Cytosolic condensates rich in polyserine define subcellular sites of tau aggregation.

Tau aggregates are a hallmark of multiple neurodegenerative diseases and can contain RNAs and RNA-binding proteins, including serine/arginine repetitive matrix protein 2 (SRRM2) and pinin (PNN). However, how these nuclear proteins mislocalize and their influence on the prion-like propagation of tau aggregates is unknown. We demonstrate that polyserine repeats in SRRM2 and PNN are necessary and sufficient for recruitment to tau aggregates. Moreover, we show tau aggregates preferentially grow in association with endogenous cytoplasmic assemblies-mitotic interchromatin granules and cytoplasmic speckles (CSs)-which contain SRRM2 and PNN. Polyserine overexpression in cells nucleates assemblies that are sites of tau aggregate growth. Further, modulating the levels of polyserine-containing proteins results in a corresponding change in tau aggregation. These findings define a specific protein motif, and cellular condensates, that promote tau aggregate propagation. As CSs form in induced pluripotent stem cell (iPSC) derived neurons under inflammatory or hyperosmolar stress, they may affect tau aggregate propagation in neurodegenerative disease.

Impact of vaccination and variants of concern on long COVID clinical phenotypes.

BACKGROUND: Defining patterns of symptoms in long COVID is necessary to advance therapies for this heterogeneous condition. Here we aimed to describe clusters of symptoms in individuals with long COVID and explore the impact of the emergence of variants of concern (VOCs) and vaccination on these clusters. METHODS: In a prospective, multi centre cohort study, individuals with symptoms persisting > 4 weeks from acute COVID-19 were divided into two groups based on timing of acute infection; pre-Alpha VOC, denoted wild type (WT) group and post-Alpha VOC (incorporating alpha and delta dominant periods) denoted VOC group. We used multiple correspondence analysis (MCA) and hierarchical clustering in the WT and VOC groups to identify symptom clusters. We then used logistic regression to explore factors associated with individual symptoms. RESULTS: A total of 417 individuals were included in the analysis, 268 in WT and 149 in VOC groups respectively. In both groups MCA identified three similar clusters; a musculoskeletal (MSK) cluster characterised by joint pain and myalgia, a cardiorespiratory cluster and a less symptomatic cluster. Differences in characteristic symptoms were only seen in the cardiorespiratory cluster where a decrease in the frequency of palpitations (10% vs 34% p = 0.008) and an increase in cough (63% vs 17% p < 0.001) in the VOC compared to WT groups was observed. Analysis of the frequency of individual symptoms showed significantly lower frequency of both chest pain (25% vs 39% p = 0.004) and palpitations (12% vs 32% p < 0.001) in the VOC group compared to the WT group. In adjusted analysis being in the VOC group was significantly associated with a lower odds of both chest pain and palpitations, but vaccination was not associated with these symptoms. CONCLUSION: This study suggests changes in long COVID phenotype in individuals infected later in the pandemic, with less palpitations and chest pain reported. Adjusted analyses suggest that these effects are mediated through introduction of variants rather than an effect from vaccination.

A protein interaction map of the LSU processome.

Maturation of the large ribosomal subunit (LSU) in eukaryotes is a complex and highly coordinated process that requires the concerted action of a large, dynamic, ribonucleoprotein complex, the LSU processome. While we know that >80 ribosome biogenesis factors are required throughout the course of LSU assembly, little is known about how these factors interact with each other within the LSU processome. To interrogate its organization and architecture, we took a systems biology approach and performed a semi-high-throughput, array-based, directed yeast two-hybrid assay. Assaying 4800 protein-protein interactions, we identified 232 high-confidence, binary-interacting protein pairs, representing a fourfold increase from current knowledge. The resulting LSU processome interactome map has enhanced our understanding of the organization and function of the biogenesis factors within the LSU processome, revealing both novel and previously identified subcomplexes and hub proteins, including Nop4.

H/ACA snoRNA levels are regulated during stem cell differentiation.

H/ACA small nucleolar RNAs (snoRNAs) guide pseudouridylation as part of a small nucleolar ribonucleoprotein complex (snoRNP). Disruption of H/ACA snoRNA levels in stem cells impairs pluripotency, yet it remains unclear how H/ACA snoRNAs contribute to differentiation. To determine if H/ACA snoRNA levels are dynamic during differentiation, we comprehensively profiled H/ACA snoRNA abundance in multiple murine cell types and during differentiation in three cellular models, including mouse embryonic stem cells and mouse myoblasts. We determined that the profiles of H/ACA snoRNA abundance are cell-type specific, and we identified a subset of snoRNAs that are specifically regulated during differentiation. Additionally, we demonstrated that a decrease in Snora27 abundance upon differentiation corresponds to a decrease in pseudouridylation of its target site within the E-site transfer RNA (tRNA) binding region of the 28S ribosomal RNA (rRNA) in the large ribosomal subunit. Together, these data point toward a potential model in which H/ACA snoRNAs are specifically regulated during differentiation to alter pseudouridylation and fine tune ribosome function.

Driving nucleolar assembly.

In this issue of Genes & Development, Grob and colleagues (pp. 220-230) identify the minimal molecular requirements to assemble a fully functional nucleolus in human cells and demonstrate the importance of the nucleolar transcription factor upstream binding factor (UBF) as a mitotic bookmark at the ribosomal DNA (rDNA).

Long noncoding RNAs as sinks in Prader-Willi syndrome.

In this issue of Molecular Cell, Yin et al. (2012) identify a class of long noncoding RNAs (lncRNAs) and propose a new mechanism as to how they contribute to the pathogenesis of Prader-Willi syndrome.

A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization.

Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. Here we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an "L"-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conserved basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Thus, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins.

Tissue specific roles for the ribosome biogenesis factor Wdr43 in zebrafish development.

During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.

NOL11, implicated in the pathogenesis of North American Indian childhood cirrhosis, is required for pre-rRNA transcription and processing.

The fundamental process of ribosome biogenesis requires hundreds of factors and takes place in the nucleolus. This process has been most thoroughly characterized in baker's yeast and is generally well conserved from yeast to humans. However, some of the required proteins in yeast are not found in humans, raising the possibility that they have been replaced by functional analogs. Our objective was to identify non-conserved interaction partners for the human ribosome biogenesis factor, hUTP4/Cirhin, since the R565W mutation in the C-terminus of hUTP4/Cirhin was reported to cause North American Indian childhood cirrhosis (NAIC). By screening a yeast two-hybrid cDNA library derived from human liver, and through affinity purification followed by mass spectrometry, we identified an uncharacterized nucleolar protein, NOL11, as an interaction partner for hUTP4/Cirhin. Bioinformatic analysis revealed that NOL11 is conserved throughout metazoans and their immediate ancestors but is not found in any other phylogenetic groups. Co-immunoprecipitation experiments show that NOL11 is a component of the human ribosomal small subunit (SSU) processome. siRNA knockdown of NOL11 revealed that it is involved in the cleavage steps required to generate the mature 18S rRNA and is required for optimal rDNA transcription. Furthermore, abnormal nucleolar morphology results from the absence of NOL11. Finally, yeast two-hybrid analysis shows that NOL11 interacts with the C-terminus of hUTP4/Cirhin and that the R565W mutation partially disrupts this interaction. We have therefore identified NOL11 as a novel protein required for the early stages of ribosome biogenesis in humans. Our results further implicate a role for NOL11 in the pathogenesis of NAIC.

Metabolism of deoxypyrimidines and deoxypyrimidine antiviral analogs in isolated brain mitochondria.

The goal of this project was to characterize deoxypyrimidine salvage pathways used to maintain deoxynucleoside triphosphate pools in isolated brain mitochondria and to determine the extent that antiviral pyrimidine analogs utilize or affect these pathways. Mitochondria from rat brains were incubated in media with labeled and unlabeled deoxynucleosides and deoxynucleoside analogs. Products were analyzed by HPLC coupled to an inline UV monitor and liquid scintillation counter. Isolated mitochondria transported thymidine and deoxycytidine into the matrix, and readily phosphorylated both of these to mono-, di-, and tri-phosphate nucleotides. Rates of phosphorylation were much higher than rates observed in mitochondria from heart and liver. Deoxyuridine was phosphorylated much more slowly than thymidine and only to dUMP. 3'-azido-3'-deoxythymidine, zidovudine (AZT), an antiviral thymidine analog, was phosphorylated to AZT-MP as readily as thymidine was phosphorylated to TMP, but little if any AZT-DP or AZT-TP was observed. AZT at 5.5 ± 1.7 µM was shown to inhibit thymidine phosphorylation by 50%, but was not observed to inhibit deoxycytidine phosphorylation except at levels > 100 µM. Stavudine and lamivudine were inert when incubated with isolated brain mitochondria. The kinetics of phosphorylation of thymidine, dC, and AZT were significantly different in brain mitochondria compared to mitochondria from liver and heart.

Identification of Distinct Long COVID Clinical Phenotypes Through Cluster Analysis of Self-Reported Symptoms.

Background: We aimed to describe the clinical presentation of individuals presenting with prolonged recovery from coronavirus disease 2019 (COVID-19), known as long COVID. Methods: This was an analysis within a multicenter, prospective cohort study of individuals with a confirmed diagnosis of COVID-19 and persistent symptoms >4 weeks from onset of acute symptoms. We performed a multiple correspondence analysis (MCA) on the most common self-reported symptoms and hierarchical clustering on the results of the MCA to identify symptom clusters. Results: Two hundred thirty-three individuals were included in the analysis; the median age of the cohort was 43 (interquartile range [IQR], 36-54) years, 74% were women, and 77.3% reported a mild initial illness. MCA and hierarchical clustering revealed 3 clusters. Cluster 1 had predominantly pain symptoms with a higher proportion of joint pain, myalgia, and headache; cluster 2 had a preponderance of cardiovascular symptoms with prominent chest pain, shortness of breath, and palpitations; and cluster 3 had significantly fewer symptoms than the other clusters (2 [IQR, 2-3] symptoms per individual in cluster 3 vs 6 [IQR, 5-7] and 4 [IQR, 3-5] in clusters 1 and 2, respectively; P < .001). Clusters 1 and 2 had greater functional impairment, demonstrated by significantly longer work absence, higher dyspnea scores, and lower scores in SF-36 domains of general health, physical functioning, and role limitation due to physical functioning and social functioning. Conclusions: Clusters of symptoms are evident in long COVID patients that are associated with functional impairments and may point to distinct underlying pathophysiologic mechanisms of disease.

The National Children's Study Archive Model: A 3-Tier Framework for Dissemination of Data and Specimens for General Use and Secondary Analysis.

The National Children's Study (NCS) Archive was created as a repository of samples, data, and information from the NCS Vanguard Study-a longitudinal pregnancy and birth cohort evaluating approaches to study influence of environmental exposures on child health and development-to provide qualified researchers with access to NCS materials for use in secondary research. The National Children's Study Archive (NCSA) model is a 3-tiered access model designed to make the wealth of information and materials gathered during the NCS Vanguard Study available at a user appropriate level. The NCSA model was developed as a 3-tier framework, for users of varying access levels, providing intuitive data exploration and visualization tools, an end-to-end data and sample request management system, and a restricted portal for participant-level data access with a team of experts available to assist users. This platform provides a model to accelerate transformation of information and materials from existing studies into new scientific discoveries. Trial Registration: ClinicalTrials.gov Identifier: NCT00852904 (first posted February 27, 2009).

Dynamic Change and Clinical Relevance of Postinfectious SARS-CoV-2 Antibody Responses.

BACKGROUND: Although reports suggest that most individuals with coronavirus disease 2019 (COVID-19) develop detectable antibodies postinfection, the kinetics, durability, and relative differences between immunoglobulin M (IgM) and immunoglobulin G (IgG) responses beyond the first few weeks after symptom onset remain poorly understood. METHODS: Within a large, well-phenotyped, diverse, prospective cohort of subjects with and without severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction (PCR)-confirmed infection and historical controls derived from cohorts with high prevalence of viral coinfections and samples taken during prior flu seasons, we measured SARS-CoV-2 serological responses (both IgG and IgM) using commercially available assays. We calculated sensitivity, specificity, and relationship with disease severity and mapped the kinetics of antibody responses over time using generalized additive models. RESULTS: We analyzed 1001 samples from 752 subjects, 327 with confirmed SARS-CoV-2 (29.7% with severe disease) spanning a period of 90 days from symptom onset. Sensitivity was lower (44.1%-47.1%) early (<10 days) after symptom onset but increased to >80% after 10 days. IgM positivity increased earlier than IgG-targeted assays, but positivity peaked between days 32 and 38 post-onset of symptoms and declined thereafter, a dynamic that was confirmed when antibody levels were analyzed, with a more rapid decline observed with IgM. Early (<10 days) IgM but not IgG levels were significantly higher in those who subsequently developed severe disease (signal/cutoff 4.20 [0.75-17.93] vs 1.07 [0.21-5.46]; P = .048). CONCLUSIONS: This study suggests that postinfectious antibody responses in those with confirmed COVID-19 begin to decline relatively early postinfection and suggests a potential role for higher IgM levels early in infection in the prediction of subsequent disease severity.

Fine-mapping reveals novel alternative splicing of the dopamine transporter.

The dopamine transporter gene (SLC6A3, DAT) has been implicated in the pathogenesis of numerous psychiatric and neurodevelopmental disorders, including schizophrenia (SZ). We previously detected association between SZ and intronic SLC6A3 variants that replicated in two independent Caucasian samples, but had no obvious function. In follow-up analyses, we sequenced the coding and intronic regions of SLC6A3 to identify complete linkage disequilibrium patterns of common variations. We genotyped 78 polymorphisms, narrowing the potentially causal region to two correlated clusters of associated SNPs localized predominantly to introns 3 and 4. Our computational analysis of these intronic regions predicted a novel cassette exon within intron 3, designated E3b, which is conserved among primates. We confirmed alternative splicing of E3b in post-mortem human substantia nigra (SN). As E3b introduces multiple in-frame stop codons, the SLC6A3 open reading frame is truncated and the spliced product may undergo nonsense mediated decay. Thus, factors that increase E3b splicing could reduce the amount of unspliced product available for translation. Observations consistent with this prediction were made using cellular assays and in post-mortem human SN. In mini-gene constructs, the extent of splicing is also influenced by at least two common haplotypes, so the alternative splicing was evaluated in relation to SZ risk. Meta-analyses across genome-wide association studies did not support the initial associations and further post-mortem studies did not suggest case-control differences in splicing. These studies do not provide a compelling link to schizophrenia. However, the impact of the alternative splicing on other neuropsychiatric disorders should be investigated. © 2010 Wiley-Liss, Inc.

Association of Early-Life Social and Digital Media Experiences With Development of Autism Spectrum Disorder-Like Symptoms.

Importance: Despite growing evidence that parent-child interactions and time viewing digital media affect child development, these factors have rarely been studied in association with autism spectrum disorder (ASD) symptoms. Objective: To determine the association of experiential factors, including social activities and screen viewing in the first 18 months of life, perinatal factors, and demographic factors, with ASD-like symptoms and risk on the Modified Checklist for Autism in Toddlers (M-CHAT) at 2 years. Design, Setting, and Participants: Data for this cohort study were derived from the National Children's Study, a US multicenter epidemiological study of environmental influences on child health and development. A total of 2152 children were enrolled at birth from October 1, 2010, to October 31, 2012. Data were analyzed from December 1, 2017, to December 3, 2019. Exposures: Caregivers reported whether the child viewed television and/or videos (yes or no) at 12 months of age, hours of viewing at 18 months of age, time spent by the caregiver reading to the child (number of days per week compared with daily) at 12 months of age, and frequency of playing with the child (daily or less than daily) at 12 months of age. Prematurity, maternal age at birth, child sex, household income, race/ethnicity, and caregiver English-language status were included in analysis. Main Outcomes and Measures: Significant association of exposures with ASD risk by M-CHAT and/or ASD-like symptoms assessed by revised M-CHAT (M-CHAT-R) total score in multiple regression models. Results: Among the 2152 children included in the analysis (1099 boys [51.1%]), television and/or video viewing (yes or no) at 12 months of age was significantly associated with greater ASD-like symptoms at 2 years of age (change, 4.2%; 95% CI, 0.1%-8.3%) but not with ASD risk (risk prevalence rates, 8.3% vs 4.4%; adjusted odds ratio [AOR], 1.40; 95% CI, 0.86-2.29). Similarly, parent-child play daily compared with less than daily was significantly associated with fewer ASD-like symptoms at 2 years of age (change, -8.9%; 95% CI, -16.5% to -0.9%) but not with ASD risk (risk prevalence rates, 6.4% vs 14.0%; AOR, 0.58; 95% CI, 0.31-1.08). However, high screen viewing at 18 months of age was not significantly associated with ASD-like symptoms (change, 10.7%; 95% CI, -2.0% to 23.0%) or ASD risk by M-CHAT (AOR, 1.18; 95% CI, 0.56-2.49) at 2 years of age. Conclusions and Relevance: This cohort study found greater screen exposure and less caregiver-child play early in life to be associated with later ASD-like symptoms. Further research is needed to evaluate experiential factors for potential risk or protective effects in ASD.

Spectroscopic investigations and potential energy surfaces of the ground and excited electronic states of 1,3-benzodioxan.

The laser-induced fluorescence spectra (both fluorescence excitation and dispersed fluorescence) of jet-cooled 1,3-benzodioxan along with its ultraviolet absorption spectra have been recorded and analyzed in order to determine the vibrational quantum levels in both the ground and S(1)(pi,pi(*)) electronic excited states. A detailed energy map of the vibrational levels involving the six lowest frequency vibrations was established and utilized to better understand the structural and conformational differences between the ground and excited electronic states. The energies of more than a dozen vibrational excited states involving the out-of-plane ring twisting (nu(47)) and the out-of-plane ring bending (nu(48)) modes were determined for both S(0) and S(1) electronic states. Ab initio and density functional theory (DFT) calculations were also carried out to complement the experimental work. The data allowed one-dimensional potential energy functions in terms of the ring-twisting coordinate to be calculated. These show the molecule to have a twisting angle of 33 degrees and a barrier to planarity of 4300+/-500 cm(-1) for the S(0) ground state and an angle of 24 degrees and a barrier of 1500+/-200 cm(-1) for the S(1)(pi,pi(*)) excited state.

Engineering a conserved RNA regulatory protein repurposes its biological function in vivo.

PUF (PUmilio/FBF) RNA-binding proteins recognize distinct elements. In C. elegans, PUF-8 binds to an 8-nt motif and restricts proliferation in the germline. Conversely, FBF-2 recognizes a 9-nt element and promotes mitosis. To understand how motif divergence relates to biological function, we first determined a crystal structure of PUF-8. Comparison of this structure to that of FBF-2 revealed a major difference in a central repeat. We devised a modified yeast 3-hybrid screen to identify mutations that confer recognition of an 8-nt element to FBF-2. We identified several such mutants and validated structurally and biochemically their binding to 8-nt RNA elements. Using genome engineering, we generated a mutant animal with a substitution in FBF-2 that confers preferential binding to the PUF-8 element. The mutant largely rescued overproliferation in animals that spontaneously generate tumors in the absence of puf-8. This work highlights the critical role of motif length in the specification of biological function.

Diverse Regulators of Human Ribosome Biogenesis Discovered by Changes in Nucleolar Number.

Ribosome biogenesis is a highly regulated, essential cellular process. Although studies in yeast have established some of the biological principles of ribosome biogenesis, many of the intricacies of its regulation in higher eukaryotes remain unknown. To understand how ribosome biogenesis is globally integrated in human cells, we conducted a genome-wide siRNA screen for regulators of nucleolar number. We found 139 proteins whose depletion changed the number of nucleoli per nucleus from 2-3 to only 1 in human MCF10A cells. Follow-up analyses on 20 hits found many (90%) to be essential for the nucleolar functions of rDNA transcription (7), pre-ribosomal RNA (pre-rRNA) processing (16), and/or global protein synthesis (14). This genome-wide analysis exploits the relationship between nucleolar number and function to discover diverse cellular pathways that regulate the making of ribosomes and paves the way for further exploration of the links between ribosome biogenesis and human disease.

The molecular basis for ANE syndrome revealed by the large ribosomal subunit processome interactome.

ANE syndrome is a ribosomopathy caused by a mutation in an RNA recognition motif of RBM28, a nucleolar protein conserved to yeast (Nop4). While patients with ANE syndrome have fewer mature ribosomes, it is unclear how this mutation disrupts ribosome assembly. Here we use yeast as a model system and show that the mutation confers growth and pre-rRNA processing defects. Recently, we found that Nop4 is a hub protein in the nucleolar large subunit (LSU) processome interactome. Here we demonstrate that the ANE syndrome mutation disrupts Nop4's hub function by abrogating several of Nop4's protein-protein interactions. Circular dichroism and NMR demonstrate that the ANE syndrome mutation in RRM3 of human RBM28 disrupts domain folding. We conclude that the ANE syndrome mutation generates defective protein folding which abrogates protein-protein interactions and causes faulty pre-LSU rRNA processing, thus revealing one aspect of the molecular basis of this human disease.