Multiprotein bridging factor 1 is required for robust activation of the integrated stress response on collided ribosomes.

In yeast, multiprotein bridging factor 1 (Mbf1) has been proposed to function in the integrated stress response (ISR) as a transcriptional coactivator by mediating a direct interaction between general transcription machinery and the process's key effector, Gcn4. However, mounting evidence has demonstrated that Mbf1 (and its human homolog EDF1) is recruited to collided ribosomes, a known activator of the ISR. In this study, we connect these otherwise seemingly disparate functions of Mbf1.

The RNA helicase HrpA rescues collided ribosomes in .

Although many antibiotics inhibit bacterial ribosomes, loss of known factors that rescue stalled ribosomes does not lead to robust antibiotic sensitivity in , suggesting the existence of additional mechanisms. Here, we show that the RNA helicase HrpA rescues stalled ribosomes in Acting selectively on ribosomes that have collided, HrpA uses ATP hydrolysis to split stalled ribosomes into subunits. Cryo-EM structures reveal how HrpA simultaneously binds to two collided ribosomes, explaining its selectivity, and how its helicase module engages downstream mRNA, such that by exerting a pulling force on the mRNA, it would destabilize the stalled ribosome.

Theoretical infrared spectroscopy of protonated methane isotopologues.

The vibrational spectroscopy of protonated methane and its mixed hydrogen/deuterium isotopologues remains a challenge to both experimental and computational spectroscopy due to the iconic floppiness of CH. Here, we compute the finite-temperature broadband infrared spectra of CH and all its isotopologues, CHD up to CD, from path integral molecular dynamics in conjunction with interactions and dipoles computed consistently at CCSD(T) coupled cluster accuracy. The potential energy and dipole moment surfaces have been accurately represented in full dimensionality in terms of high-dimensional neural networks.

Structural basis for differential inhibition of eukaryotic ribosomes by tigecycline.

Tigecycline is widely used for treating complicated bacterial infections for which there are no effective drugs. It inhibits bacterial protein translation by blocking the ribosomal A-site. However, even though it is also cytotoxic for human cells, the molecular mechanism of its inhibition remains unclear.

SnapShot: Eukaryotic ribosome biogenesis II.

Ribosome production is essential for cell growth. Approximately 200 assembly factors drive this complicated pathway that starts in the nucleolus and ends in the cytoplasm. A large number of structural snapshots of the pre-60S pathway have revealed the principles behind large subunit synthesis.

UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER.

Reversible modification of target proteins by ubiquitin and ubiquitin-like proteins (UBLs) is widely used by eukaryotic cells to control protein fate and cell behaviour. UFM1 is a UBL that predominantly modifies a single lysine residue on a single ribosomal protein, uL24 (also called RPL26), on ribosomes at the cytoplasmic surface of the endoplasmic reticulum (ER). UFM1 conjugation (UFMylation) facilitates the rescue of 60S ribosomal subunits (60S) that are released after ribosome-associated quality-control-mediated splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER.

Mechanisms of Translation-coupled Quality Control.

Stalling of ribosomes engaged in protein synthesis can lead to significant defects in the function of newly synthesized proteins and thereby impair protein homeostasis. Consequently, partially synthesized polypeptides resulting from translation stalling are recognized and eliminated by several quality control mechanisms. First, if translation elongation reactions are halted prematurely, a quality control mechanism called ribosome-associated quality control (RQC) initiates the ubiquitination of the nascent polypeptide chain and subsequent proteasomal degradation.

B. subtilis MutS2 splits stalled ribosomes into subunits without mRNA cleavage.

Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through the recruitment of SmrB, a nuclease that cleaves the mRNA.

RNF14-dependent atypical ubiquitylation promotes translation-coupled resolution of RNA-protein crosslinks.

Reactive aldehydes are abundant endogenous metabolites that challenge homeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA damage requires repair to prevent cancer and premature aging, but it is unknown whether cells also possess mechanisms that resolve aldehyde-induced RNA lesions. Here, we establish photoactivatable ribonucleoside-enhanced crosslinking (PAR-CL) as a model system to study RNA crosslinking damage in the absence of confounding DNA damage in human cells.

Structural insights into coordinating 5S RNP rotation with ITS2 pre-RNA processing during ribosome formation.

The rixosome defined in Schizosaccharomyces pombe and humans performs diverse roles in pre-ribosomal RNA processing and gene silencing. Here, we isolate and describe the conserved rixosome from Chaetomium thermophilum, which consists of two sub-modules, the sphere-like Rix1-Ipi3-Ipi1 and the butterfly-like Las1-Grc3 complex, connected by a flexible linker. The Rix1 complex of the rixosome utilizes Sda1 as landing platform on nucleoplasmic pre-60S particles to wedge between the 5S rRNA tip and L1-stalk, thereby facilitating the 180° rotation of the immature 5S RNP towards its mature conformation.

Structure of nascent 5S RNPs at the crossroad between ribosome assembly and MDM2-p53 pathways.

The 5S ribonucleoprotein (RNP) is assembled from its three components (5S rRNA, Rpl5/uL18 and Rpl11/uL5) before being incorporated into the pre-60S subunit. However, when ribosome synthesis is disturbed, a free 5S RNP can enter the MDM2-p53 pathway to regulate cell cycle and apoptotic signaling. Here we reconstitute and determine the cryo-electron microscopy structure of the conserved hexameric 5S RNP with fungal or human factors.

MutS2 splits stalled ribosomes into subunits without mRNA cleavage.

Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In , these pathways are triggered by ribosome collisions through recruitment of SmrB, a nuclease that cleaves the mRNA. In , the related protein MutS2 was recently implicated in ribosome rescue.

SnapShot: Eukaryotic ribosome biogenesis I.

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.

Molecular basis for recognition and deubiquitination of 40S ribosomes by Otu2.

In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal subunit recycling. Despite its importance for translation efficiency the exact role and structural basis for this translational reset is poorly understood. Here, structural analysis by cryo-electron microscopy of native and reconstituted Otu2-bound ribosomal complexes reveals that Otu2 engages 40S subunits mainly between ribosome recycling and initiation stages.

Mechanism of 5S RNP recruitment and helicase-surveilled rRNA maturation during pre-60S biogenesis.

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.

The dynamic architecture of Map1- and NatB-ribosome complexes coordinates the sequential modifications of nascent polypeptide chains.

Cotranslational modification of the nascent polypeptide chain is one of the first events during the birth of a new protein. In eukaryotes, methionine aminopeptidases (MetAPs) cleave off the starter methionine, whereas N-acetyl-transferases (NATs) catalyze N-terminal acetylation. MetAPs and NATs compete with other cotranslationally acting chaperones, such as ribosome-associated complex (RAC), protein targeting and translocation factors (SRP and Sec61) for binding sites at the ribosomal tunnel exit.

Concurrent remodelling of nucleolar 60S subunit precursors by the Rea1 ATPase and Spb4 RNA helicase.

Biogenesis intermediates of nucleolar ribosomal 60S precursor particles undergo a number of structural maturation steps before they transit to the nucleoplasm and are finally exported into the cytoplasm. The AAA-ATPase Rea1 participates in the nucleolar exit by releasing the Ytm1-Erb1 heterodimer from the evolving pre-60S particle. Here, we show that the DEAD-box RNA helicase Spb4 with its interacting partner Rrp17 is further integrated into this maturation event.

Deciphering the Impact of Helium Tagging on Flexible Molecules: Probing Microsolvation Effects of Protonated Acetylene by Quantum Configurational Entropy.

Helium, the lightest and most weakly interacting noble gas, is well-known for its unsurpassed chemical inertness. In many applications of helium in experimental techniques, such as tagging, messenger, or nanodroplet isolation action spectroscopy of molecules or complexes, it is assumed that the interaction of helium with the respective species, and thus the resulting interaction-induced perturbation, is small enough not to affect their structure and dynamics. Here, we probe the impact of one up to many attached helium atoms on protonated acetylene─an important nonclassical carbocation subject to three-center two-electron bonding in its ground state structure─using highly accurate interaction potentials in conjunction with entropy-based higher-order nonlinear correlation analysis.

Molecular basis of eIF5A-dependent CAT tailing in eukaryotic ribosome-associated quality control.

Ribosome-associated quality control (RQC) is a conserved process degrading potentially toxic truncated nascent peptides whose malfunction underlies neurodegeneration and proteostasis decline in aging. During RQC, dissociation of stalled ribosomes is followed by elongation of the nascent peptide with alanine and threonine residues, driven by Rqc2 independently of mRNA, the small ribosomal subunit and guanosine triphosphate (GTP)-hydrolyzing factors. The resulting CAT tails (carboxy-terminal tails) and ubiquitination by Ltn1 mark nascent peptides for proteasomal degradation.

Structural basis for clearing of ribosome collisions by the RQT complex.

Translation of aberrant messenger RNAs can cause stalling of ribosomes resulting in ribosomal collisions. Collided ribosomes are specifically recognized to initiate stress responses and quality control pathways. Ribosome-associated quality control facilitates the degradation of incomplete translation products and requires dissociation of the stalled ribosomes.

Two modes of Cue2-mediated mRNA cleavage with distinct substrate recognition initiate no-go decay.

Ribosome collisions are recognized by E3 ubiquitin ligase Hel2/ZNF598, leading to RQC (ribosome-associated quality control) and to endonucleolytic cleavage and degradation of the mRNA termed NGD (no-go decay). NGD in yeast requires the Cue2 endonuclease and occurs in two modes, either coupled to RQC (NGDRQC+) or RQC uncoupled (NGDRQC-). This is mediated by an unknown mechanism of substrate recognition by Cue2.

Nuclear factor erythroid 2-related factor 2 (Nrf2) deficiency causes age-dependent progression of female osteoporosis.

Background: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial transcription factor for cellular redox homeostasis. The association of Nrf2 with elderly female osteoporotic has yet to be fully described. The aim was to elucidate a potential age-dependent Nrf2 contribution to female osteoporosis in mice.

Cms1 coordinates stepwise local 90S pre-ribosome assembly with timely snR83 release.

Ribosome synthesis begins in the nucleolus with 90S pre-ribosome construction, but little is known about how the many different snoRNAs that modify the pre-rRNA are timely guided to their target sites. Here, we report a role for Cms1 in such a process. Initially, we discovered CMS1 as a null suppressor of a nop14 mutant impaired in Rrp12-Enp1 factor recruitment to the 90S.

IKKβ primes inflammasome formation by recruiting NLRP3 to the trans-Golgi network.

The NLRP3 inflammasome plays a central role in antimicrobial defense as well as in the context of sterile inflammatory conditions. NLRP3 activity is governed by two independent signals: the first signal primes NLRP3, rendering it responsive to the second signal, which then triggers inflammasome formation. Our understanding of how NLRP3 priming contributes to inflammasome activation remains limited.

The nucleoplasmic phase of pre-40S formation prior to nuclear export.

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.