The transition of the 90S to the pre-40S pre-ribosome is a decisive step in eukaryotic small subunit biogenesis leading to a first pre-40S intermediate (state Dis-C or primordial pre-40S), where the ...U3 snoRNA keeps the nascent 18S rRNA locally immature. We in vitro reconstitute the ATP-dependent U3 release from this particle, catalyzed by the helicase Dhr1, and follow this process by cryo-EM revealing two successive pre-40S intermediates, Dis-D and Dis-E. The latter has lost not only U3 but all residual 90S factors including the GTPase Bms1. In vitro remodeling likewise induced the formation of the central pseudoknot, a universally conserved tertiary RNA structure that comprises the core of the small subunit decoding center. Thus, we could structurally reveal a key tertiary RNA folding step that is essential to form the active 40S subunit.
Gene expression in metazoans is controlled by promoter-proximal pausing of RNA polymerase II, which can undergo productive elongation or promoter-proximal termination. Integrator-PP2A (INTAC) plays a ...crucial role in determining the fate of paused polymerases, but the underlying mechanisms remain unclear. Here, we establish a rapid degradation system to dissect the functions of INTAC RNA endonuclease and phosphatase modules. We find that both catalytic modules function at most if not all active promoters and enhancers, yet differentially affect polymerase fate. The endonuclease module induces promoter-proximal termination, with its disruption leading to accumulation of elongation-incompetent polymerases and downregulation of highly expressed genes, while elongation-competent polymerases accumulate at lowly expressed genes and non-coding elements, leading to their upregulation. The phosphatase module primarily prevents the release of paused polymerases and limits transcriptional activation, especially for highly paused genes. Thus, both INTAC catalytic modules have unexpectedly general yet distinct roles in dynamic transcriptional control.
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•The INTAC endonuclease and phosphatase modules target most if not all active genes•The endonuclease module primarily induces promoter-proximal RNA Pol II termination•Endonuclease loss has different consequences for highly and lowly expressed genes•The phosphatase module suppresses RNA Pol II release and attenuates transcription
Hu et al. reveal that the two catalytic modules of INTAC have unexpectedly general yet distinct roles in transcription: the endonuclease module cleaves nascent RNAs to induce promoter-proximal RNA Pol II termination, while the phosphatase module primarily dephosphorylates RNA Pol II, suppresses RNA Pol II release, and attenuates transcription.
Huntingtin (HTT) is a large (348 kDa) protein that is essential for embryonic development and is involved in diverse cellular activities such as vesicular transport, endocytosis, autophagy and the ...regulation of transcription. Although an integrative understanding of the biological functions of HTT is lacking, the large number of identified HTT interactors suggests that it serves as a protein-protein interaction hub. Furthermore, Huntington's disease is caused by a mutation in the HTT gene, resulting in a pathogenic expansion of a polyglutamine repeat at the amino terminus of HTT. However, only limited structural information regarding HTT is currently available. Here we use cryo-electron microscopy to determine the structure of full-length human HTT in a complex with HTT-associated protein 40 (HAP40; encoded by three F8A genes in humans) to an overall resolution of 4 Å. HTT is largely α-helical and consists of three major domains. The amino- and carboxy-terminal domains contain multiple HEAT (huntingtin, elongation factor 3, protein phosphatase 2A and lipid kinase TOR) repeats arranged in a solenoid fashion. These domains are connected by a smaller bridge domain containing different types of tandem repeats. HAP40 is also largely α-helical and has a tetratricopeptide repeat-like organization. HAP40 binds in a cleft and contacts the three HTT domains by hydrophobic and electrostatic interactions, thereby stabilizing the conformation of HTT. These data rationalize previous biochemical results and pave the way for improved understanding of the diverse cellular functions of HTT.
DNA methylation is an important epigenetic modification. Ten-eleven translocation (TET) proteins are involved in DNA demethylation through iteratively oxidizing 5-methylcytosine (5mC) into ...5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Here we show that human TET1 and TET2 are more active on 5mC-DNA than 5hmC/5fC-DNA substrates. We determine the crystal structures of TET2-5hmC-DNA and TET2-5fC-DNA complexes at 1.80 Å and 1.97 Å resolution, respectively. The cytosine portion of 5hmC/5fC is specifically recognized by TET2 in a manner similar to that of 5mC in the TET2-5mC-DNA structure, and the pyrimidine base of 5mC/5hmC/5fC adopts an almost identical conformation within the catalytic cavity. However, the hydroxyl group of 5hmC and carbonyl group of 5fC face towards the opposite direction because the hydroxymethyl group of 5hmC and formyl group of 5fC adopt restrained conformations through forming hydrogen bonds with the 1-carboxylate of NOG and N4 exocyclic nitrogen of cytosine, respectively. Biochemical analyses indicate that the substrate preference of TET2 results from the different efficiencies of hydrogen abstraction in TET2-mediated oxidation. The restrained conformation of 5hmC and 5fC within the catalytic cavity may prevent their abstractable hydrogen(s) adopting a favourable orientation for hydrogen abstraction and thus result in low catalytic efficiency. Our studies demonstrate that the substrate preference of TET2 results from the intrinsic value of its substrates at their 5mC derivative groups and suggest that 5hmC is relatively stable and less prone to further oxidation by TET proteins. Therefore, TET proteins are evolutionarily tuned to be less reactive towards 5hmC and facilitate the generation of 5hmC as a potentially stable mark for regulatory functions.
Eukaryotic ribosome biogenesis involves RNA folding and processing that depend on assembly factors and small nucleolar RNAs (snoRNAs). The 90S (SSU-processome) is the earliest pre-ribosome ...structurally analyzed, which was suggested to assemble stepwise along the growing pre-rRNA from 5′ > 3′, but this directionality may not be accurate. Here, by analyzing the structure of a series of 90S assembly intermediates from Chaetomium thermophilum, we discover a reverse order of 18S rRNA subdomain incorporation. Large parts of the 18S rRNA 3′ and central domains assemble first into the 90S before the 5′ domain is integrated. This final incorporation depends on a contact between a heterotrimer Enp2-Bfr2-Lcp5 recruited to the flexible 5′ domain and Kre33, which reconstitutes the Kre33-Enp-Brf2-Lcp5 module on the compacted 90S. Keeping the 5′ domain temporarily segregated from the 90S scaffold could provide extra time to complete the multifaceted 5′ domain folding, which depends on a distinct set of snoRNAs and processing factors.
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•Series of cryo-EM structures of the 90S pre-ribosome depicting its assembly order•5′ domain of the 18S rRNA is integrated into the 90S pre-ribosome at later stages•Kre33-Enp2-Brf2-Lcp5 module mediates the final compaction of 90S pre-ribosome•Temporal rRNA segregation from the 90S scaffold provides time for 5′ domain folding
Cheng et al. report a series of 90S pre-ribosome structures that reveal a reverse order of 18S rRNA subdomain incorporation. Accordingly, the 5′ domain is stably assembled only after the 3′ and central domains have been integrated into the 90S. This final compaction depends on the conserved Kre33-Enp2-Bfr2-Lcp5 module.
In eukaryotes, most secretory and membrane proteins are targeted by an N‐terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein‐conducting channel ...(PCC). In the post‐translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre‐opened state. Here, we present a cryo‐EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N‐terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C‐terminus of Sec63. Together, we obtained a near‐complete and integrated model of the active Sec complex.
SYNOPSIS
The overall architecture of the eukaryotic SecY/Sec61 channel for post‐translational protein translocation, and how it recognizes N‐terminal signal sequences in transported proteins, remain unclear. Here, structural analyses reveal the molecular architecture of the engaged yeast Sec complex, which binds the signal sequence in a unique open conformation.
Cryo‐EM structures of the heptameric Sec complex reveal the conformational transition from the partially open apo‐state to the fully open signal sequence‐bound state.
The signal sequence binds the lateral gate of the Sec61 complex, which is stabilized by the transmembrane helices of the Sec62 subunit.
The X‐ray structure of the N‐terminal cytoplasmic domain of Sec62 reveals a novel fold that is coordinated by the phosphorylated C‐terminal peptide of the Sec63 subunit.
Cryo‐electron microscopy and X‐ray crystallography studies reveal the molecular architecture of the engaged eukaryotic heptameric Sec complex, binding the protein signal sequence in an open conformation.
UHRF1 is an important epigenetic regulator connecting DNA methylation and histone methylations. UHRF1 is required for maintenance of DNA methylation through recruiting DNMT1 to DNA replication forks. ...Recent studies have shown that the plant homeodomain (PHD) of UHRF1 recognizes the N terminus of unmodified histone H3, and the interaction is inhibited by methylation of H3R2, whereas the tandem tudor domain (TTD) of UHRF1 recognizes trimethylated histone H3 lysine 9 (H3K9me3). However, how the two domains of UHRF1 coordinately recognize histone methylations remains elusive. In this report, we identified that PHD largely enhances the interaction between TTD and H3K9me3. We present the crystal structure of UHRF1 containing both TTD and PHD (TTD-PHD) in complex with H3K9m3 peptide at 3.0 Å resolution. The structure shows that TTD-PHD binds to the H3K9me3 peptide with 1:1 stoichiometry with the two domains connected by the H3K9me3 peptide and a linker region. The TTD interacts with residues Arg-8 and trimethylated Lys-9, and the PHD interacts with residues Ala-1, Arg-2, and Lys-4 of the H3K9me3 peptide. The biochemical experiments indicate that PHD-mediated recognition of unmodified H3 is independent of the TTD, whereas TTD-mediated recognition of H3K9me3 PHD. Thus, both TTD and PHD are essential for specific recognition of H3K9me3 by UHRF1. Interestingly, the H3K9me3 peptide induces conformational changes of TTD-PHD, which do not affect the autoubiquitination activity or hemimethylated DNA binding affinity of UHRF1 in vitro. Taken together, our studies provide structural insight into the coordinated recognition of H3K9me3 by the TTD and PHD of UHRF1.
Background: UHRF1 is an important epigenetic regulator connecting DNA methylation and histone methylations.
Results: PHD-H3 interaction is independent of the TTD, whereas TTD-H3K9me3 interaction the PHD.
Conclusion: Both TTD and PHD are essential for specific recognition of H3K9me3 by human UHRF1.
Significance: This work reveals how UHRF1 recognizes H3K9me3, which is important for its cellular localization and DNA methylation.
How ribosomes are made
The 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.
1477
The steps that drive the stepwise dissociation of factors in the transition from the 90
S
to the 40
S
ribosome subunit are observed.
Production of small ribosomal subunits initially requires the formation of a 90
S
precursor followed by an enigmatic process of restructuring into the primordial pre-40
S
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 90
S
pre-ribosome, followed by Utp24 endonuclease–driven RNA cleavage at site A
1
, thereby separating the 5′-external transcribed spacer (ETS) from 18
S
ribosomal RNA. Next, the 5′-ETS and 90
S
assembly factors become dislodged, but this occurs sequentially, not en bloc. Eventually, the primordial pre-40
S
emerges, still retaining some 90
S
factors including Dhr1, now ready to unwind the final small nucleolar U3–18
S
RNA hybrid. Our data shed light on the elusive 90
S
to pre-40
S
transition and clarify the principles of assembly and remodeling of large ribonucleoproteins.
In eukaryotic translation, termination and ribosome recycling phases are linked to subsequent initiation of a new round of translation by persistence of several factors at ribosomal sub‐complexes. ...These comprise/include the large eIF3 complex, eIF3j (Hcr1 in yeast) and the ATP‐binding cassette protein ABCE1 (Rli1 in yeast). The ATPase is mainly active as a recycling factor, but it can remain bound to the dissociated 40S subunit until formation of the next 43S pre‐initiation complexes. However, its functional role and native architectural context remains largely enigmatic. Here, we present an architectural inventory of native yeast and human ABCE1‐containing pre‐initiation complexes by cryo‐EM. We found that ABCE1 was mostly associated with early 43S, but also with later 48S phases of initiation. It adopted a novel hybrid conformation of its nucleotide‐binding domains, while interacting with the N‐terminus of eIF3j. Further, eIF3j occupied the mRNA entry channel via its ultimate C‐terminus providing a structural explanation for its antagonistic role with respect to mRNA binding. Overall, the native human samples provide a near‐complete molecular picture of the architecture and sophisticated interaction network of the 43S‐bound eIF3 complex and the eIF2 ternary complex containing the initiator tRNA.
Synopsis
Function and native architecture of ribosomal complexes with recycling factor ATPase ABCE1 have remained unclear. Here, a cryo‐EM‐based structural inventory of native ABCE1‐bound translation initiation complexes from human and yeast reveal a novel hybrid conformation of the ABCE1 stabilized by a dimer of translation initiation factor eIF3j.
Cryo‐EM structures reveal the near‐complete molecular architecture of eIF3 and eIF2 ternary complexes in the context of the 43S pre‐initiation complex.
Under native conditions, ABCE1 is present in all captured stages of translation initiation and displays a hybrid conformation with ADP and ATP bound concurrently.
In the new ABCE1 conformation, an eIF3j dimer stabilizes the nucleotide binding site of ABCE1 and occludes the mRNA entry channel via the C‐terminus of one monomer.
Cryo‐EM structures of native human and yeast translation initiation complexes reveal a novel hybrid conformation of the recycling factor ATPase ABCE1, which is stabilized by a dimer of translation initiation factor eIF3j.
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