Donnelly Centre for Cellular and Biomolecular Research

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A novel protein domain in an ancestral splicing factor drove the evolution of neural microexons.

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A novel protein domain in an ancestral splicing factor drove the evolution of neural microexons.

Nat Ecol Evol. 2019 04;3(4):691-701

Authors: Torres-Méndez A, Bonnal S, Marquez Y, Roth J, Iglesias M, Permanyer J, Almudí I, O'Hanlon D, Guitart T, Soller M, Gingras AC, Gebauer F, Rentzsch F, Blencowe BJ, Valcárcel J, Irimia M

Abstract
The mechanisms by which entire programmes of gene regulation emerged during evolution are poorly understood. Neuronal microexons represent the most conserved class of alternative splicing in vertebrates, and are critical for proper brain development and function. Here, we discover neural microexon programmes in non-vertebrate species and trace their origin to bilaterian ancestors through the emergence of a previously uncharacterized 'enhancer of microexons' (eMIC) protein domain. The eMIC domain originated as an alternative, neural-enriched splice isoform of the pan-eukaryotic Srrm2/SRm300 splicing factor gene, and subsequently became fixed in the vertebrate and neuronal-specific splicing regulator Srrm4/nSR100 and its paralogue Srrm3. Remarkably, the eMIC domain is necessary and sufficient for microexon splicing, and functions by interacting with the earliest components required for exon recognition. The emergence of a novel domain with restricted expression in the nervous system thus resulted in the evolution of splicing programmes that qualitatively expanded the neuronal molecular complexity in bilaterians.

PMID: 30833759 [PubMed - indexed for MEDLINE]



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Patterns of joint involvement in juvenile idiopathic arthritis and prediction of disease course: A prospective study with multilayer non-negative matrix factorization.

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Patterns of joint involvement in juvenile idiopathic arthritis and prediction of disease course: A prospective study with multilayer non-negative matrix factorization.

PLoS Med. 2019 Feb;16(2):e1002750

Authors: Eng SWM, Aeschlimann FA, van Veenendaal M, Berard RA, Rosenberg AM, Morris Q, Yeung RSM, ReACCh-Out Research Consortium

Abstract
BACKGROUND: Joint inflammation is the common feature underlying juvenile idiopathic arthritis (JIA). Clinicians recognize patterns of joint involvement currently not part of the International League of Associations for Rheumatology (ILAR) classification. Using unsupervised machine learning, we sought to uncover data-driven joint patterns that predict clinical phenotype and disease trajectories.
METHODS AND FINDINGS: We analyzed prospectively collected clinical data, including joint involvement using a standard 71-joint homunculus, for 640 discovery patients with newly diagnosed JIA enrolled in a Canada-wide study who were followed serially for five years, treatment-naïve except for nonsteroidal anti-inflammatory drugs (NSAIDs) and diagnosed within one year of symptom onset. Twenty-one patients had systemic arthritis, 300 oligoarthritis, 125 rheumatoid factor (RF)-negative polyarthritis, 16 RF-positive polyarthritis, 37 psoriatic arthritis, 78 enthesitis-related arthritis (ERA), and 63 undifferentiated arthritis. At diagnosis, we observed global hierarchical groups of co-involved joints. To characterize these patterns, we developed sparse multilayer non-negative matrix factorization (NMF). Model selection by internal bi-cross-validation identified seven joint patterns at presentation, to which all 640 discovery patients were assigned: pelvic girdle (57 patients), fingers (25), wrists (114), toes (48), ankles (106), knees (283), and indistinct (7). Patterns were distinct from clinical subtypes (P < 0.001 by χ2 test) and reproducible through external data set validation on a 119-patient, prospectively collected independent validation cohort (reconstruction accuracy Q2 = 0.55 for patterns; 0.35 for groups). Some patients matched multiple patterns. To determine whether their disease outcomes differed, we further subdivided the 640 discovery patients into three subgroups by degree of localization-the percentage of their active joints aligning with their assigned pattern: localized (≥90%; 359 patients), partially localized (60%-90%; 124), or extended (<60%; 157). Localized patients more often maintained their baseline patterns (P < 0.05 for five groups by permutation test) than nonlocalized patients (P < 0.05 for three groups by permutation test) over a five-year follow-up period. We modelled time to zero joints in the discovery cohort using a multivariate Cox proportional hazards model considering joint pattern, degree of localization, and ILAR subtype. Despite receiving more intense treatment, 50% of nonlocalized patients had zero joints at one year compared to six months for localized patients. Overall, localized patients required less time to reach zero joints (partial: P = 0.0018 versus localized by log-rank test; extended: P = 0.0057). Potential limitations include the requirement for patients to be treatment naïve (except NSAIDs), which may skew the patient cohorts towards milder disease, and the validation cohort size precluded multivariate analyses of disease trajectories.
CONCLUSIONS: Multilayer NMF identified patterns of joint involvement that predicted disease trajectory in children with arthritis. Our hierarchical unsupervised approach identified a new clinical feature, degree of localization, which predicted outcomes in both cohorts. Detailed assessment of every joint is already part of every musculoskeletal exam for children with arthritis. Our study supports both the continued collection of detailed joint involvement and the inclusion of patterns and degrees of localization to stratify patients and inform treatment decisions. This will advance pediatric rheumatology from counting joints to realizing the potential of using data available from uncovering patterns of joint involvement.

PMID: 30807586 [PubMed - in process]



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Complex modifier landscape underlying genetic background effects.

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Complex modifier landscape underlying genetic background effects.

Proc Natl Acad Sci U S A. 2019 03 12;116(11):5045-5054

Authors: Hou J, Tan G, Fink GR, Andrews BJ, Boone C

Abstract
The phenotypic consequence of a given mutation can be influenced by the genetic background. For example, conditional gene essentiality occurs when the loss of function of a gene causes lethality in one genetic background but not another. Between two individual Saccharomyces cerevisiae strains, S288c and Σ1278b, ∼1% of yeast genes were previously identified as "conditional essential." Here, in addition to confirming that some conditional essential genes are modified by a nonchromosomal element, we show that most cases involve a complex set of genomic modifiers. From tetrad analysis of S288C/Σ1278b hybrid strains and whole-genome sequencing of viable hybrid spore progeny, we identified complex sets of multiple genomic regions underlying conditional essentiality. For a smaller subset of genes, including CYS3 and CYS4, each of which encodes components of the cysteine biosynthesis pathway, we observed a segregation pattern consistent with a single modifier associated with conditional essentiality. In natural yeast isolates, we found that the CYS3/CYS4 conditional essentiality can be caused by variation in two independent modifiers, MET1 and OPT1, each with roles associated with cellular cysteine physiology. Interestingly, the OPT1 allelic variation appears to have arisen independently from separate lineages, with rare allele frequencies below 0.5%. Thus, while conditional gene essentiality is usually driven by genetic interactions associated with complex modifier architectures, our analysis also highlights the role of functionally related, genetically independent, and rare variants.

PMID: 30804202 [PubMed - indexed for MEDLINE]



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Dimerization of a ubiquitin variant leads to high affinity interactions with a ubiquitin interacting motif.

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Dimerization of a ubiquitin variant leads to high affinity interactions with a ubiquitin interacting motif.

Protein Sci. 2019 Feb 21;:

Authors: Manczyk N, Veggiani G, Gish GD, Yates BP, Ernst A, Sidhu SS, Sicheri F

Abstract
We previously described structural and functional characterization of the first ubiquitin variant (UbV), UbV.v27.1, engineered by phage display to bind with high affinity to a specific ubiquitin interacting motif (UIM). We identified two substitutions relative to ubiquitin (Gly10Val/His68Tyr) that were critical for enhancing binding affinity but could only rationalize the mechanism of action of the Tyr68 substitution. Here we extend our characterization and uncover the mechanism by which the Val10 substitution enhances binding affinity. We show that Val10 in UbV.v27.1 drives UbV dimerization through an intermolecular β-strand exchange. Dimerization serves to increase the contact surface between the UIM and UbV and also affords direct contacts between two UIMs through an overall 2:2 binding stoichiometry. Our identification of the role of Val10 in UbV dimerization suggests a general means for the development of dimeric UbVs with improved affinity and specificity relative to their monomeric UbV counterparts. This article is protected by copyright. All rights reserved.

PMID: 30793400 [PubMed - as supplied by publisher]



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Analysis of Mutants Suggests Kamin Blocking in C. elegans is Due to Interference with Memory Recall Rather than Storage.

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Analysis of Mutants Suggests Kamin Blocking in C. elegans is Due to Interference with Memory Recall Rather than Storage.

Sci Rep. 2019 Feb 20;9(1):2371

Authors: Merritt DM, Melkis JG, Kwok B, Tran C, van der Kooy D

Abstract
Higher-order conditioning phenomena, including context conditioning and blocking, occur when conditioning to one set of stimuli interacts with conditioning to a second set of stimuli to modulate the strength of the resultant memories. Here we analyze higher-order conditioning in the nematode worm Caenorhabditis elegans, demonstrating for the first time the presence of blocking in this animal, and dissociating it from context conditioning. We present an initial genetic dissection of these phenomena in a model benzaldehyde/NH4Cl aversive learning system, and suggest that blocking may involve an alteration of memory retrieval rather than storage. These findings offer a fundamentally different explanation for blocking than traditional explanations, and position C. elegans as a powerful model organism for the study of higher order conditioning.

PMID: 30787354 [PubMed - in process]



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Yeast Two-Hybrid Analysis for Ubiquitin-Variant Inhibitors of Human Deubiquitinases.

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Yeast Two-Hybrid Analysis for Ubiquitin-Variant Inhibitors of Human Deubiquitinases.

J Mol Biol. 2019 Feb 11;:

Authors: Pascoe N, Seetharaman A, Teyra J, Manczyk N, Satori MA, Tjandra D, Makhnevych T, Schwerdtfeger C, Brasher BB, Moffat J, Costanzo M, Boone C, Sicheri F, Sidhu S

Abstract
We applied a Yeast-Two-Hybrid (Y2H) analysis to screen for ubiquitin variant (UbV) inhibitors of a human deubiquitinase (DUB), ubiquitin-specific protease 2 (USP2). The Y2H screen used USP2 as the bait, and a prey library consisting of UbVs randomized at four specific positions, which were known to interact with USP2 from phage display analysis. The screen yielded numerous UbVs that bound to USP2 both as a Y2H interaction in vivo and as purified proteins in vitro. The Y2H-derived UbVs inhibited the catalytic activity of USP2 in vitro with nanomolar-range potencies, and they bound and inhibited USP2 in human cells. Mutational and structural analysis showed that potent and selective inhibition could be achieved by just two substitutions in a UbV, which exhibited improved hydrophobic and hydrophilic contacts compared to the wild-type ubiquitin interaction with USP2. Our results establish Y2H as an effective platform for the development of UbV inhibitors of DUBs in vivo, providing an alternative strategy for the analysis of DUBs that are recalcitrant to phage display and other in vitro methods.

PMID: 30763569 [PubMed - as supplied by publisher]



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Transplantation of Directly Reprogrammed Human Neural Precursor Cells Following Stroke Promotes Synaptogenesis and Functional Recovery.

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Transplantation of Directly Reprogrammed Human Neural Precursor Cells Following Stroke Promotes Synaptogenesis and Functional Recovery.

Transl Stroke Res. 2019 Feb 12;:

Authors: Vonderwalde I, Azimi A, Rolvink G, Ahlfors JE, Shoichet MS, Morshead CM

Abstract
Stroke is one of the leading causes of long-term disability. Cell transplantation is a promising strategy to treat stroke. We explored the efficacy of directly reprogrammed human neural precursor cell (drNPC) transplants to promote functional recovery in a model of focal ischemic stroke in the mouse sensorimotor cortex. We show that drNPCs express neural precursor cell markers and are neurally committed at the time of transplantation. Mice that received drNPC transplants recovered motor function, irrespective of transplant vehicle or recipient sex, and with no correlation to lesion volume or glial scarring. The majority of drNPCs found in vivo, at the time of functional recovery, remained undifferentiated. Notably, no correlation between functional recovery and long-term xenograft survival was observed, indicating that drNPCs provide therapeutic benefits beyond their survival. Furthermore, increased synaptophysin expression in transplanted brains suggests that drNPCs promote neuroplasticity through enhanced synaptogenesis. Our findings provide insight into the mechanistic underpinnings of drNPC-mediated recovery for stroke and support the notion that drNPCs may have clinical applications for stroke therapy.

PMID: 30747366 [PubMed - as supplied by publisher]



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CNTN5-/+or EHMT2-/+human iPSC-derived neurons from individuals with autism develop hyperactive neuronal networks.

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CNTN5-/+or EHMT2-/+human iPSC-derived neurons from individuals with autism develop hyperactive neuronal networks.

Elife. 2019 Feb 12;8:

Authors: Deneault E, Faheem M, White SH, Rodrigues DC, Sun S, Wei W, Piekna A, Thompson T, Howe JL, Chalil L, Kwan V, Walker S, Pasceri P, Roth FP, Yuen RK, Singh KK, Ellis J, Scherer SW

Abstract
Induced pluripotent stem cell (iPSC)-derived neurons are increasingly used to model Autism Spectrum Disorder (ASD), which is clinically and genetically heterogeneous. To study the complex relationship of penetrant and weaker polygenic risk variants to ASD, 'isogenic' iPSC-derived neurons are critical. We developed a set of procedures to control for heterogeneity in reprogramming and differentiation, and generated 53 different iPSC-derived glutamatergic neuronal lines from 25 participants from 12 unrelated families with ASD. Heterozygous de novo and rare-inherited presumed-damaging variants were characterized in ASD risk genes/loci. Combinations of putative etiologic variants (GLI3/KIF21A or EHMT2/UBE2I) in separate families were modeled. We used a multi-electrode array, with patch-clamp recordings, to determine a reproducible synaptic phenotype in 25% of the individuals with ASD (other relevant data on the remaining lines was collected). Our most compelling new results revealed a consistent spontaneous network hyperactivity in neurons deficient for CNTN5 or EHMT2. The biobank of iPSC-derived neurons and accompanying genomic data are available to accelerate ASD research.
Editorial note: This article has been through an editorial process in which authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

PMID: 30747104 [PubMed - in process]



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Identification and Functional Testing of Novel Interacting Protein Partners for the Stress Sensors Wsc1p and Mid2p of Saccharomyces cerevisiae.

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Identification and Functional Testing of Novel Interacting Protein Partners for the Stress Sensors Wsc1p and Mid2p of Saccharomyces cerevisiae.

G3 (Bethesda). 2019 Feb 07;:

Authors: Santiago-Cartagena E, González-Crespo S, Vélez V, Martínez N, Snider J, Jessulat M, Aoki H, Minic Z, Akamine P, Mejías I, Pérez LM, Rymond BC, Babu M, Stagljar I, Rodríguez-Medina JR

Abstract
Wsc1p and Mid2p are transmembrane signaling proteins of cell wall stress in the budding yeast Saccharomyces cerevisiae. When an environmental stress compromises cell wall integrity, they activate a cell response through the Cell Wall Integrity (CWI) pathway. Studies have shown that the cytoplasmic domain of Wsc1p initiates the CWI signaling cascade by interacting with Rom2p, a Rho1-GDP-GTP exchange factor. Binding of Rom2p to the cytoplasmic tail of Wsc1p requires dephosphorylation of specific serine residues but the mechanism by which the sensor is dephosphorylated and how it subsequently interacts with Rom2p remains unclear. We hypothesize that Wsc1p and Mid2p must be physically associated with interacting proteins other than Rom2p that facilitate its interaction and regulate the activation of CWI pathway. To address this, a cDNA plasmid library of yeast proteins was expressed in bait strains bearing membrane yeast two-hybrid (MYTH) reporter modules of Wsc1p and Mid2p, and their interacting preys were recovered and sequenced. 14 previously unreported interactors were confirmed for Wsc1p and 29 for Mid2p. The interactors' functionality were assessed by cell growth assays and CWI pathway activation by western blot analysis of Slt2p/Mpk1p phosphorylation in null mutants of each interactor under defined stress conditions. The susceptibility of these strains to different stresses were tested against antifungal agents and chemicals. This study reports important novel protein interactions of Wsc1p and Mid2p that are associated with the cellular response to oxidative stress induced by Hydrogen Peroxide and cell wall stress induced by Caspofungin.

PMID: 30733383 [PubMed - as supplied by publisher]



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Rad5 Recruits Error-Prone DNA Polymerases for Mutagenic Repair of ssDNA Gaps on Undamaged Templates.

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Rad5 Recruits Error-Prone DNA Polymerases for Mutagenic Repair of ssDNA Gaps on Undamaged Templates.

Mol Cell. 2019 03 07;73(5):900-914.e9

Authors: Gallo D, Kim T, Szakal B, Saayman X, Narula A, Park Y, Branzei D, Zhang Z, Brown GW

Abstract
Post-replication repair (PRR) allows tolerance of chemical- and UV-induced DNA base lesions in both an error-free and an error-prone manner. In classical PRR, PCNA monoubiquitination recruits translesion synthesis (TLS) DNA polymerases that can replicate through lesions. We find that PRR responds to DNA replication stress that does not cause base lesions. Rad5 forms nuclear foci during normal S phase and after exposure to types of replication stress where DNA base lesions are likely absent. Rad5 binds to the sites of stressed DNA replication forks, where it recruits TLS polymerases to repair single-stranded DNA (ssDNA) gaps, preventing mitotic defects and chromosome breaks. In contrast to the prevailing view of PRR, our data indicate that Rad5 promotes both mutagenic and error-free repair of undamaged ssDNA that arises during physiological and exogenous replication stress.

PMID: 30733119 [PubMed - indexed for MEDLINE]



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