Biology subjects

Korennykh, A.

Publications and source records attributed to Korennykh, A..

6 recordsLinked to original sources

bioRxiv2024

Cryo-EM Structure of HRSL Domain Reveals Activating Crossed Helices at the Core of GCN2

GCN2 is a conserved receptor kinase activating the Integrated Stress Response (ISR) in eukaryotic cells. The ISR kinases detect accumulation of stress molecules and reprogram translation from basal tasks to preferred production of cytoprotective proteins. GCN2 stands out evolutionarily among all protein kinases due to the presence of a histidyl tRNA synthetase-like (HRSL) domain, which arises only in GCN2 and is located next to the kinase domain. How HRSL contributes to GCN2 signaling remains unknown. Here we report a 3.2 [A] cryo-EM structure of HRSL from thermotolerant yeast Kluyveromyces marxianus. This structure shows a constitutive symmetrical homodimer featuring a compact helical-bundle structure at the junction between HRSL and kinase domains, in the core of the receptor. Mutagenesis demonstrates that this junction structure activates GCN2 and indicates that our cryo-EM structure captures the active signaling state of HRSL. Based on these results, we put forward a GCN2 regulation mechanism, where HRSL drives the formation of activated kinase dimers. Remaining domains of GCN2 have the opposite role and in the absence of stress they help keep GCN2 basally inactive. This autoinhibitory activity is relieved upon stress ligand binding. We propose that the opposing action of HRSL and additional GCN2 domains thus yields a regulated ISR receptor. Significance statementRegulation of protein synthesis (translation) is a central mechanism by which eukaryotic cells adapt to stressful conditions. In starving cells, this translational adaptation is achieved via the receptor kinase GCN2, which stays inactive under normal conditions, but is switched on under stress. The molecular mechanism of GCN2 switching is not well understood due to the presence of a structurally and biochemically uncharacterized histidyl tRNA synthetase-like domain (HRSL) at the core of GCN2. Here we use single-particle cryo-EM and biochemistry to elucidate the structure and function of HRSL. We identify a structure at the kinase/HRSL interface, which forms crossed helices and helps position GCN2 kinase domains for activation. These data clarify the molecular mechanism of GCN2 regulation.

biochemistry↗

bioRxiv2019

The Metabolites NADP+ and NADPH are the Targets of the Circadian Protein Nocturnin (Curled)

Nocturnin (NOCT) is a rhythmically expressed protein that regulates metabolism under the control of circadian clock. It has been proposed that NOCT deadenylates and regulates metabolic enzyme mRNAs. However, in contrast to other deadenylases, purified NOCT lacks the deadenylase activity. To identify the substrate of NOCT, we conducted a mass spectrometry screen and report that NOCT specifically and directly converts the dinucleotide NADP+ into NAD+ and NADPH into NADH. Further, we demonstrate that the Drosophila NOCT ortholog, Curled, has the same enzymatic activity. We obtained the 2.7 [A] crystal structure of the human NOCT*NADPH complex, which revealed that NOCT recognizes the chemically unique ribose-phosphate backbone of the metabolite, placing the 2-terminal phosphate productively for removal. We provide evidence for NOCT targeting to mitochondria and propose that NADP(H) regulation, which takes place at least in part in mitochondria, establishes the molecular link between circadian clock and metabolism.

biophysics↗

bioRxiv2018

Concerted 2-5A-Mediated mRNA Decay and Transcription Reprogram Protein Synthesis in dsRNA Response

RNA degradation by RNase L during 2-5A-mediated decay (2-5AMD) is a conserved mammalian stress response to viral and endogenous double-stranded RNA (dsRNA). 2-5AMD onsets rapidly and facilitates a switch of protein synthesis from homeostasis to production of interferons (IFNs). To understand the mechanism of this protein synthesis reprogramming, we examined 2-5AMD in human cells. 2-5AMD triggers polysome collapse characteristic of a translation initiation defect, but translation initiation complexes and ribosomes purified from the translation-arrested cells remain functional. Using spike-in RNA-seq we found that basal messenger RNAs (mRNAs) rapidly decay, while mRNAs encoding IFNs and IFN-stimulated genes evade 2-5AMD and accumulate. The IFN evasion results from the combined effect of better mRNA stability and positive feedback amplification in the IFN response. Therefore, 2-5AMD and transcription act in concert to revamp the cellular mRNA composition. The resulting preferential accumulation of innate immune mRNAs establishes \"prioritized\" synthesis of defense proteins.

cell biology↗

bioRxiv2018

Realtime 2-5A kinetics suggests interferons β and {lambda} evade global arrest of translation by RNase L

Cells of all mammals recognize double-stranded RNA (dsRNA) as a foreign material. In response, they release interferons (IFNs) and activate a ubiquitously expressed pseudokinase/endoribonuclease RNase L. RNase L executes regulated RNA decay and halts global translation. Here we developed a biosensor for 2,5-oligoadenylate (2-5A), the natural activator of RNase L. We found that 2-5A was acutely synthesized by cells in response to dsRNA sensing, which immediately triggered cellular RNA cleavage by RNase L and arrested host protein synthesis. However, translation-arrested cells still transcribed IFN-stimulated genes (ISGs) and secreted IFNs of types I and III (IFN-{beta} and IFN-{lambda}). Our data suggests that IFNs escape from the action of RNase L on translation. We propose that 2-5A/RNase L pathway serves to rapidly and accurately suppress basal protein synthesis, preserving privileged production of defense proteins of the innate immune system.\n\nSignificanceRNase L is a mammalian enzyme that can stop global protein synthesis during interferon response. Cells must balance the need to make interferons (which are proteins) with the risk to lose cell-wide translation due to RNase L. This balance can most simply be achieved if RNase L was activated late in the interferon response. However, we show by engineering a biosensor for the RNase L pathway, that on the contrary, RNase L activation precedes interferon synthesis. Further, translation of interferons evades the action of RNase L. Our data suggest that RNase L facilitates a switch of protein synthesis from homeostasis to specific needs of innate immune signaling.

cell biology↗

bioRxiv2018

Crystal Structure of Human Nocturnin Catalytic Domain

Nocturnin (NOCT) helps the circadian clock to adjust metabolism according to day and night activity. NOCT is upregulated in early evening and it has been proposed to serve as a deadenylase for metabolic enzyme mRNAs. We present a 2.7-[A] crystal structure of the catalytic domain of human NOCT. Our structure shows that NOCT has a close overall similarity to CCR4 deadenylase family members, PDE12 and CNOT6L, and to a DNA repair enzyme TDP2. All the key catalytic residues present in PDE12, CNOT6L and TDP2 are conserved in NOCT and have the same conformations. However, we observe substantial differences in the surface properties of NOCT, an unexpectedly narrow active site pocket, and conserved structural elements in the vicinity of the catalytic center, which are unique to NOCT and absent in the deadenylases PDE12/CNOT6L. Our work thus reveals the structure of an intriguing circadian protein and suggests that NOCT has considerable differences from the related deadenylases, which may point to a unique cellular function of this enzyme.

biophysics↗

bioRxiv2016

Y-RNA and tRNA Cleavage by RNase L Mediates Terminal dsRNA Response

Double-stranded RNA (dsRNA) is a danger signal that triggers endonucleolytic degradation of RNA inside infected and stressed mammalian cells. This mechanism inhibits growth and ultimately removes problematic cells via apoptosis. To elucidate the molecular functions of this program and understand the connection between RNA cleavage and programmed cell death, we visualized dsRNA-induced degradation of human small RNAs using RtcB ligase-assisted RNA sequencing (RtcB RNA-seq). RtcB RNA-seq revealed strong cleavage of select transfer RNAs (tRNAs) and autoantigenic Y-RNAs, and identified the innate immune receptor RNase L as the responsible endoribonuclease. RNase L cleaves the non-coding RNA (ncRNA) targets site-specifically, releasing abundant ncRNA fragments, and downregulating full-length tRNAs and Y-RNAs. The depletion of a single Y-RNA, RNY1, appears particularly important and the loss of this Y-RNA is sufficient to initiate apoptosis. Site-specific cleavage of small ncRNA by RNase L thus emerges as an important terminal step in dsRNA surveillance.

immunology↗