Set1 is the catalytic subunit of SET1C or COMPASS, which methylates histone H3K4 and serves as a scaffold for the association of seven tightly bound polypeptides. We have employed yeast two-hybrid screenings to determine the interactome of Set1 and each subunit, providing a unique resource for exploring known and novel roles of the complex. Our screenings identified a multitude of potential interactors involved in chromatin regulation, DNA replication, meiotic breaks, and Ty transposition, processes previously associated with SET1C. Consistent with Set1 being an RNA-binding protein, the screens link SET1C to multiple aspects of RNA biogenesis, including pre-mRNA splicing and polyadenylation. The results reveal that several importins are candidate interactors of Set1, along with RGG motif-containing proteins, providing insights into the mechanisms by which Set1 moves between cytoplasmic and nuclear compartments. We further reveal that reconstituted SET1C interacts with the AT hook domain of the chromatin remodeler Snf2 and methylates multiple arginines within this domain. In vivo, we report that the ARTSTRGR AT-hook motif is methylated in a Set1-dependent manner revealing new interplay between lysine and arginine methylation.

Set1 is the catalytic subunit of SET1C or COMPASS, which methylates histone H3K4 and serves as a scaffold for the association of seven tightly bound polypeptides. We have employed yeast two-hybrid screenings to determine the interactome of Set1 and each subunit, providing a unique resource for exploring known and novel roles of the complex. Our screenings identified a multitude of interactors involved in chromatin regulation, DNA replication, meiotic breaks, and Ty transposition, processes previously associated with SET1C. Consistent with Set1 being an RNA-binding protein, the screens link SET1C to multiple aspects of RNA biogenesis, including pre-mRNA splicing and polyadenylation. The results reveal that Set1 interacts with several importins and with RGG motif-containing proteins, providing insights into the mechanisms by which Set1 moves between cytoplasmic and nuclear compartments. We demonstrate that the transcriptional corepressor Nrm1 is methylated by SET1C in vitro suggesting that H3K4-like domains may represent a class of non-histone substrates for SET1C. We further reveal that reconstituted SET1C interacts with the AT hook domain of the chromatin remodeler Snf2 and methylates multiple arginines within this domain. In vivo , we report that the ARTSTRGR AT-hook motif is methylated in a Set1-dependent manner revealing new interplay between lysine and arginine methylation. ### Competing Interest Statement The authors have declared no competing interest.

Protein UFMylation regulates numerous cellular processes including ribosome quality control and nuclear DNA repair. Here, we present a technique to isolate nuclei and purify UFMylated proteins under denaturing non-reducing conditions from commonly used mammalian cell line models such as hTERT-RPE1, HEK293, U2OS, and HCT116 cells. We then describe procedures for identifying and analyzing purified UFMylated proteins using mass spectrometry and western blot.For complete details on the use and execution of this protocol, please refer to Panichnantakul et al.1

Senescent cells, which accumulate with age, exhibit a pro-inflammatory senescence-associated secretory phenotype (SASP) that includes the secretion of cytokines, lipids, and extracellular vesicles (EVs). Here, we established an in vitro model of senescence induced by Raf-1 oncogene in RAW 264.7 murine macrophages (MΦ) and compared them to senescent MΦ found in mouse lung tumors or primary macrophages treated with hydrogen peroxide. The transcriptomic analysis of senescent MΦ revealed an important inflammatory signature regulated by NFkB. We observed an increased secretion of EVs in senescent MΦ, and these EVs presented an enrichment for ribosomal proteins, major vault protein, pro-inflammatory miRNAs, including miR-21a, miR-155, and miR-132, and several mRNAs. The secretion of senescent MΦ allowed senescent murine embryonic fibroblasts to restart cell proliferation. This antisenescence function of the macrophage secretome may explain their pro-tumorigenic activity and suggest that senolytic treatment to eliminate senescent MΦ could potentially prevent these deleterious effects.

The highly repetitive and transcriptionally active ribosomal DNA (rDNA) genes are exceedingly susceptible to genotoxic stress. Induction of DNA double-strand breaks (DSBs) in rDNA repeats is associated with ataxia-telangiectasia-mutated (ATM)-dependent rDNA silencing and nucleolar reorganization where rDNA is segregated into nucleolar caps. However, the regulatory events underlying this response remain elusive. Here, we identify protein UFMylation as essential for rDNA-damage response in human cells. We further show the only ubiquitin-fold modifier 1 (UFM1)-E3 ligase UFL1 and its binding partner DDRGK1 localize to nucleolar caps upon rDNA damage and that UFL1 loss impairs ATM activation and rDNA transcriptional silencing, leading to reduced rDNA segregation. Moreover, analysis of nuclear and nucleolar UFMylation targets in response to DSB induction further identifies key DNA-repair factors including ATM, in addition to chromatin and actin network regulators. Taken together, our data provide evidence of an essential role for UFMylation in orchestrating rDNA DSB repair.

ABSTRACT The highly repetitive and transcriptionally active ribosomal DNA (rDNA) genes are exceedingly susceptible to genotoxic stress. Induction of DNA double-strand breaks (DSBs) in rDNA repeats is associated with ATM-dependent rDNA silencing and nucleolar reorganization where rDNA is segregated into nucleolar caps. However, the regulatory events underlying this response remain elusive. Here, we identify protein UFMylation as essential for rDNA damage response in human cells. We further show the only UFM1-E3-ligase UFL1 and its binding partner DDRGK1 localize to nucleolar caps upon rDNA damage, and that UFL1 loss impairs ATM activation and rDNA transcriptional silencing, leading to reduced rDNA segregation. A first-ever analysis of nuclear and nucleolar UFMylation targets in response to DSBs induction further identified key DNA repair factors including ATM, in addition to chromatin and actin network regulators. Taken together, our data provides the first evidence of an essential role for UFMylation in orchestrating rDNA DSB repair.

Partitioning of active gene loci to the nuclear envelope (NE) is a mechanism by which organisms increase the speed of adaptation and metabolic robustness to fluctuating resources in the environment. In the yeast Saccharomyces cerevisiae, adaptation to nutrient depletion or other stresses, manifests as relocalization of active gene loci from nucleoplasm to the NE, resulting in more efficient transport and translation of mRNA. The mechanism by which this partitioning occurs remains a mystery. Here, we demonstrate that the yeast inositol depletion-responsive gene locus INO1 partitions to the nuclear envelope, driven by local histone acetylation-induced polymer-polymer phase separation from the nucleoplasmic phase. This demixing is consistent with recent evidence for chromatin phase separation by acetylation-mediated dissolution of multivalent histone association and fits a physical model where increased bending stiffness of acetylated chromatin polymer causes its phase separation from de-acetylated chromatin. Increased chromatin spring stiffness could explain nucleation of transcriptional machinery at active gene loci.

Two isoforms of the nuclear pore complex (NPC) have been identified in the yeast S. cerevisiae, which coexist at the periphery of the nucleus and differ by the presence or absence of a nuclear basket. Here, we present a protocol to isolate the two types of NPCs from the same cell extract and dissect their interactomes. We describe steps for powder preparation and magnetic bead conjunction and detail differential affinity purification and outcome evaluation through SDS-PAGE, silver staining, and mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Bensidoun et al.1

For over 40 years, ribosomes were considered monolithic machines that translate the genetic code indiscriminately. However, over the past two decades, there have been a growing number of studies that suggest ribosomes to have a degree of compositional and functional adaptability in response to tissue type, cell environment and stimuli, cell cycle or development state. In such form, ribosomes themselves take an active part in translation regulation through an intrinsic adaptability provided by evolution, which furnished ribosomes with a dynamic plasticity that confers another layer of gene expression regulation. Yet despite the identification of various sources that give rise to ribosomal heterogeneity both at the protein and RNA level, its functional relevance is still debated, and many questions remain. Here, we will review aspects, including evolutionary ones, of ribosome heterogeneity emerging at the nucleic acid level, and aim to reframe ribosome 'heterogeneity' as an adaptive and dynamic process of plasticity.The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

In all cells and tissues, protein production is carried out by a ubiquitous machine – the ribosome, a three Mega Dalton macromolecular complex that is essential across all domains of life. It was 65 years ago, in 1958, that the ribosome as protein production machinery was first named by Richard Roberts [1–3]. Five years later, in January 1963, Jonathan Warner, Paul Knopf and Alex Rich published the first paper on the characterization of polyribosomes, or polysomes, from rabbit reticulocyte lysate [4]. The very same year, work by Alfred Gierer in Tubingen, also using reticulocytes [5] and Hans Noll in Pittsburgh, using rat liver [6] independently suggested that protein synthesis was conducted on structures with multiple ribosomes scanning a single mRNA, and in 1964, description of the A(cceptor) and P(eptidyl) sites (the E, or exit, site was only identified by Knut Nierhaus in 1981 [7]) and mRNA and tRNA, followed [8–14]. In the span of a little over a year and a half, views of protein synthesis evolved from a vague idea of an interaction between mRNA, tRNA and ribosomes to a basic model that guides ongoing research into mechanisms of translation regulation to this day: ribosomes move from one end of an mRNA to another, and each has a site for a tRNA attached to the growing polypeptide and a site for the incoming tRNA attached to an amino acid. It also became clear very early on that the ribosome itself is composed of both RNA and proteins and investigation into its composition and biogenesis began in the 1950s and 60s, in bacteria and eukaryotes, respectively [3]. In 1962, Klaus Scherrer, Harriet Latham, and James E. Darnell demonstrated the existence of precursor ribosomal RNAs (rRNAs) in HeLa cells [15], while R. J. Britten, B. J. McCarthy, and Richard Roberts suggested that ribosomes are formed in a stepwise manner [16,17]. The nucleolus was identified as the site of ribosome biogenesis only 2 years later [18], the same year as rRNA base methylation [19] and both rRNA and ‘nascent’ ribosomes of different molecular mass were isolated from human nuclei for the first time in 1967 [15,19,20]. It was in 1972 that a study by Kumar & Warner on nuclear vs cytoplasmic ribosomes in human cells revealed a considerable difference in protein content [21]. At the time, these ‘extra proteins’ within nuclear ribosomes were – in retrospect, correctly – hypothesized to function in the processing of rRNA precursors, and while ribosomes were moved to the cytoplasm, these ‘extra proteins’ stayed behind and were surmised to be re-utilized in further rounds of ribosome production [5]. These ‘extra’ proteins are now known to be the many ribosome biogenesis factors that are required to modify, process, and fold the ribosomal RNAs (rRNA) and assemble them with the ribosomal proteins (r-proteins) into mature ribosomes prior to their transport into the cytoplasm, to the site of translation [22,23]. Without doubt, the ribosome has intrigued many scientists in the decades since its initial characterization, either in its function as a protein synthesis machinery or as a complex macromolecular machine that requires a carefully orchestrated pathway and hundreds of proteins to ensure its assembly. Ribosome research has also come of age with the advances in biochemistry and mass spectrometry in the 2000s and the deep sequencing, high-resolution and cryoelectron microscopy approaches of the last decade. These enabled not only the identification and subsequent characterization of many of the ribosome biogenesis factors but also provided insights into many aspects of the ribosome life cycle: from translation regulation, non-canonical translation initiation and localized translation to ribosome heterogeneity, ribosome and pre-ribosome structures across several species, as well as the intricate link between cell stress and ribosome production, and numerous ribosome-linked disease etiologies or ribosomopathies. Yet despite our growing understanding of ribosome maturation and function over the years, there are many questions that still remain unanswered. The main purpose of this Special Focus on the Ribosome Life cycle is to review some of these questions and discuss recent findings as well as emerging concepts in ribosome biogenesis, translation, and ribosome biology as a whole. Collectively genetic, biochemical, and structural studies have revealed many different aspects of ribosomes in bacteria and eukaryotes to near-atomic levels and have provided a detailed understanding of what are now considered well-established models for ribosome biogenesis and function both in terms of model organisms and ribosome biology (encompassing biogenesis, composition, and function) as viewed from a ‘classical’ perspective [24]. However, as both functional and compositional diversity of ribosomes have been suggested in recent years, it may become increasingly important to step outside the framework of these models to explore the functional and structural diversity of both ribosome biogenesis and function across the biological diversity as it exists in cells, tissues, and different organisms. In his point-of-view, Ferreira-Cerca discusses how studying the ribosome in the ‘third domain of life’, archaea, as well as in many non-model bacterial and eukaryotic organisms, which have so far been largely neglected, will not only be important to provide an as yet untapped window into the evolution of ribosome biogenesis and function but also to unravel fundamental principles of the molecular adaptation of these central cellular processes [25]. One such example is the mitochondrial ribosome. Initially identified in 1958 and then more thoroughly described in RNA BIOLOGY 2023, VOL. 20, NO. 1, 307–310 https://doi.org/10.1080/15476286.2023.2221946

The mitochondrial ribosome (mitoribosome) has diverged drastically from its evolutionary progenitor, the bacterial ribosome. Structural and compositional diversity is particularly striking in the phylum Euglenozoa, with an extraordinary protein gain in the mitoribosome of kinetoplastid protists. Here we report an even more complex mitoribosome in diplonemids, the sister-group of kinetoplastids. Affinity pulldown of mitoribosomal complexes from Diplonema papillatum, the diplonemid type species, demonstrates that they have a mass of > 5 MDa, contain as many as 130 integral proteins, and exhibit a protein-to-RNA ratio of 11:1. This unusual composition reflects unprecedented structural reduction of ribosomal RNAs, increased size of canonical mitoribosomal proteins, and accretion of three dozen lineage-specific components. In addition, we identified >50 candidate assembly factors, around half of which contribute to early mitoribosome maturation steps. Because little is known about early assembly stages even in model organisms, our investigation of the diplonemid mitoribosome illuminates this process. Together, our results provide a foundation for understanding how runaway evolutionary divergence shapes both biogenesis and function of a complex molecular machine.

Protein cross-linking mass spectrometry (XL-MS) has been developed into a powerful and robust tool that is now well implemented and routinely used by an increasing number of laboratories. While bulk cross-linking of complexes provides useful information on whole complexes, it is limiting for the probing of specific protein “neighbourhoods,” or vicinity interactomes. For example, it is not unusual to find cross-linked peptide pairs that are disproportionately overrepresented compared to the surface areas of complexes, while very few or no cross-links are identified in other regions. When studying dynamic complexes along their pathways, some vicinity cross-links may be of too low abundance in the pool of heterogenous complexes of interest to be efficiently identified by standard XL-MS. In this chapter, we describe a targeted XL-MS approach from single-step affinity purified (ssAP) complexes that enables the investigation of specific protein “neighbourhoods” within molecular complexes in yeast, using a small cross-linker anchoring tag, the CH-tag. One advantage of this method over a general cross-linking strategy is the possibility to significantly enrich for localized anchored-cross-links within complexes, thus yielding a higher sensitivity to detect highly dynamic or low abundance protein interactions within a specific protein “neighbourhood” occurring along the pathway of a selected bait protein. Moreover, many variations of the method can be employed; the ssAP-tag and the CH-tag can either be fused to the same or different proteins in the complex, or the CH-tag can be fused to multiple protein components in the same cell line to explore dynamic vicinity interactions along a pathway.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1A) is a nuclear transcriptional coregulator that plays a major role in the control of metabolism and mitochondrial biogenesis. We showed that a diabetes-linked single nucleotide polymorphism (SNP rs8192678) within the coding region of PPARGC1A gene (Gly482Ser) results in decreased protein stability and half-life in liver and beta-cell lines and human induced pluripotent stem cells (iPSCs) . Our mass spectrometry data suggest that degradation of the S482 variant is mediated by phosphorylation at amino acid 482 when a serine is present. A kinome screen identified three potential kinases (NEK2, MARK4 and S6K2) that can modify this site. We found that mutation of the mouse homolog at the conserved site (serine to glycine) similarly stabilized mouse PGC1A. To study the physiological consequences of this SNP, we generated whole-body homozygous glycine (G/G) , serine (S/S) and heterozygous (S/G) mice. We analyzed various metabolic parameters in male (N=10-per genotype) and female (N=8-9) mice subjected to standard chow or high fat, high fructose diet (HFHF) for 14 or 24 weeks. We found that G/G male and female mice had increased caloric intake using metabolic cages without increased body weight. Male mice on a HFHF diet carrying a glycine allele (G/G or S/G) were more resistant to insulin in an insulin tolerance test (ITT) , secreted less insulin in response to a mixed-meal challenge and had decreased glucose uptake in muscle and adipose tissue. In line in humans, non-diabetic subjects with G/G (N=20) or G/S (N=22) variants oxidize more fat while those with S/S variant (N=6) oxidize more carbohydrate over 6-hours following the ingestion of a high-fat meal (68% fat, 18% carbohydrate) . In summary, these results demonstrate that differences in PGC1A stability associated with phosphorylation at site 482 may lead to differences in glucose and fat metabolism, which could explain the link between this SNP and metabolic diseases. Disclosure M.Galipeau: None. M.Faraj: None. M.Oeffinger: None. F.C.Lynn: None. R.A.Screaton: None. T.Alquier: None. J.L.Estall: None. N.Jouvet: None. E.Courty: None. R.Vandenbeek: None. N.P.Khan: None. K.Bouyakdan: None. C.Nian: None. C.Iorio: None. D.D.Scott: None. Funding CIHR (PJT-148771) CIHR (PJT-168853)

The control of RNA metabolism is an important aspect of molecular biology with wide-ranging impacts on cells. Central to processing of coding RNAs is the addition of the methyl-7 guanosine (m7G) "cap" on their 5' end. The eukaryotic translation initiation factor eIF4E directly binds the m7G cap and through this interaction plays key roles in many steps of RNA metabolism including nuclear RNA export and translation. eIF4E also stimulates capping of many transcripts through its ability to drive the production of the enzyme RNMT which methylates the G-cap to form the mature m7G cap. Here, we found that eIF4E also physically associated with RNMT in human cells. Moreover, eIF4E directly interacted with RNMT in vitro. eIF4E is only the second protein reported to directly bind the methyltransferase domain of RNMT, the first being its co-factor RAM. We combined high-resolution NMR methods with biochemical studies to define the binding interfaces for the RNMT-eIF4E complex. Further, we found that eIF4E competes for RAM binding to RNMT and conversely, RNMT competes for binding of well-established eIF4E-binding partners such as the 4E-BPs. RNMT uses novel structural means to engage eIF4E. Finally, we observed that m7G cap-eIF4E-RNMT trimeric complexes form, and thus RNMT-eIF4E complexes may be employed so that eIF4E captures newly capped RNA. In all, we show for the first time that the cap-binding protein eIF4E directly binds to the cap-maturation enzyme RNMT.

RbgA is an essential protein for the assembly of the 50S subunit in Bacillus subtilis. Depletion of RbgA leads to the accumulation of the 45S intermediate. A strain expressing a RbgA variant with reduced GTPase activity generates spontaneous suppressor mutations in uL6. Each suppressor strain accumulates a unique 44S intermediate. We reasoned that characterizing the structure of these mutant 44S intermediates may explain why RbgA is required to catalyze the folding of the 50S functional sites. We found that in the 44S particles, rRNA helices H42 and H97, near the binding site of uL6, adopt a flexible conformation and allow the central protuberance and functional sites in the mutant 44S particles to mature in any order. Instead, the wild-type 45S particles exhibit a stable H42-H97 interaction and their functional sites always mature last. The dependence on RbgA was also less pronounced in the 44S particles. We concluded that the binding of uL6 pauses the maturation of the functional sites, but the central protuberance continues to fold. RbgA exclusively binds intermediates with a formed central protuberance and licenses the folding of the functional sites. Through this mechanism, RbgA ensures that the functional sites of the 50S mature last.

Cellular functions are mostly defined by the dynamic interactions of proteins within macromolecular networks. Deciphering the composition of macromolecular complexes and their dynamic rearrangements is the key to getting a comprehensive picture of cellular behavior and to understanding biological systems. In the last decade, affinity purification coupled to mass spectrometry has emerged as a powerful tool to comprehensively study interaction networks and their assemblies. However, the study of these interactomes has been hampered by severe methodological limitations. In particular, the affinity purification of intact complexes from cell lysates suffers from protein and RNA degradation, loss of transient interactors, and poor overall yields. In this chapter, we describe a rapid single-step affinity purification method for the efficient isolation of dynamic macromolecular complexes. The technique employs cell lysis by cryo-milling, which ensures nondegraded starting material in the submicron range, and magnetic beads, which allow for dense antibody-conjugation and thus rapid complex isolation, while avoiding loss of transient interactions. The method is epitope tag-independent, and overcomes many of the previous limitations to produce large interactomes with almost no contamination. The protocol described here has been optimized for the yeast S. cerevisiae.

Growth factor indepdendent 1 (GFI1) is a SNAG-domain, DNA binding transcriptional repressor which controls myeloid differentiation through molecular mechanisms and co-factors that still remain to be clearly identified. Here we show that GFI1 associates with the chromodomain helicase DNA binding protein 4 (CHD4) and other components of the Nucleosome remodeling and deacetylase (NuRD) complex. In granulo-monocytic precursors, GFI1, CHD4 or GFI1/CHD4 complexes occupy sites enriched for histone marks associated with active transcription suggesting that GFI1 recruits the NuRD complex to target genes regulated by active or bivalent promoters and enhancers. GFI1 and GFI1/CHD4 complexes occupy promoters that are either enriched for IRF1 or SPI1 consensus binding sites, respectively. During neutrophil differentiation, chromatin closure and depletion of H3K4me2 occurs at different degrees depending on whether GFI1, CHD4 or both are present, indicating that GFI1 is more efficient in depleting of H3K4me2 and -me1 marks when associated with CHD4. Our data suggest that GFI1/CHD4 complexes regulate histone modifications differentially to enable regulation of target genes affecting immune response, nucleosome organization or cellular metabolic processes and that both the target gene specificity and the activity of GFI1 during myeloid differentiation depends on the presence of chromatin remodeling complexes.