Systematic VCP-UBXD Adaptor Network Proteomics Identifies a Role for UBXN10 in Regulating Ciliogenesis

The AAA-ATPase VCP (also known as p97 or CDC48) uses ATP hydrolysis to “segregate” ubiquitinated proteins from their binding partners. VCP acts via UBX-domain containing adaptors that provide target specificity, but targets and functions of UBXD proteins remain poorly understood. Through systematic proteomic analysis of UBXD proteins in human cells, we reveal a network of over 195 interacting proteins, implicating VCP in diverse cellular pathways. We have explored one such complex between an unstudied adaptor UBXN10 and the intraflagellar transport B (IFT-B) complex, which regulates anterograde transport into cilia. UBXN10 localizes to cilia in a VCP-dependent manner and both VCP and UBXN10 are required for ciliogenesis. Pharmacological inhibition of VCP destabilized the IFT-B complex and increased trafficking rates. Depletion of UBXN10 in zebrafish embryos causes defects in left-right asymmetry, which depends on functional cilia. This study provides a resource for exploring the landscape of UBXD proteins in biology and identifies an unexpected requirement for VCP-UBXN10 in ciliogenesis

Sgs1 and Exo1 Redundantly Inhibit Break-Induced Replication and De Novo Telomere Addition at Broken Chromosome Ends

In budding yeast, an HO endonuclease-inducible double-strand break (DSB) is efficiently repaired by several homologous recombination (HR) pathways. In contrast to gene conversion (GC), where both ends of the DSB can recombine with the same template, break-induced replication (BIR) occurs when only the centromere-proximal end of the DSB can locate homologous sequences. Whereas GC results in a small patch of new DNA synthesis, BIR leads to a nonreciprocal translocation. The requirements for completing BIR are significantly different from those of GC, but both processes require 5′ to 3′ resection of DSB ends to create single-stranded DNA that leads to formation of a Rad51 filament required to initiate HR. Resection proceeds by two pathways dependent on Exo1 or the BLM homolog, Sgs1. We report that Exo1 and Sgs1 each inhibit BIR but have little effect on GC, while overexpression of either protein severely inhibits BIR. In contrast, overexpression of Rad51 markedly increases the efficiency of BIR, again with little effect on GC. In sgs1Δ exo1Δ strains, where there is little 5′ to 3′ resection, the level of BIR is not different from either single mutant; surprisingly, there is a two-fold increase in cell viability after HO induction whereby 40% of all cells survive by formation of a new telomere within a few kb of the site of DNA cleavage. De novo telomere addition is rare in wild-type, sgs1Δ, or exo1Δ cells. In sgs1Δ exo1Δ, repair by GC is severely inhibited, but cell viaiblity remains high because of new telomere formation. These data suggest that the extensive 5′ to 3′ resection that occurs before the initiation of new DNA synthesis in BIR may prevent efficient maintenance of a Rad51 filament near the DSB end. The severe constraint on 5′ to 3′ resection, which also abrogates activation of the Mec1-dependent DNA damage checkpoint, permits an unprecedented level of new telomere addition

Competitive Interactions between Invasive Nile Tilapia and Native Fish: The Potential for Altered Trophic Exchange and Modification of Food Webs

Recent studies have highlighted both the positive and negative impacts of species invasions. Most of these studies have been conducted on either immobile invasive plants or sessile fauna found at the base of food webs. Fewer studies have examined the impacts of vagile invasive consumers on native competitors. This is an issue of some importance given the controlling influence that consumers have on lower order plants and animals. Here, we present results of laboratory experiments designed to assess the impacts of unintended aquaculture releases of the Nile tilapia (Oreochromis niloticus), in estuaries of the Gulf of Mexico, on the functionally similar redspotted sunfish (Lepomis miniatus). Laboratory choice tests showed that tilapia prefer the same structured habitat that native sunfish prefer. In subsequent interspecific competition experiments, agonistic tilapia displaced sunfish from their preferred structured habitats. When a piscivore (largemouth bass) was present in the tank with both species, the survival of sunfish decreased. Based on these findings, if left unchecked, we predict that the proliferation of tilapia (and perhaps other aggressive aquaculture fishes) will have important detrimental effects on the structure of native food webs in shallow, structured coastal habitats. While it is likely that the impacts of higher trophic level invasive competitors will vary among species, these results show that consequences of unintended releases of invasive higher order consumers can be important

Conservation Genetics of a Critically Endangered Limpet Genus and Rediscovery of an Extinct Species

A third of all known freshwater mollusk extinctions worldwide have occurred within a single medium-sized American drainage. The Mobile River Basin (MRB) of Alabama, a global hotspot of temperate freshwater biodiversity, was intensively industrialized during the 20(th) century, driving 47 of its 139 endemic mollusk species to extinction. These include the ancylinid limpet Rhodacmea filosa, currently classified as extinct (IUCN Red List), a member of a critically endangered southeastern North American genus reduced to a single known extant population (of R. elatior) in the MRB.We document here the tripling of known extant populations of this North American limpet genus with the rediscovery of enduring Rhodacmea filosa in a MRB tributary and of R. elatior in its type locality: the Green River, Kentucky, an Ohio River Basin (ORB) tributary. Rhodacmea species are diagnosed using untested conchological traits and we reassessed their systematic and conservation status across both basins using morphometric and genetic characters. Our data corroborated the taxonomic validity of Rhodacmea filosa and we inferred a within-MRB cladogenic origin from a common ancestor bearing the R. elatior shell phenotype. The geographically-isolated MRB and ORB R. elatior populations formed a cryptic species complex: although overlapping morphometrically, they exhibited a pronounced phylogenetic disjunction that greatly exceeded that of within-MRB R. elatior and R. filosa sister species.Rhodacmea filosa, the type species of the genus, is not extinct. It persists in a Coosa River tributary and morphometric and phylogenetic analyses confirm its taxonomic validity. All three surviving populations of the genus Rhodacmea merit specific status. They collectively contain all known survivors of a phylogenetically highly distinctive North American endemic genus and therefore represent a concentrated fraction of continental freshwater gastropod biodiversity. We recommend the establishment of a proactive targeted conservation program that may include their captive propagation and reintroduction

Break-induced replication requires all essential DNA replication factors except those specific for pre-RC assembly

Break-induced replication (BIR) is an efficient homologous recombination (HR) pathway employed to repair a DNA double-strand break (DSB) when homology is restricted to one end. All three major replicative DNA polymerases are required for BIR, including the otherwise nonessential Pol32 subunit. Here we show that BIR requires the replicative DNA helicase (Cdc45, the GINS, and Mcm2–7 proteins) as well as Cdt1. In contrast, both subunits of origin recognition complex (ORC) and Cdc6, which are required to create a prereplication complex (pre-RC), are dispensable. The Cdc7 kinase, required for both initiation of DNA replication and post-replication repair (PRR), is also required for BIR. Ubiquitination and sumoylation of the DNA processivity clamp PCNA play modest roles; in contrast, PCNA alleles that suppress pol32Δ's cold sensitivity fail to suppress its role in BIR, and are by themselves dominant inhibitors of BIR. These results suggest that origin-independent BIR involves cross-talk between normal DNA replication factors and PRR

The Structure of DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide

In the late 1950s the drug thalidomide (Contergan) was administered as a mild sedative to pregnant women leading to the birth of thousands of children with multiple birth defects including frequent limb deformities. Despite its teratogenicity, thalidomide and its derivatives have recently re-emerged as effective clinical drugs in treating multiple myeloma and 5q-dysplasias. The primary target of thalidomide is the CUL4(RBX1)-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase. We present the molecular architecture of the DDB1-CRBN complex bound to the small molecule inhibitors thalidomide, lenalidomide and pomalidomide. All compounds recognize a common binding site on CRBN. The CRL4CRBN architecture implicates CRBN in substrate binding and suggests that thalidomide and its derivatives can block substrate access to the receptor. Using an unbiased biochemical screen for CRL4CRBN substrates, we identify the homeobox transcription factor MEIS2 required for limb development as a CRL4CRBN ligase target, and show that its cellular levels are altered by thalidomide. One Sentence Summary: We present the molecular architecture of the CRL4CRBN ligase in complex with thalidomide and establish the homeobox transcription factor MEIS2 a substrate for the ligase

Structure of DDB1-CRBN bound to thalidomide: insights into CRL4 inhibition by small molecules

In the 1950s, the drug thalidomide, administered as a sedative to pregnant woman led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-asociated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4CRBN) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4CRBN. How IMiD binding affects CRL4CRBN at the molecular level remained unclear