STAT nuclear export (16McBride K.M. Reich N.C. Sci. STKE 2003. 2003; : RE13Google Scholar, 17Vinkemeier U. J. Cell Biol. PubMed Scopus Google Scholar, T. T. N. Nat. Rev. Immunol. 2005; 5: PubMed Scopus Google Scholar). a of STATs in the the of nuclear import and export is (16McBride K.M. Reich N.C. Sci. STKE 2003. 2003; : RE13Google Scholar, 17Vinkemeier U. J. Cell Biol. PubMed Scopus Google Scholar, S. Schindler C. J. 2003; PubMed Scopus Google Scholar). The for inactive STATs has been to a where nuclear import is by nuclear This appears to by nuclear export and nuclear elements. the is toward nuclear and signal toward nuclear The were identified as STAT that STAT in a (reviewed in Ref. Rev. Immunol. PubMed Scopus Google Scholar). studies have the important role that and play in responses to and STATs several well in to tyrosine including and roles for and more L. Bhattacharya S. Schindler C. 2006; PubMed Scopus Google Scholar, W. S. I. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus (61) Google Scholar). STATs except are on at one in their (reviewed in Ref. T. Kovarik P. Oncogene. PubMed Scopus Google Scholar, T. Müller M. Kovarik P. T. Levy D.E. and of and Google Scholar). phosphorylation a in and and in a in in and a in D. R. D. N. S. J. J. PubMed Google Scholar). and at one phosphorylation in their TAD, and STAT kinases have been identified through the of and in kinase STATs 1, 3, T. T. S. Zhao S. T. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google and and T. Kovarik P. Oncogene. PubMed Scopus Google Scholar, T. Müller M. Kovarik P. T. Levy D.E. and of and Google Scholar, A. T. Science. PubMed Scopus Google role for these kinases has been confirmed by gene knock-out in a number of an of as phosphorylation of dimers appears to exclusively in the STAT phosphorylation transcriptional (see Consistent with mice a exhibit L. M. Kolbe T. Muller M. Decker T. Immunity. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). mice exhibit a in gene expression but an Y. X. F. Levy D.E. Darnell Jr., J.E. Mol. Cell. Biol. PubMed Scopus Google Scholar). By contrast, expression of in growth and biological defects not in Y. X. F. Levy D.E. Darnell Jr., J.E. Mol. Cell. Biol. PubMed Scopus Google Scholar). of IFN-γ is impaired in A. Gadina M. W. R. A. C. R. O'Shea J.J. Proc. Natl. Acad. Sci. U. S. 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Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to increase their coding potential and regulate gene expression through multiple mechanisms. Recent transcriptome-wide analysis of AS using RNA sequencing has revealed that AS is highly pervasive in plants. Pre-mRNAs from over 60% of intron-containing genes undergo AS to produce a vast repertoire of mRNA isoforms. The functions of most splice variants are unknown. However, emerging evidence indicates that splice variants increase the functional diversity of proteins. Furthermore, AS is coupled to transcript stability and translation through nonsense-mediated decay and microRNA-mediated gene regulation. Widespread changes in AS in response to developmental cues and stresses suggest a role for regulated splicing in plant development and stress responses. Here, we review recent progress in uncovering the extent and complexity of the AS landscape in plants, its regulation, and the roles of AS in gene regulation. The prevalence of AS in plants has raised many new questions that require additional studies. New tools based on recent technological advances are allowing genome-wide analysis of RNA elements in transcripts and of chromatin modifications that regulate AS. Application of these tools in plants will provide significant new insights into AS regulation and crosstalk between AS and other layers of gene regulation.

Landscape genomics is an emerging research field that aims to identify the environmental factors that shape adaptive genetic variation and the gene variants that drive local adaptation. Its development has been facilitated by next-generation sequencing, which allows for screening thousands to millions of single nucleotide polymorphisms in many individuals and populations at reasonable costs. In parallel, data sets describing environmental factors have greatly improved and increasingly become publicly accessible. Accordingly, numerous analytical methods for environmental association studies have been developed. Environmental association analysis identifies genetic variants associated with particular environmental factors and has the potential to uncover adaptive patterns that are not discovered by traditional tests for the detection of outlier loci based on population genetic differentiation. We review methods for conducting environmental association analysis including categorical tests, logistic regressions, matrix correlations, general linear models and mixed effects models. We discuss the advantages and disadvantages of different approaches, provide a list of dedicated software packages and their specific properties, and stress the importance of incorporating neutral genetic structure in the analysis. We also touch on additional important aspects such as sampling design, environmental data preparation, pooled and reduced-representation sequencing, candidate-gene approaches, linearity of allele-environment associations and the combination of environmental association analyses with traditional outlier detection tests. We conclude by summarizing expected future directions in the field, such as the extension of statistical approaches, environmental association analysis for ecological gene annotation, and the need for replication and post hoc validation studies.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to take at least 12–18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose–response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2–4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from H9 human embryonic stem cell lines, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19. A screen of the ReFRAME library of approximately 12,000 known drugs for antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) identified several candidate compounds with suitable activities and pharmacological profiles, which could potentially expedite the deployment of therapies for coronavirus disease 2019 (COVID-19).

Alternative splicing (AS) is a key regulatory mechanism that contributes to transcriptome and proteome diversity. As very few genome-wide studies analyzing AS in plants are available, we have performed high-throughput sequencing of a normalized cDNA library which resulted in a high coverage transcriptome map of Arabidopsis. We detect ∼150,000 splice junctions derived mostly from typical plant introns, including an eightfold increase in the number of U12 introns (2069). Around 61% of multiexonic genes are alternatively spliced under normal growth conditions. Moreover, we provide experimental validation of 540 AS transcripts (from 256 genes coding for important regulatory factors) using high-resolution RT-PCR and Sanger sequencing. Intron retention (IR) is the most frequent AS event (∼40%), but many IRs have relatively low read coverage and are less well-represented in assembled transcripts. Additionally, ∼51% of Arabidopsis genes produce AS transcripts which do not involve IR. Therefore, the significance of IR in generating transcript diversity was generally overestimated in previous assessments. IR analysis allowed the identification of a large set of cryptic introns inside annotated coding exons. Importantly, a significant fraction of these cryptic introns are spliced out in frame, indicating a role in protein diversity. Furthermore, we show extensive AS coupled to nonsense-mediated decay in AFC2, encoding a highly conserved LAMMER kinase which phosphorylates splicing factors, thus establishing a complex loop in AS regulation. We provide the most comprehensive analysis of AS to date which will serve as a valuable resource for the plant community to study transcriptome complexity and gene regulation.

BACKGROUND & AIMS: Recent studies have highlighted the role of noncoding RNAs (ncRNAs) in carcinogenesis, and suggested that this class of genes might be used as biomarkers in cancer. We searched the human genome for novel genes including ncRNAs related to hepatocellular carcinoma (HCC). METHODS: An HCC-specific gene library was generated and screened for deregulated genes with 46 HCCs, 4 focal nodular hyperplasias, and 7 cirrhoses utilizing cDNA arrays. Sequencing of library clones identified a novel ncRNA as the most up-regulated gene in HCC. This gene was also cloned from different monkeys and characterized by quantitative RT-PCR, Northern blot analysis and in situ hybridization. Structural and functional studies included comparative sequence and protein expression analyses, quantitative RT-PCR of polysomal preparations, and siRNA-mediated knockdown experiments. RESULTS: The most up-regulated gene in HCC named highly up-regulated in liver cancer (HULC) was characterized as a novel mRNA-like ncRNA. HULC RNA is spliced and polyadenlyated, and resembles the mammalian LTR transposon 1A. It does not contain substantial open reading frames, and no native translation product was detected. HULC is present in the cytoplasm, where it copurifies with ribosomes. siRNA-mediated knockdown of HULC RNA in 2 HCC cell lines altered the expression of several genes, 5 of which were known to be affected in HCC, suggesting a role for HULC in post-transcriptional modulation of gene expression. CONCLUSIONS: HULC is the first ncRNA with highly specific up-regulation in HCC. Because HULC was detected in blood of HCC patients, a potential use as novel biomarker can be envisaged.

Selective autophagy of damaged mitochondria requires autophagy receptors optineurin (OPTN), NDP52 (CALCOCO2), TAX1BP1, and p62 (SQSTM1) linking ubiquitinated cargo to autophagic membranes. By using quantitative proteomics, we show that Tank-binding kinase 1 (TBK1) phosphorylates all four receptors on several autophagy-relevant sites, including the ubiquitin- and LC3-binding domains of OPTN and p62/SQSTM1 as well as the SKICH domains of NDP52 and TAX1BP1. Constitutive interaction of TBK1 with OPTN and the ability of OPTN to bind to ubiquitin chains are essential for TBK1 recruitment and kinase activation on mitochondria. TBK1 in turn phosphorylates OPTN's UBAN domain at S473, thereby expanding the binding capacity of OPTN to diverse Ub chains. In combination with phosphorylation of S177 and S513, this posttranslational modification promotes recruitment and retention of OPTN/TBK1 on ubiquitinated, damaged mitochondria. Moreover, phosphorylation of OPTN on S473 enables binding to pS65 Ub chains and is also implicated in PINK1-driven and Parkin-independent mitophagy. Thus, TBK1-mediated phosphorylation of autophagy receptors creates a signal amplification loop operating in selective autophagy of damaged mitochondria.

Abstract IQ-TREE ( http://www.iqtree.org ) is a user-friendly and widely used software package for phylogenetic inference using maximum likelihood. Since the release of version 1 in 2014, we have continuously expanded IQ-TREE to integrate a plethora of new models of sequence evolution and efficient computational approaches of phylogenetic inference to deal with genomic data. Here, we describe notable features of IQ-TREE version 2 and highlight the key advantages over other software.

RNA editing by members of the ADAR (adenosine deaminases acting on RNA) family leads to site-specific conversion of adenosine to inosine (A-to-I) in precursor messenger RNAs. Editing by ADARs is believed to occur in all metazoa, and is essential for mammalian development. Currently, only a limited number of human ADAR substrates are known, whereas indirect evidence suggests a substantial fraction of all pre-mRNAs being affected. Here we describe a computational search for ADAR editing sites in the human transcriptome, using millions of available expressed sequences. We mapped 12,723 A-to-I editing sites in 1,637 different genes, with an estimated accuracy of 95%, raising the number of known editing sites by two orders of magnitude. We experimentally validated our method by verifying the occurrence of editing in 26 novel substrates. A-to-I editing in humans primarily occurs in noncoding regions of the RNA, typically in Alu repeats. Analysis of the large set of editing sites indicates the role of editing in controlling dsRNA stability.

The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform.