XBP1u, a central component of the unfolded protein response (UPR), is a mammalian protein containing a functionally critical translational arrest peptide (AP). Here, we present a 3 Å cryo-EM ...structure of the stalled human XBP1u AP. It forms a unique turn in the ribosomal exit tunnel proximal to the peptidyl transferase center where it causes a subtle distortion, thereby explaining the temporary translational arrest induced by XBP1u. During ribosomal pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan of the XBP1u AP revealed that only 8 out of 20 mutagenized positions are optimal; in the remaining 12 positions, we identify 55 different mutations increase the level of translational arrest. Thus, the wildtype XBP1u AP induces only an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel.
Protein degradation, a major eukaryotic response to cellular signals, is subject to numerous layers of regulation. In yeast, the evolutionarily conserved GID E3 ligase mediates glucose-induced ...degradation of fructose-1,6-bisphosphatase (Fbp1), malate dehydrogenase (Mdh2), and other gluconeogenic enzymes. "GID" is a collection of E3 ligase complexes; a core scaffold, RING-type catalytic core, and a supramolecular assembly module together with interchangeable substrate receptors select targets for ubiquitylation. However, knowledge of additional cellular factors directly regulating GID-type E3s remains rudimentary. Here, we structurally and biochemically characterize Gid12 as a modulator of the GID E3 ligase complex. Our collection of cryo-EM reconstructions shows that Gid12 forms an extensive interface sealing the substrate receptor Gid4 onto the scaffold, and remodeling the degron binding site. Gid12 also sterically blocks a recruited Fbp1 or Mdh2 from the ubiquitylation active sites. Our analysis of the role of Gid12 establishes principles that may more generally underlie E3 ligase regulation.
Dear Editor, The ubiquitin specific protease 7 （USP7）, also known as herpes virus associated ubiquitin specific protease （HAUSP）, is a well-characterized deubiquitinase （Reyes- Turcu et al., 2009）. ...USP7 plays important roles in various biological processes, including cell survival, proliferation, apoptosis, tumorigenesis, viral infection, and epigenetic regulation through regulating the protein stability of tumor suppressors （p53, PTEN, FOXO, claspin）, E3 ligases （MDM2, Mule, viral proteins ICP0）, epigenetic regulators （DNMT1, Tip60, UHRF1） （Pfoh et al., 2015）. USP7 is comprised of three recognizable domains： the N-terminal TRAF domain, the catalytic domain, and the C-terminal Tandem UBL domain （designated TUDusPT） （Fig. 1A）. Previous study shows that the TRAF domain binds to P/AxxS motif （from p53 and MDM2） and is responsible for substrate recognition （Sheng et al., 2006）. However, how USP7 recognizes other substrates remains largely unknown.
How ribosomes are madeThe formation of eukaryotic ribosomes is a complex process that starts with transcription of a large precursor RNA that assembles into a large 90S preribosome, which matures to ...finally give the 40S small subunit of the ribosome. Cheng et al. and Du et al. give insight into this process, using cryo–electron microscopy to look at intermediates along the pathway. Together, these studies reveal how a cast of molecular players act to coordinate the compositional and structural changes that transform the 90S preribosome into a pre-40S subunit.Science, this issue p. 1470, p. 1477Production of small ribosomal subunits initially requires the formation of a 90S precursor followed by an enigmatic process of restructuring into the primordial pre-40S subunit. We elucidate this process by biochemical and cryo–electron microscopy analysis of intermediates along this pathway in yeast. First, the remodeling RNA helicase Dhr1 engages the 90S pre-ribosome, followed by Utp24 endonuclease–driven RNA cleavage at site A1, thereby separating the 5′-external transcribed spacer (ETS) from 18S ribosomal RNA. Next, the 5′-ETS and 90S assembly factors become dislodged, but this occurs sequentially, not en bloc. Eventually, the primordial pre-40S emerges, still retaining some 90S factors including Dhr1, now ready to unwind the final small nucleolar U3–18S RNA hybrid. Our data shed light on the elusive 90S to pre-40S transition and clarify the principles of assembly and remodeling of large ribonucleoproteins.
Ribosome assembly is driven by numerous assembly factors, including the Rix1 complex and the AAA ATPase Rea1. These two assembly factors catalyze 60S maturation at two distinct states, triggering ...poorly understood large-scale structural transitions that we analyzed by cryo-electron microscopy. Two nuclear pre-60S intermediates were discovered that represent previously unknown states after Rea1-mediated removal of the Ytm1-Erb1 complex and reveal how the L1 stalk develops from a pre-mature nucleolar to a mature-like nucleoplasmic state. A later pre-60S intermediate shows how the central protuberance arises, assisted by the nearby Rix1-Rea1 machinery, which was solved in its pre-ribosomal context to molecular resolution. This revealed a Rix12-Ipi32 tetramer anchored to the pre-60S via Ipi1, strategically positioned to monitor this decisive remodeling. These results are consistent with a general underlying principle that temporarily stabilized immature RNA domains are successively remodeled by assembly factors, thereby ensuring failsafe assembly progression.
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•Pre-60S cryo-EM structures show L1 stalk formation after Erb1-Ytm1 release•L1 stalk maturation sets the basis for central protuberance construction•Nucleoplasmic Rix1-Rea1 pre-60S particle reveals architecture of the Rix1 complex•Ipi1 serves as anchor of the Rix1 complex to the pre-60S particle
Kater et al. show structures of pre-60S assembly intermediates, revealing how two Rix1-Rea1 machinery-dependent transitions result in L1 stalk formation and construction of the central protuberance. A molecular model of the Rix1 complex shows how its Ipi1 subunit anchors it to the pre-60S subunit, in turn permitting Rea1 recruitment.
Ribosome assembly is catalyzed by numerous trans-acting factors and coupled with irreversible pre-rRNA processing, driving the pathway toward mature ribosomal subunits. One decisive step early in ...this progression is removal of the 5′ external transcribed spacer (5′-ETS), an RNA extension at the 18S rRNA that is integrated into the huge 90S pre-ribosome structure. Upon endo-nucleolytic cleavage at an internal site, A1, the 5′-ETS is separated from the 18S rRNA and degraded. Here we present biochemical and cryo-electron microscopy analyses that depict the RNA exosome, a major 3′-5′ exoribonuclease complex, in a super-complex with the 90S pre-ribosome. The exosome is docked to the 90S through its co-factor Mtr4 helicase, a processive RNA duplex-dismantling helicase, which strategically positions the exosome at the base of 5′-ETS helices H9-H9’, which are dislodged in our 90S-exosome structures. These findings suggest a direct role of the exosome in structural remodeling of the 90S pre-ribosome to drive eukaryotic ribosome synthesis.
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•Cryo-EM structures show the 90S pre-ribosome with the RNA exosome•The exosome is docked to the 90S through its co-factor Mtr4 helicase•The exosome is close to the base of 5′-ETS helices H9-H9’•RNA exosome has a role in 90S pre-ribosome remodeling
Lau et al. show cryo-EM structures of the RNA exosome docked to the 90S pre-ribosome, indicating that the exosome directs 90S maturation and 5′-ETS degradation. A molecular model of the 90S-bound exosome reveals how the exosome could remodel the early pre-ribosome at the stage of 90S pre-A1-to-post-A1 transition.
TET proteins oxidize 5-methylcytosine (5mC) on DNA and play important roles in various biological processes. Mutations of TET2 are frequently observed in myeloid malignance. Here, we present the ...crystal structure of human TET2 bound to methylated DNA at 2.02 Å resolution. The structure shows that two zinc fingers bring the Cys-rich and DSBH domains together to form a compact catalytic domain. The Cys-rich domain stabilizes the DNA above the DSBH core. TET2 specifically recognizes CpG dinucleotide and shows substrate preference for 5mC in a CpG context. 5mC is inserted into the catalytic cavity with the methyl group orientated to catalytic Fe(II) for reaction. The methyl group is not involved in TET2-DNA contacts so that the catalytic cavity allows TET2 to accommodate 5mC derivatives for further oxidation. Mutations of Fe(II)/NOG-chelating, DNA-interacting, and zinc-chelating residues are frequently observed in human cancers. Our studies provide a structural basis for understanding the mechanisms of TET-mediated 5mC oxidation.
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•The Cys-rich and DSBH domains form a compact catalytic domain•TET2-dsDNA structure shows specific recognition of CpG dinucleotide by TET2•5mC is inserted into the catalytic cavity through a base-flipping mechanism•The catalytic cavity can accommodate 5mC and its derivatives for oxidation
The crystal structure of TET2 with dsDNA shows that TET2 specifically recognizes CpG dinucleotide through a base-flipping mechanism and that the catalytic cavity allows TET2 to accommodate 5mC and its derivatives for oxidation.
Biogenesis of the small ribosomal subunit in eukaryotes starts in the nucleolus with the formation of a 90S precursor and ends in the cytoplasm. Here, we elucidate the enigmatic structural ...transitions of assembly intermediates from human and yeast cells during the nucleoplasmic maturation phase. After dissociation of all 90S factors, the 40S body adopts a close-to-mature conformation, whereas the 3' major domain, later forming the 40S head, remains entirely immature. A first coordination is facilitated by the assembly factors TSR1 and BUD23-TRMT112, followed by re-positioning of RRP12 that is already recruited early to the 90S for further head rearrangements. Eventually, the uS2 cluster, CK1 (Hrr25 in yeast) and the export factor SLX9 associate with the pre-40S to provide export competence. These exemplary findings reveal the evolutionary conserved mechanism of how yeast and humans assemble the 40S ribosomal subunit, but reveal also a few minor differences.
Ribosome production is vital for every cell, and failure causes human diseases. It is driven by ∼200 assembly factors functioning along an ordered pathway from the nucleolus to the cytoplasm. ...Structural snapshots of biogenesis intermediates from the earliest 90S pre-ribosomes to mature 40S subunits unravel the mechanisms of small ribosome synthesis. To view this SnapShot, open or download the PDF.
Ribosome production is vital for every cell, and failure causes human diseases. It is driven by ∼200 assembly factors functioning along an ordered pathway from the nucleolus to the cytoplasm. Structural snapshots of biogenesis intermediates from the earliest 90S pre-ribosomes to mature 40S subunits unravel the mechanisms of small ribosome synthesis. To view this SnapShot, open or download the PDF.
Ribosome biogenesis proceeds along a multifaceted pathway from the nucleolus to the cytoplasm that is extensively coupled to several quality control mechanisms. However, the mode by which 5S ...ribosomal RNA is incorporated into the developing pre‐60S ribosome, which in humans links ribosome biogenesis to cell proliferation by surveillance by factors such as p53–MDM2, is poorly understood. Here, we report nine nucleolar pre‐60S cryo‐EM structures from Chaetomium thermophilum, one of which clarifies the mechanism of 5S RNP incorporation into the early pre‐60S. Successive assembly states then represent how helicases Dbp10 and Spb4, and the Pumilio domain factor Puf6 act in series to surveil the gradual folding of the nearby 25S rRNA domain IV. Finally, the methyltransferase Spb1 methylates a universally conserved guanine nucleotide in the A‐loop of the peptidyl transferase center, thereby licensing further maturation. Our findings provide insight into the hierarchical action of helicases in safeguarding rRNA tertiary structure folding and coupling to surveillance mechanisms that culminate in local RNA modification.
Synopsis
During pre‐60S assembly, the 5S RNP is stably incorporated in a fixed orientation, before the 25S rRNA domain IV develops further under the hierarchical action of helicases and assembly factors, which together surveil local RNA modification.
Nucleolar pre‐60S structures from C. thermophilum reveal the early pre‐60S assembly pathway.
5S RNP incorporation in the early pre‐60S uncovers a new rotational state.
Hierarchical action of Dbp10, Spb4, Rrp17 and Puf6 safeguards Spb1‐mediated methylation in the A‐loop of the 25S rRNA.
Conserved and unique maturation steps are discovered for the thermophile pre‐60S biogenesis pathway.
During pre‐60S assembly, the 5S RNP is stably incorporated in a fixed orientation, before the 25S rRNA domain IV develops further under the hierarchical action of helicases and assembly factors, which together surveil local RNA modification.
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