Crystal Structure of a 9-Subunit Archaeal Exosome in Pre-Catalytic States of the Phosphorolytic Reaction

The RNA exosome is an important protein complex that functions in the 3′ processing and degradation of RNA in archaeal and eukaryotic organisms. The archaeal exosome is functionally similar to bacterial polynucleotide phosphorylase (PNPase) and RNase PH enzymes as it uses inorganic phosphate (Pi) to processively cleave RNA substrates releasing nucleoside diphosphates. To shed light on the mechanism of catalysis, we have determined the crystal structures of mutant archaeal exosome in complex with either Pi or with both RNA and Pi at resolutions of 1.8 Å and 2.5 Å, respectively. These structures represent views of precatalytic states of the enzyme and allow the accurate determination of the substrate binding geometries. In the structure with both Pi and RNA bound, the Pi closely approaches the phosphate of the 3′-end nucleotide of the RNA and is in a perfect position to perform a nucleophilic attack. The presence of negative charge resulting from the close contacts between the phosphates appears to be neutralized by conserved positively charged residues in the active site of the archaeal exosome. The high degree of structural conservation between the archaeal exosome and the PNPase including the requirement for divalent metal ions for catalysis is discussed

Intraflagellar transport complex structure and cargo interactions

Intraflagellar transport (IFT) is required for the assembly and maintenance of cilia, as well as the proper function of ciliary motility and signaling. IFT is powered by molecular motors that move along the axonemal microtubules, carrying large complexes of IFT proteins that travel together as so-called trains. IFT complexes likely function as adaptors that mediate interactions between anterograde/retrograde motors and ciliary cargoes, facilitating cargo transport between the base and tip of the cilium. Here, we provide an up-to-date review of IFT complex structure and architecture, and discuss how interactions with cargoes and motors may be achieved

Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis

We previously reported that Parkinson's disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies

A WDR35-dependent coat protein complex transports ciliary membrane cargo vesicles to cilia

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß′ COPI coatomer subunits and demonstrate an accumulation of ‘coat-less’ vesicles that fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation

Nucleoside analogue activators of cyclic AMP-independent protein kinase A of Trypanosoma

Protein kinase A (PKA), the main effector of cAMP in eukaryotes, is a paradigm for the mechanisms of ligand-dependent and allosteric regulation in signalling. Here we report the orthologous but cAMP-independent PKA of the protozoan Trypanosoma and identify 7-deazanucleosides as potent activators (EC50 >= 6.5 nM) and high affinity ligands (K-D >= 8 nM). A co-crystal structure of trypanosome PKA with 7-cyano-7-deazainosine and molecular docking show how substitution of key amino acids in both CNB domains of the regulatory subunit and its unique C-terminal alpha D helix account for this ligand swap between trypanosome PKA and canonical cAMP-dependent PKAs. We propose nucleoside-related endogenous activators of Trypanosoma brucei PKA (TbPKA). The existence of eukaryotic CNB domains not associated with binding of cyclic nucleotides suggests that orphan CNB domains in other eukaryotes may bind undiscovered signalling molecules. Phosphoproteome analysis validates 7-cyano-7-deazainosine as powerful cell-permeable inducer to explore cAMP-independent PKA signalling in medically important neglected pathogens

IFT81, encoding an IFT-B core protein, as a very rare cause of a ciliopathy phenotype

Background: Bidirectional intraflagellar transport (IFT) consists of two major protein complexes, IFT-A and IFT-B. In contrast to the IFT-B complex, all components of IFT-A have recently been linked to human ciliopathies when defective. We therefore hypothesised that mutations in additional IFT-B encoding genes can be found in patients with multisystemic ciliopathies. Methods: We screened 1628 individuals with reno-ocular ciliopathies by targeted next-generation sequencing of ciliary candidate genes, including all IFT-B encoding genes. Results: Consequently, we identified a homozygous mutation in IFT81 affecting an obligatory donor splice site in an individual with nephronophthisis and polydactyly. Further, we detected a loss-of-stop mutation with extension of the deduced protein by 10 amino acids in an individual with neuronal ceroid lipofuscinosis-1. This proband presented with retinal dystrophy and brain lesions including cerebellar atrophy, a phenotype to which the IFT81 variant might contribute. Cultured fibroblasts of this latter affected individual showed a significant decrease in ciliated cell abundance compared with controls and increased expression of the transcription factor GLI2 suggesting deranged sonic hedgehog signalling. Conclusions: This work describes identification of mutations of IFT81 in individuals with symptoms consistent with the clinical spectrum of ciliopathies. It might represent the rare case of a core IFT-B complex protein found associated with human disease. Our data further suggest that defects in the IFT-B core are an exceedingly rare finding, probably due to its indispensable role for ciliary assembly in development

Mechanisms of RNA recruitment by the exosome

Exosome-like protein complexes are essential 3'-> 5' ribonucleases involved in processing and degradation of many RNAs. They are conserved in the three domains of life and share a common architecture comprised of a ring-like core structure organized around a central channel. RNA degradation by bacterial and archaeal exosome-like complexes requires threading through this single-stranded RNA specific channel to reach the phosphorolytic active sites buried deep within the barrel-shaped complex. In contrast most eukaryotic exosomes appear to have lost phosphorolytic activity and instead rely on hydrolytic RNases for catalytic activity raising the question of the degree of conservation of RNA recruitment mechanisms between prokaryotic and eukaryotic complexes. Recent single particle electron microscopy reconstructions of apo and RNA bound yeast exosomes provide the first direct structural evidence for a channeling mechanism by a eukaryotic exosome suggesting that this mechanism is conserved between all exosome-like complexes

A single subunit, Dis3, is essentially responsible for yeast exosome core activity.

The conserved core of the exosome, the major eukaryotic 3' --> 5' exonuclease, contains nine subunits that form a ring similar to the phosphorolytic bacterial PNPase and archaeal exosome, as well as Dis3. Dis3 is homologous to bacterial RNase II, a hydrolytic enzyme. Previous studies have suggested that all subunits are active 3' --> 5' exoRNases. We show here that Dis3 is responsible for exosome core activity. The purified exosome core has a hydrolytic, processive and Mg(2+)-dependent activity with characteristics similar to those of recombinant Dis3. Moreover, a catalytically inactive Dis3 mutant has no exosome core activity in vitro and shows in vivo RNA degradation phenotypes similar to those resulting from exosome depletion. In contrast, mutations in Rrp41, the only subunit carrying a conserved phosphorolytic site, appear phenotypically not different from wild-type yeast. We observed that the yeast exosome ring mediates interactions with protein partners, providing an explanation for its essential function