The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription. It is not clear how the N protein ...mediates such distinct functions. The N protein contains two RNA-binding domains surrounded by regions of intrinsic disorder. Phosphorylation of the central disordered region promotes the protein’s transcriptional function, but the underlying mechanism is not known. Here, we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates. Unmodified N protein forms partially ordered gel-like condensates and discrete 15-nm particles based on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces these interactions, generating a more liquid-like droplet. We propose that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing.
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•Nucleocapsid protein of SARS-CoV-2 forms biomolecular condensates with viral RNA•Unmodified N protein forms gel-like condensates containing discrete RNP particles•Phosphorylated N protein forms dynamic, liquid-like condensates•The two condensate forms are well suited for the two major functions of N protein
Carlson et al. demonstrate that the nucleocapsid (N) protein of SARS-CoV-2, together with viral RNA, forms gel-like biomolecular condensates and particles that are consistent with its genome-packaging role. Phosphorylation transforms condensates into liquid-like droplets, which may provide a cytoplasmic compartment to support the protein’s function in viral genome transcription.
Translation is the set of mechanisms by which ribosomes decode genetic messages as they synthesize polypeptides of a defined amino acid sequence. While the ribosome has been honed by evolution for ...high-fidelity translation, errors are inevitable. Aberrant mRNAs, mRNA structure, defective ribosomes, interactions between nascent proteins and the ribosomal exit tunnel, and insufficient cellular resources, including low tRNA levels, can lead to functionally irreversible stalls. Life thus depends on quality control mechanisms that detect, disassemble and recycle stalled translation intermediates.
R
ibosome-associated
Q
uality
C
ontrol (RQC) recognizes aberrant ribosome states and targets their potentially toxic polypeptides for degradation. Here we review recent advances in our understanding of RQC in bacteria, fungi, and metazoans. We focus in particular on an unusual modification made to the nascent chain known as a "CAT tail", or
C
arboxy-terminal
A
lanine and
T
hreonine tail, and the mechanisms by which ancient RQC proteins catalyze CAT-tail synthesis.
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BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Eukaryotic cells employ the ribosome-associated quality control complex (RQC) to maintain homeostasis despite defects that cause ribosomes to stall. The RQC comprises the E3 ubiquitin ligase Ltn1p, ...the ATPase Cdc48p, Rqc1p, and Rqc2p. Upon ribosome stalling and splitting, the RQC assembles on the 60S species containing unreleased peptidyl-tRNA (60S:peptidyl-tRNA). Ltn1p and Rqc1p facilitate ubiquitination of the incomplete nascent chain, marking it for degradation. Rqc2p stabilizes Ltn1p on the 60S and recruits charged tRNAs to the 60S to catalyze elongation of the nascent protein with carboxy-terminal alanine and threonine extensions (CAT tails). By mobilizing the nascent chain, CAT tailing can expose lysine residues that are hidden in the exit tunnel, thereby supporting efficient ubiquitination. If the ubiquitin-proteasome system is overwhelmed or unavailable, CAT-tailed nascent chains can aggregate in the cytosol or within organelles like mitochondria. Here we identify Vms1p as a tRNA hydrolase that releases stalled polypeptides engaged by the RQC.
Various kinases, including a cyclin-dependent kinase (CDK) family member, regulate the growth and functions of primary cilia, which perform essential roles in signaling and development. Neurological ...disorders linked to CDK-Like (CDKL) proteins suggest that these underexplored kinases may have similar functions. Here, we present the crystal structures of human CDKL1, CDKL2, CDKL3, and CDKL5, revealing their evolutionary divergence from CDK and mitogen-activated protein kinases (MAPKs), including an unusual αJ helix important for CDKL2 and CDKL3 activity. C. elegans CDKL-1, most closely related to CDKL1–4 and localized to neuronal cilia transition zones, modulates cilium length; this depends on its kinase activity and αJ helix-containing C terminus. Human CDKL5, linked to Rett syndrome, also localizes to cilia, and it impairs ciliogenesis when overexpressed. CDKL5 patient mutations modeled in CDKL-1 cause localization and/or cilium length defects. Together, our studies establish a disease model system suggesting cilium length defects as a pathomechanism for neurological disorders, including epilepsy.
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•CDKL2 and CDKL3 structures have distinct C-terminal αJ helix important for activity•Human CDKL5 and C. elegans CDKL-1 localize to and have roles in cilia•CDKL-1 cilia length control depends on kinase activity and C terminus with αJ helix•CDKL5 disease-linked mutations cause defects in CDKL-1 cilia length regulation
Canning et al. reveal distinct structural features of CDKL kinases and a role for C. elegans CDKL-1 in cilium length control, which is lost in a kinase-dead mutant and by introducing CDKL5 disease-linked mutations. The study suggests ciliary length impairment as a potential mechanism that contributes to human neurological disorders.
The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 is responsible for compaction of the ∼30-kb RNA genome in the ∼90-nm virion. Previous studies suggest that each virion ...contains 35 to 40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex. Here, we show that N protein, when combined in vitro with short fragments of the viral genome, forms 15-nm particles similar to the vRNP structures observed within virions. These vRNPs depend on regions of N protein that promote protein–RNA and protein–protein interactions. Phosphorylation of N protein in its disordered serine/arginine region weakens these interactions to generate less compact vRNPs. We propose that unmodified N protein binds structurally diverse regions in genomic RNA to form compact vRNPs within the nucleocapsid, while phosphorylation alters vRNP structure to support other N protein functions in viral transcription.
Ribosomes can stall during translation due to defects in the mRNA template or translation machinery, leading to the production of incomplete proteins. The Ribosome-associated Quality control Complex ...(RQC) engages stalled ribosomes and targets nascent polypeptides for proteasomal degradation. However, how each RQC component contributes to this process remains unclear. Here we demonstrate that key RQC activities-Ltn1p-dependent ubiquitination and Rqc2p-mediated Carboxy-terminal Alanine and Threonine (CAT) tail elongation-can be recapitulated in vitro with a yeast cell-free system. Using this approach, we determined that CAT tailing is mechanistically distinct from canonical translation, that Ltn1p-mediated ubiquitination depends on the poorly characterized RQC component Rqc1p, and that the process of CAT tailing enables robust ubiquitination of the nascent polypeptide. These findings establish a novel system to study the RQC and provide a framework for understanding how RQC factors coordinate their activities to facilitate clearance of incompletely synthesized proteins.
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.
Convergent evolutionary events in independent lineages provide an opportunity to understand why evolution favors certain outcomes over others. We studied such a case where a large set of genes-those ...coding for the ribosomal proteins-gained
-regulatory sequences for a particular transcription regulator (Mcm1) in independent fungal lineages. We present evidence that these gains occurred because Mcm1 shares a mechanism of transcriptional activation with an ancestral regulator of the ribosomal protein genes, Rap1. Specifically, we show that Mcm1 and Rap1 have the inherent ability to cooperatively activate transcription through contacts with the general transcription factor TFIID. Because the two regulatory proteins share a common interaction partner, the presence of one ancestral
-regulatory sequence can 'channel' random mutations into functional sites for the second regulator. At a genomic scale, this type of intrinsic cooperativity can account for a pattern of parallel evolution involving the fixation of hundreds of substitutions.
About the Authors: Emily D. Crawford Affiliations Chan Zuckerberg Biohub, San Francisco, California, United States of America, University of California San Francisco, Department of Microbiology and ...Immunology, San Francisco, California, United States of America Irene Acosta Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America Vida Ahyong Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America Erika C. Anderson Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Shaun Arevalo Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Daniel Asarnow Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Shannon Axelrod Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America Patrick Ayscue Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America Camillia S. Azimi Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Caleigh M. Azumaya Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Stefanie Bachl Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Iris Bachmutsky Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Aparna Bhaduri Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Jeremy Bancroft Brown Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Joshua Batson Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America Astrid Behnert Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Ryan M. Boileau Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Saumya R. Bollam Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Alain R. Bonny Affiliation: University of California San Francisco, Department of Biochemistry and Biophysics, San Francisco, California, United States of America David Booth Affiliation: University of California San Francisco, Department of Biochemistry and Biophysics, San Francisco, California, United States of America Michael Jerico B. Borja Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America David Brown Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Bryan Buie Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Cassandra E. Burnett Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Lauren E. Byrnes Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Katelyn A. Cabral Affiliations University of California San Francisco, School of Medicine, San Francisco, California, United States of America, University of California San Francisco, Institute for Neurodegenerative Diseases, San Francisco, California, United States of America Joana P. Cabrera Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America Saharai Caldera Affiliations Chan Zuckerberg Biohub, San Francisco, California, United States of America, University of California San Francisco, Division of Infectious Disease, San Francisco, California, United States of America Gabriela Canales Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Gloria R. Castañeda Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America Agnes Protacio Chan Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Christopher R. Chang Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Arthur Charles-Orszag Affiliations University of California San Francisco, School of Medicine, San Francisco, California, United States of America, Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of America Carly Cheung Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America Unseng Chio Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Eric D. Chow Affiliation: University of California San Francisco, Department of Biochemistry and Biophysics, San Francisco, California, United States of America Y. Rose Citron Affiliation: University of California, Berkeley, California, United States of America Allison Cohen Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Lillian B. Cohn Affiliations Chan Zuckerberg Biohub, San Francisco, California, United States of America, University of California San Francisco, Department of Experimental Medicine, San Francisco, California, United States of America Charles Chiu Affiliation: University of California San Francisco, Department of Laboratory Medicine, San Francisco, California, United States of America Mitchel A. Cole Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Daniel N. Conrad Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Angela Constantino Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Andrew Cote Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Tre’Jon Crayton-Hall Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America Spyros Darmanis Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America Angela M. Detweiler Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America Rebekah L. Dial Affiliation: University of California San Francisco, Department of Biochemistry and Biophysics, San Francisco, California, United States of America Shen Dong Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Elias M. Duarte Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America David Dynerman Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America Rebecca Egger Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America Alison Fanton Affiliation: University of California, Berkeley, California, United States of America Stacey M. Frumm Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Becky Xu Hua Fu Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Valentina E. Garcia Affiliation: University of California San Francisco, Department of Biochemistry and Biophysics, San Francisco, California, United States of America Julie Garcia Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Christina Gladkova Affiliations University of California San Francisco, School of Medicine, San Francisco, California, United States of America, Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of America Miriam Goldman Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Rafael Gomez-Sjoberg Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America M. Grace Gordon Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America James C. R. Grove Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Shweta Gupta Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Alexis Haddjeri-Hopkins Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Pierce Hadley Affiliations University of California San Francisco, School of Medicine, San Francisco, California, United States of America, University of California San Francisco, Institute for Neurodegenerative Diseases, San Francisco, California, United States of America John Haliburton Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America Samantha L. Hao Affiliation: Chan Zuckerberg Biohub, San Francisco, California, United States of America George Hartoularos Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Nadia Herrera Affiliation: University of California San Francisco, School of Medicine, San Francisco, California, United States of America Melissa Hilberg Affiliation: University of California San Francisco, Departm
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Comprehension of degraded speech requires higher-order expectations informed by prior knowledge. Accurate top-down expectations of incoming degraded speech cause a subjective semantic ...‘pop-out’ or conscious breakthrough experience. Indeed, the same stimulus can be perceived as meaningless when no expectations are made in advance. We investigated the event-related potential (ERP) correlates of these top-down expectations, their error signals and the subjective pop-out experience in healthy participants. We manipulated expectations in a word-pair priming degraded (noise-vocoded) speech task and investigated the role of top-down expectation with a between-groups attention manipulation. Consistent with the role of expectations in comprehension, repetition priming significantly enhanced perceptual intelligibility of the noise-vocoded degraded targets for attentive participants. An early ERP was larger for mismatched (i.e. unexpected) targets than matched targets, indicative of an initial error signal not reliant on top-down expectations. Subsequently, a P3a-like ERP was larger to matched targets than mismatched targets only for attending participants—i.e. a pop-out effect—while a later ERP was larger for mismatched targets and did not significantly interact with attention. Rather than relying on complex post hoc interactions between prediction error and precision to explain this apredictive pattern, we consider our data to be consistent with prediction error minimization accounts for early stages of processing followed by Global Neuronal Workspace-like breakthrough and processing in service of task goals.
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