Reconstitution of the SARS-CoV-2 ribonucleosome provides insights into genomic RNA packaging and regulation by phosphorylation.

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.

The proximity of the N- and C- termini of bovine knob domains enable engineering of target specificity into polypeptide chains.

Cysteine-rich knob domains can be isolated from the ultralong heavy-chain complementarity-determining region (CDR) 3, which are unique to a subset of bovine antibodies, to create antibody fragments of ~4 kDa. Advantageously, the N- and C- termini of these small binding domains are in close proximity, and we propose that this may offer a practical route to engineer extrinsic binding specificity into proteins. To test this, we transplanted knob domains into various loops of rat serum albumin, targeting sites that were distal to the interface with the neonatal Fc receptor. Using knob domains raised against the clinically validated drug target complement component C5, we produced potent inhibitors, which exhibit an extended plasma half-life in vivo via attenuated renal clearance and neonatal Fc receptor-mediated avoidance of lysosomal catabolism. The same approach was also used to modify a Camelid VHH, targeting a framework loop situated at the opposing end of the domain to the CDRs, to produce a small, single-chain bispecific antibody and a dual inhibitor of Complement C3 and C5. This study presents new protein inhibitors of the complement cascade and demonstrates a broadly applicable method to engineer target specificity within polypeptide chains, using bovine knob domains.

Ribosome-associated quality control and CAT tailing.

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. Ribosome-associated Quality Control (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 Carboxy-terminal Alanine and Threonine tail, and the mechanisms by which ancient RQC proteins catalyze CAT-tail synthesis.

Phosphoregulation of Phase Separation by the SARS-CoV-2 N Protein Suggests a Biophysical Basis for its Dual Functions.

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.

Phosphorylation modulates liquid-liquid phase separation of the SARS-CoV-2 N protein.

The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription in the infected cell 1-3 . The N protein contains two globular RNA-binding domains surrounded by regions of intrinsic disorder 4 . Phosphorylation of the central disordered region is required for normal viral genome transcription 5,6 , which occurs in a cytoplasmic structure called the replication transcription complex (RTC) 7-11 . It is not known how phosphorylation controls N protein function. Here we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates 12-15 . Unmodified N protein forms partially ordered gel-like structures that depend on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces a subset of these interactions, generating a more liquid-like droplet. We speculate 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. Inhibitors of N protein phosphorylation could therefore serve as antiviral therapy.

Intrinsic cooperativity potentiates parallel cis-regulatory evolution.

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 cis-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 cis-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.

Vms1p is a release factor for the ribosome-associated quality control complex.

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.

CDKL Family Kinases Have Evolved Distinct Structural Features and Ciliary Function.

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 ?J 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 ?J 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.

In vitro analysis of RQC activities provides insights into the mechanism and function of CAT tailing.

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.

Ancestral resurrection reveals evolutionary mechanisms of kinase plasticity.

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.