Proteome-scale movements and compartment connectivity during the eukaryotic cell cycle.

Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of ∼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control and changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.

FilamentID reveals the composition and function of metabolic enzyme polymers during gametogenesis.

Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a "filament identification" (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of partially lysed cells or organelles. FilamentID identified the mitochondrial filaments as being composed of the conserved aldehyde dehydrogenase Ald4ALDH2 and the nucleoplasmic/cytoplasmic filaments as consisting of acetyl-coenzyme A (CoA) synthetase Acs1ACSS2. Structural characterization further revealed the mechanism underlying polymerization and enabled us to genetically perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores and, more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts.

Translation and mRNA Stability Control.

Messenger RNA (mRNA) stability and translational efficiency are two crucial aspects of the post-transcriptional process that profoundly impact protein production in a cell. While it is widely known that ribosomes produce proteins, studies during the past decade have surprisingly revealed that ribosomes also control mRNA stability in a codon-dependent manner, a process referred to as codon optimality. Therefore, codons, the three-nucleotide words read by the ribosome, have a potent effect on mRNA stability and provide cis-regulatory information that extends beyond the amino acids they encode. While the codon optimality molecular mechanism is still unclear, the translation elongation rate appears to trigger mRNA decay. Thus, transfer RNAs emerge as potential master gene regulators affecting mRNA stability. Furthermore, while few factors related to codon optimality have been identified in yeast, the orthologous genes in vertebrates do not necessary share the same functions. Here, we discuss codon optimality findings and gene regulation layers related to codon composition in different eukaryotic species.

3'-End Processing of Eukaryotic mRNA: Machinery, Regulation, and Impact on Gene Expression.

Formation of the 3' end of a eukaryotic mRNA is a key step in the production of a mature transcript. This process is mediated by a number of protein factors that cleave the pre-mRNA, add a poly(A) tail, and regulate transcription by protein dephosphorylation. Cleavage and polyadenylation specificity factor (CPSF) in humans, or cleavage and polyadenylation factor (CPF) in yeast, coordinates these enzymatic activities with each other, with RNA recognition, and with transcription. The site of pre-mRNA cleavage can strongly influence the translation, stability, and localization of the mRNA. Hence, cleavage site selection is highly regulated. The length of the poly(A) tail is also controlled to ensure that every transcript has a similar tail when it is exported from the nucleus. In this review, we summarize new mechanistic insights into mRNA 3'-end processing obtained through structural studies and biochemical reconstitution and outline outstanding questions in the field.

Strength in diversity: Intra-cellular metabolite sharing enhances longevity.

Much of our foundational knowledge of cellular biology comes from studies in budding yeast, often described as a simple unicellular eukaryotic model. In this issue of Cell, Correia-Melo et al. describe an unappreciated feature of yeast biology involving intra-cellular metabolite exchange, where cells adapt and respond as part of a community, and go on to show that sharing of resources linked to methionine metabolism enhances longevity of cooperating cells.

Cell-cell metabolite exchange creates a pro-survival metabolic environment that extends lifespan.

Metabolism is deeply intertwined with aging. Effects of metabolic interventions on aging have been explained with intracellular metabolism, growth control, and signaling. Studying chronological aging in yeast, we reveal a so far overlooked metabolic property that influences aging via the exchange of metabolites. We observed that metabolites exported by young cells are re-imported by chronologically aging cells, resulting in cross-generational metabolic interactions. Then, we used self-establishing metabolically cooperating communities (SeMeCo) as a tool to increase metabolite exchange and observed significant lifespan extensions. The longevity of the SeMeCo was attributable to metabolic reconfigurations in methionine consumer cells. These obtained a more glycolytic metabolism and increased the export of protective metabolites that in turn extended the lifespan of cells that supplied them with methionine. Our results establish metabolite exchange interactions as a determinant of cellular aging and show that metabolically cooperating cells can shape the metabolic environment to extend their lifespan.

The proteomic landscape of genome-wide genetic perturbations.

Functional genomic strategies have become fundamental for annotating gene function and regulatory networks. Here, we combined functional genomics with proteomics by quantifying protein abundances in a genome-scale knockout library in Saccharomyces cerevisiae, using data-independent acquisition mass spectrometry. We find that global protein expression is driven by a complex interplay of (1) general biological properties, including translation rate, protein turnover, the formation of protein complexes, growth rate, and genome architecture, followed by (2) functional properties, such as the connectivity of a protein in genetic, metabolic, and physical interaction networks. Moreover, we show that functional proteomics complements current gene annotation strategies through the assessment of proteome profile similarity, protein covariation, and reverse proteome profiling. Thus, our study reveals principles that govern protein expression and provides a genome-spanning resource for functional annotation.

Reconstruction of a fatty acid synthesis cycle from acyl carrier protein and cofactor structural snapshots.

Fatty acids (FAs) play a central metabolic role in living cells as constituents of membranes, cellular energy reserves, and second messenger precursors. A 2.6 MDa FA synthase (FAS), where the enzymatic reactions and structures are known, is responsible for FA biosynthesis in yeast. Essential in the yeast FAS catalytic cycle is the acyl carrier protein (ACP) that actively shuttles substrates, biosynthetic intermediates, and products from one active site to another. We resolve the S. cerevisiae FAS structure at 1.9 Å, elucidating cofactors and water networks involved in their recognition. Structural snapshots of ACP domains bound to various enzymatic domains allow the reconstruction of a full yeast FA biosynthesis cycle. The structural information suggests that each FAS functional unit could accommodate exogenous proteins to incorporate various enzymatic activities, and we show proof-of-concept experiments where ectopic proteins are used to modulate FAS product profiles.

Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast.

Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) levels. 5-InsP7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism.

A replication fork determinant for the establishment of sister chromatid cohesion.

Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Cohesion establishment requires acetylation of conserved cohesin lysine residues by Eco1 acetyltransferase. Here, we explore how cohesin acetylation is linked to DNA replication. Biochemical reconstitution of replication-coupled cohesin acetylation reveals that transient DNA structures, which form during DNA replication, control the acetylation reaction. As polymerases complete lagging strand replication, strand displacement synthesis produces DNA flaps that are trimmed to result in nicked double-stranded DNA. Both flaps and nicks stimulate cohesin acetylation, while subsequent nick ligation to complete Okazaki fragment maturation terminates the acetylation reaction. A flapped or nicked DNA substrate constitutes a transient molecular clue that directs cohesin acetylation to a window behind the replication fork, next to where cohesin likely entraps both sister chromatids. Our results provide an explanation for how DNA replication is linked to sister chromatid cohesion establishment.

DNA-Protein Crosslinks and Their Resolution.

Covalent DNA-protein crosslinks (DPCs) are pervasive DNA lesions that interfere with essential chromatin processes such as transcription or replication. This review strives to provide an overview of the sources and principles of cellular DPC formation. DPCs are caused by endogenous reactive metabolites and various chemotherapeutic agents. However, in certain conditions DPCs also arise physiologically in cells. We discuss the cellular mechanisms resolving these threats to genomic integrity. Detection and repair of DPCs require not only the action of canonical DNA repair pathways but also the activity of specialized proteolytic enzymes-including proteases of the SPRTN/Wss1 family-to degrade the crosslinked protein. Loss of DPC repair capacity has dramatic consequences, ranging from genome instability in yeast and worms to cancer predisposition and premature aging in mice and humans.

Role of the TOM Complex in Protein Import into Mitochondria: Structural Views.

Mitochondria are central to energy production, metabolism and signaling, and apoptosis. To make new mitochondria from preexisting mitochondria, the cell needs to import mitochondrial proteins from the cytosol into the mitochondria with the aid of translocators in the mitochondrial membranes. The translocase of the outer membrane (TOM) complex, an outer membrane translocator, functions as an entry gate for most mitochondrial proteins. Although high-resolution structures of the receptor subunits of the TOM complex were deposited in the early 2000s, those of entire TOM complexes became available only in 2019. The structural details of these TOM complexes, consisting of the dimer of the ß-barrel import channel Tom40 and four α-helical membrane proteins, revealed the presence of several distinct paths and exits for the translocation of over 1,000 different mitochondrial precursor proteins. High-resolution structures of TOM complexes now open up a new era of studies on the structures, functions, and dynamics of the mitochondrial import system.

Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation.

How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5' UTR structures and upstream near-cognate start codons.

Structure of RNA polymerase II pre-initiation complex at 2.9 Å defines initial DNA opening.

Transcription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of the yeast PIC and define the mechanism of initial DNA opening. We trap the PIC in an intermediate state that contains half a turn of open DNA located 30-35 base pairs downstream of the TATA box. The initially opened DNA region is flanked and stabilized by the polymerase "clamp head loop" and the TFIIF "charged region" that both contribute to promoter-initiated transcription. TFIIE facilitates initiation by buttressing the clamp head loop and by regulating the TFIIH translocase. The initial DNA bubble is then extended in the upstream direction, leading to the open promoter complex and enabling start-site scanning and RNA synthesis. This unique mechanism of DNA opening may permit more intricate regulation than in the Pol I and Pol III systems.

Dynamic 3D proteomes reveal protein functional alterations at high resolution in situ.

Biological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other 'omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology.

An in vitro system to silence mitochondrial gene expression.

The human mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation system, and defects in mitochondrial gene expression lead to severe neuromuscular disorders. However, the mechanisms of mitochondrial gene expression remain poorly understood due to a lack of experimental approaches to analyze these processes. Here, we present an in vitro system to silence translation in purified mitochondria. In vitro import of chemically synthesized precursor-morpholino hybrids allows us to target translation of individual mitochondrial mRNAs. By applying this approach, we conclude that the bicistronic, overlapping ATP8/ATP6 transcript is translated through a single ribosome/mRNA engagement. We show that recruitment of COX1 assembly factors to translating ribosomes depends on nascent chain formation. By defining mRNA-specific interactomes for COX1 and COX2, we reveal an unexpected function of the cytosolic oncofetal IGF2BP1, an RNA-binding protein, in mitochondrial translation. Our data provide insight into mitochondrial translation and innovative strategies to investigate mitochondrial gene expression.

A phosphate starvation response-centered network regulates mycorrhizal symbiosis.

To secure phosphorus (P) from soil, most land plants use a direct phosphate uptake pathway via root hairs and epidermis and an indirect phosphate uptake pathway via mycorrhizal symbiosis. The interaction between these two pathways is unclear. Here, we mapped a network between transcription factors and mycorrhizal symbiosis-related genes using Y1H. Intriguingly, this gene regulatory network is governed by the conserved P-sensing pathway, centered on phosphate starvation response (PHR) transcription factors. PHRs are required for mycorrhizal symbiosis and regulate symbiosis-related genes via the P1BS motif. SPX-domain proteins suppress OsPHR2-mediated induction of symbiosis-related genes and inhibit mycorrhizal infection. In contrast, plants overexpressing OsPHR2 show improved mycorrhizal infection and are partially resistant to P-mediated inhibition of symbiosis. Functional analyses of network nodes revealed co-regulation of hormonal signaling and mycorrhizal symbiosis. This network deciphers extensive regulation of mycorrhizal symbiosis by endogenous and exogenous signals and highlights co-option of the P-sensing pathway for mycorrhizal symbiosis.

Synthetic Genomes.

DNA synthesis technology has progressed to the point that it is now practical to synthesize entire genomes. Quite a variety of methods have been developed, first to synthesize single genes but ultimately to massively edit or write from scratch entire genomes. Synthetic genomes can essentially be clones of native sequences, but this approach does not teach us much new biology. The ability to endow genomes with novel properties offers special promise for addressing questions not easily approachable with conventional gene-at-a-time methods. These include questions about evolution and about how genomes are fundamentally wired informationally, metabolically, and genetically. The techniques and technologies relating to how to design, build, and deliver big DNA at the genome scale are reviewed here. A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.

Checkpoint Responses to DNA Double-Strand Breaks.

Cells confront DNA damage in every cell cycle. Among the most deleterious types of DNA damage are DNA double-strand breaks (DSBs), which can cause cell lethality if unrepaired or cancers if improperly repaired. In response to DNA DSBs, cells activate a complex DNA damage checkpoint (DDC) response that arrests the cell cycle, reprograms gene expression, and mobilizes DNA repair factors to prevent the inheritance of unrepaired and broken chromosomes. Here we examine the DDC, induced by DNA DSBs, in the budding yeast model system and in mammals.

RNA Splicing by the Spliceosome.

The spliceosome removes introns from messenger RNA precursors (pre-mRNA). Decades of biochemistry and genetics combined with recent structural studies of the spliceosome have produced a detailed view of the mechanism of splicing. In this review, we aim to make this mechanism understandable and provide several videos of the spliceosome in action to illustrate the intricate choreography of splicing. The U1 and U2 small nuclear ribonucleoproteins (snRNPs) mark an intron and recruit the U4/U6.U5 tri-snRNP. Transfer of the 5' splice site (5'SS) from U1 to U6 snRNA triggers unwinding of U6 snRNA from U4 snRNA. U6 folds with U2 snRNA into an RNA-based active site that positions the 5'SS at two catalytic metal ions. The branch point (BP) adenosine attacks the 5'SS, producing a free 5' exon. Removal of the BP adenosine from the active site allows the 3'SS to bind, so that the 5' exon attacks the 3'SS to produce mature mRNA and an excised lariat intron.