Summary An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its pathogenic mechanism remains unclear. Here we use human induced motor neurons (iMNs) to show that repeat-expanded C9ORF72 is haploinsufficient in ALS. We show that C9ORF72 interacts with endosomes and is required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduces C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms leads to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescues patient neuron survival and ameliorates neurodegenerative processes in both gain- and loss-of function C9ORF72 mouse models. Thus, modulating vesicle trafficking can rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN, and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS/FTD.
Many cellular processes are carried out by molecular ‘machines’— assemblies of multiple differentiated proteins that physically interact to execute biological functions1–8. Despite much speculation, strong evidence of the mechanisms by which these assemblies evolved is lacking. Here we use ancestral gene resurrection9–11and manipulative genetic experiments to determine how the complexity of an essential molecular machine—the hexameric transmembrane ring of the eukaryotic V-ATPase proton pump—increased hundreds of millions of years ago. We show that the ring of Fungi, which is composed of three paralogous proteins, evolved from a more ancient two-paralogue complex because of a gene duplication that was followed by loss in each daughter copy of specific interfaces by which it interacts with other ring proteins. These losses were complementary, so both copies became obligate components with restricted spatial roles in the complex. Reintroducing a single historical mutation from each paralogue lineage into the resurrected ancestral proteins is sufficient to recapitulate their asymmetric degeneration and trigger the requirement for the more elaborate three-component ring. Our experiments show that increased complexity in an essential molecular machine evolved because of simple, high-probability evolutionary processes, without the apparent evolution of novel functions. They point to a plausible mechanism for the evolution of complexity in other multi-paralogue protein complexes.
Ancestral sequence reconstruction (ASR) is widely used to formulate and test hypotheses about the sequences, functions, and structures of ancient genes. Ancestral sequences are usually inferred from an alignment of extant sequences using a maximum likelihood (ML) phylogenetic algorithm, which calculates the most likely ancestral sequence assuming a probabilistic model of sequence evolution and a specific phylogeny—typically the tree with the ML. The true phylogeny is seldom known with certainty, however. ML methods ignore this uncertainty, whereas Bayesian methods incorporate it by integrating the likelihood of each ancestral state over a distribution of possible trees. It is not known whether Bayesian approaches to phylogenetic uncertainty improve the accuracy of inferred ancestral sequences. Here, we use simulation-based experiments under both simplified and empirically derived conditions to compare the accuracy of ASR carried out using ML and Bayesian approaches. We show that incorporating phylogenetic uncertainty by integrating over topologies very rarely changes the inferred ancestral state and does not improve the accuracy of the reconstructed ancestral sequence. Ancestral state reconstructions are robust to uncertainty about the underlying tree because the conditions that produce phylogenetic uncertainty also make the ancestral state identical across plausible trees; conversely, the conditions under which different phylogenies yield different inferred ancestral states produce little or no ambiguity about the true phylogeny. Our results suggest that ML can produce accurate ASRs, even in the face of phylogenetic uncertainty. Using Bayesian integration to incorporate this uncertainty is neither necessary nor beneficial.
Sequence-specific DNA-binding proteins are among the most important classes of gene regulatory proteins, controlling changes in transcription that underlie many aspects of biology. In this work, we identify a transcriptional regulator from the human fungal pathogen Candida albicans that binds DNA specifically but has no detectable homology with any previously described DNA-or RNA-binding protein. This protein, named White-Opaque Regulator 3 (Wor3), regulates white-opaque switching, the ability of C. albicans to switch between two heritable cell types. We demonstrate that ectopic overexpression of WOR3 results in mass conversion of white cells to opaque cells and that deletion of WOR3 affects the stability of opaque cells at physiological temperatures. Genome-wide chromatin immunoprecipitation of Wor3 and gene expression profiling of a wor3 deletion mutant strain indicate that Wor3 is highly integrated into the previously described circuit regulating white-opaque switching and that it controls a subset of the opaque transcriptional program. We show by biochemical, genetic, and microfluidic experiments that Wor3 binds directly to DNA in a sequence-specific manner, and we identify the set of cis-regulatory sequences recognized by Wor3. Bioinformatic analyses indicate that the Wor3 family arose more recently in evolutionary time than most previously described DNA-binding domains; it is restricted to a small number of fungi that include the major fungal pathogens of humans. These observations show that new families of sequence-specific DNA-binding proteins may be restricted to small clades and suggest that current annotations-which rely on deep conservation-underestimate the fraction of genes coding for transcriptional regulators.transcriptional regulation | transcription factor | transcription networks | epigenetic switch
Most models of gene duplication assume that the ancestral functions of the preduplication gene are independent and can therefore be neatly partitioned between descendant paralogs. However, many gene products, such as transcriptional regulators, are components within cooperative assemblies; here, we show that a natural consequence of duplication and divergence of such proteins can be competitive interference between the paralogs. Our example is based on the duplication of the essential MADS-box transcriptional regulator Mcm1, which is found in all fungi and regulates a large set of genes. We show that a set of historical amino acid sequence substitutions minimized paralog interference in contemporary species and, in doing so, increased the molecular complexity of this gene regulatory network. We propose that paralog interference is a common constraint on gene duplicate evolution, and its resolution, which can generate additional regulatory complexity, is needed to stabilize duplicated genes in the genome.
Changes in expression patterns may occur when organisms are presented with new environmental challenges, for example following migration or genetic changes. To elucidate the mechanisms by which the translational machinery adapts to such changes, we perturbed the tRNA pool of Saccharomyces cerevisiae by tRNA gene deletion. We then evolved the deletion strain and observed that the genetic adaptation was recurrently based on a strategic mutation that changed the anticodon of other tRNA genes to match that of the deleted one. Strikingly, a systematic search in hundreds of genomes revealed that anticodon mutations occur throughout the tree of life. We further show that the evolution of the tRNA pool also depends on the need to properly couple translation to protein folding. Together, our observations shed light on the evolution of the tRNA pool, demonstrating that mutation in the anticodons of tRNA genes is a common adaptive mechanism when meeting new translational demands.DOI: http://dx.doi.org/10.7554/eLife.01339.001
To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which – the evolution of GKPID’s capacity to bind the cortical marker protein – can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals.DOI: http://dx.doi.org/10.7554/eLife.10147.001
Protein kinases have evolved diverse specificities to enable cellular information processing. To gain insight into the mechanisms underlying kinase diversification, we studied the CMGC protein kinases using ancestral reconstruction. Within this group, the cyclin dependent kinases (CDKs) and mitogen activated protein kinases (MAPKs) require proline at the +1 position of their substrates, while Ime2 prefers arginine. The resurrected common ancestor of CDKs, MAPKs, and Ime2 could phosphorylate substrates with +1 proline or arginine, with preference for proline. This specificity changed to a strong preference for +1 arginine in the lineage leading to Ime2 via an intermediate with equal specificity for proline and arginine. Mutant analysis revealed that a variable residue within the kinase catalytic cleft, DFGx, modulates +1 specificity. Expansion of Ime2 kinase specificity by mutation of this residue did not cause dominant deleterious effects in vivo. Tolerance of cells to new specificities likely enabled the evolutionary divergence of kinases.DOI: http://dx.doi.org/10.7554/eLife.04126.001
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