Biology subjects

Albert, B.

Publications and source records attributed to Albert, B..

15 recordsLinked to original sources

bioRxiv2026

RNA helicase Drs1 gates 25S rRNA domain III incorporation during early nucleolar pre-60S maturation

Assembly of eukaryotic large ribosomal subunits (LSU) requires coordinated structural and compositional transitions within pre-60S particles, yet the underlying mechanisms remain poorly understood. Here, we show that the DEAD-box helicase Drs1 promotes early maturation across distinct regions of the pre-60S particle. Loss of Drs1 function causes accumulation of co-transcriptional intermediates retaining SSU processome components, indicating that Drs1 promotes timely separation of nascent LSU precursors from the small-subunit assembly pathway. Cryo-EM analyses reveal both a redistribution toward early nucleolar maturation states upon loss of Drs1, including Nsa1-deficient intermediates, and a confinement of Drs1-associated particles to states preceding stable incorporation of 25S rRNA domain III. Drs1 directly engages Erb1 through its unstructured N-terminal extension, promoting stable assembly of the Nop7-Erb1-Ytm1 module associated with domain III maturation. CRAC analysis localizes Drs1 to spatially clustered sites spanning the 5.8S and 25S rRNAs, encompassing domains I-IV. Together, these findings support a model in which Drs1 couples stabilization of assembly-factors with pre-rRNA remodeling across the pre-60S particle, thereby driving ordered early LSU maturation and the timed integration of domain III.

biochemistry↗

bioRxiv2025

Sni445 recruits box C/D snoRNPs snR4 and snR45 to guide ribosomal RNA acetylation by Kre33

Eukaryotic ribosome synthesis is a highly complex, multistep process that is best characterized in the yeast Saccharomyces cerevisiae. It is orchestrated by over 200 ribosome assembly factors and 75 small nucleolar ribonucleoproteins (snoRNPs), which guide site-specific chemical modifications of precursor ribosomal RNA (pre-rRNA). While canonical box C/D snoRNPs direct 2-O-methylation, the atypical box C/D snoRNPs snR4 and snR45 mediate acetylation of 18S rRNA residues C1280 and C1773, respectively, catalyzed by the acetyltransferase Kre33. Here, we identify and characterize Ynl050c/Sni445 as a novel ribosome assembly factor and previously unrecognized auxiliary component of the snR4 and snR45 box C/D snoRNPs. Sni445 associates with snR4 and snR45 in their free form and is required for their stable incorporation into 90S pre-ribosomes. Genetic interactions link Sni445 and the snR4 and snR45 snoRNAs to ribosomal proteins Rps20 (uS10) and Rps14 (uS11), which are positioned near the respective acetylation sites in the 40S subunit. Moreover, Sni445 physically interacts with Kre33 within the 90S pre-ribosome, and its absence abolishes acetylation of C1280 and C1773. Our findings suggest that Sni445 facilitates the recruitment of snR4 and snR45 snoRNPs to 90S particles and might promote their interaction with Kre33, thereby enabling the site-specific acetylation of 18S rRNA by Kre33.

molecular biology↗

bioRxiv2025

New Insights into nuclear import and nucleolar localization of yeast RNA exosome subunits

The RNA exosome is a multiprotein complex essential for RNA maturation and degradation. In budding yeast, a nine-subunit protein complex (Exo9) associated with Rrp44 forms Exo10 in the cytoplasm and, in complex with Rrp6, Exo11 in the nucleus. Depending on its subcellular localization, the exosome interacts with different cofactors and RNA substrates. In the cytoplasm, Exo10 associates with the SKI complex via Ski7, while in the nucleus, Exo11 interacts with the TRAMP complex. Within the nucleolus, the exosome participates in ribosomal RNA (rRNA) processing, facilitated by Mtr4-dependent adaptors Utp18 and Nop53. In this manuscript, we have performed a comprehensive study that addresses the targeting mechanism and precise subcellular localization of all members of the Exo11 complex. We observed a high concentration of all Exo11 subunits in the nucleolus and identified the importins Srp1 () and Kap95 ({beta}) as responsible for the nuclear import of Exo9 subunits. Notably, Exo9 subunits localization was not significantly disrupted in the simultaneous absence of NLS-containing subunits Rrp6 and Rrp44, suggesting redundant nuclear import pathways for Exo9. Additionally, we show evidence that Ski7 may play a role in the Exo9 retention in the cytoplasm. To explore the exosome sub-nucleolar localization, we compared Rrp43 with nuclear exosome cofactors and show that it is enriched in the same nucleolar region as Mtr4 and Nop53. In conclusion, our findings provide a detailed characterization of Exo11 distribution, highlight the primary nuclear import mechanisms for Exo9, and reveal the specific localization of the exosome within the granular component (GC) of the yeast nucleolus, suggesting a spatial regulation of the RNA processing pathway. Highlights- Comprehensive study of the localization of all exosome subunits in yeast. - Identification of karyopherins involved in exosome nuclear import. - Ski7 dependent cytoplasmic retention of exosome. - Yeast sub-nucleolar organization of exosome and its cofactors.

molecular biology↗

bioRxiv2024

The snoRNP chaperone snR190 and the Npa1 complex form a macromomecular assembly required for 60S ribosomal subunit maturation

The early steps of large-ribosomal-subunit assembly feature among the least understood steps of ribosome synthesis in eukaryotes. In Saccharomyces cerevisiae, the snR190 box C/D snoRNP chaperone and the Npa1 complex, composed of the -solenoid scaffold proteins Npa1 and Npa2, the DEAD-box helicase Dbp6, the RNA-binding protein Nop8 and Rsa3, are likely involved in early 25S rRNA folding events. Here, we report for the first time the existence outside pre-ribosomal particles of an independent macromolecular assembly constituted by the Npa1 complex and the snR190 snoRNP chaperone. Nop8 mediates the formation of this assembly and can associate on its own with free snR190. Moreover, Nop8 RRM domain helps tether the snR190 snoRNP to pre-ribosomal particles. snR190 features a specific central stem-loop structure, which is required for high-affinity binding between free snR190 and the Npa1 complex. Deleting this extension does not prevent snR190 association with pre-ribosomal particles but impairs snR190 activity in early pre-rRNA processing events. This work establishes the importance of association with auxiliary protein complexes for optimum snoRNP chaperone activity during rRNA folding events.

molecular biology↗

bioRxiv2024

Stm1 regulates Ifh1 activity revealing crosstalk between ribosome biogenesis and ribosome dormancy

Ribosome abundance in changing environments is governed by biogenesis and degradation, but the underlying mechanisms regulating these opposing processes remain unknown. Here we show that Suppressor of Tom1 (Stm1), a dormancy factor protecting cytosolic ribosomes during starvation, has an unexpected function to promote ribosome biogenesis during exponential growth conditions. Indeed, Stm1 transiently localizes to the nucleolus and engages with pre-ribosomal particles. Stm1 upregulates transcription of ribosomal protein genes by directly binding the activation domain (AD) of the transcription factor Ifh1. These novel Stm1 functions confer rapamycin-sensitivity and are mediated by its C-terminal intrinsically disordered region (IDR), which is dispensable for ribosome hibernation. We conclude that Stm1 regulates ribosome homeostasis linking ribosome biogenesis and ribosome dormancy.

cell biology↗

bioRxiv2023

K29-linked unanchored polyubiquitin chains disrupt ribosome biogenesis and direct ribosomal proteins to the Intranuclear Quality control compartment (INQ)

Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with Ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs), Ubp2 and Ubp14, and E3 ligases, Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the Ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the Intranuclear Quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with Ribosomopathies.

cell biology↗

bioRxiv2022

Not1 and Not4 inversely determine mRNA solubility that sets the dynamics of co-translational events

BackgroundThe Ccr4-Not complex is most well known as the major eukaryotic deadenylase. However, several studies have uncovered roles of the complex, in particular of the Not subunits, unrelated to deadenylation and relevant for translation. In particular, the existence of Not condensates that regulate translation elongation dynamics have been reported. Typical studies that evaluate translation efficiency rely on soluble extracts obtained after disruption of cells and ribosome profiling. Yet cellular mRNAs in condensates can be actively translated and may not be present in such extracts. ResultsIn this work, by analyzing soluble and insoluble mRNA decay intermediates in yeast, we determine that insoluble mRNAs are enriched for ribosomes dwelling at non-optimal codons compared to soluble mRNAs. mRNA decay is higher for soluble RNAs, but the proportion of co-translational degradation relative to the overall mRNA decay is higher for insoluble mRNAs. We show that depletion of Not1 and Not4 inversely impact mRNA solubilities and, for soluble mRNAs, ribosome dwelling according to codon optimality. Depletion of Not4 solubilizes mRNAs with lower non-optimal codon content and higher expression that are rendered insoluble by Not1 depletion. By contrast, depletion of Not1 solubilizes mitochondrial mRNAs, which are rendered insoluble upon Not4 depletion. ConclusionOur results reveal that mRNA solubility defines dynamics of co-translation events and is oppositely regulated by Not1 and Not4, a mechanism that we additionally determine may already be set by Not1 promoter association in the nucleus.

molecular biology↗

bioRxiv2021

ChEC-seq: a robust method to identify protein-DNA interactions genome-wide

Mittal et al. (2021; first brought to our attention in May 2019) have raised concerns regarding the Chromatin Endogenous Cleavage-sequencing (ChEC-seq) technique (Zentner et al., 2015) that may create a false impression that this method has fundamental flaws which prevent one from distinguishing between signal and noise. Although Mittal et al. focus on studies of the global co-activators SAGA, TFIID and Mediator that we were not involved in, we feel obliged to highlight here several of our own publications (Albert et al., 2019; Bruzzone et al., 2018; Hafner et al., 2018; Kubik et al., 2019; Kubik et al., 2018), as well as recent unpublished data, that employed ChEC-seq and directly addressed the observation raised by Mittal et al. that cleavage maps for various MNase fusion proteins often qualitatively resemble each other and those generated by "free" (unfused) MNase. Our studies lay out a clear path for determining sites of preferential factor localization by normalization of ChEC-seq experimental data to matched free-MNase controls. They also demonstrate the use of in vivo functional assays to assess ChEC-seq reliability and reveal examples where ChEC-seq identifies functional binding sites missed by conventional ChIP-seq analysis.

molecular biology↗

bioRxiv2020

Mechanisms Coordinating Ribosomal Protein Gene Transcription in Response to Stress

While expression of ribosomal protein genes (RPGs) in the budding yeast Saccharomyces cerevisiae has been extensively studied, a longstanding enigma persists regarding their co-regulation under fluctuating nutrient and stress conditions. Most (<90%) of RPG promoters display one of two distinct arrangements of a core set of transcription factors (TFs; Rap1, Fhl1 and Ifh1) and are further differentiated by the presence or absence of the HMGB box protein Hmo1. However, a third group of promoters appears not to bind any of these proteins, raising the question of how the whole suite of genes is co-regulated. We demonstrate that all RPGs are regulated by two distinct, but complementary mechanisms driven by the TFs Ifh1 and Sfp1, both of which are required for maximal expression in optimal conditions and coordinated down-regulation upon stress. At the majority of RPG promoters Ifh1-dependent regulation predominates, whereas Sfp1 plays the major role at all other genes. We also uncovered an unexpected, protein homeostasis-dependent binding property of Hmo1 at a large subset of RPG promoters. Finally, we show that the Ifh1 paralog Crf1, previously described as a transcriptional repressor, can act as a constitutive RPG activator in the W303 strain background when overexpressed. Our study thus provides a more complete picture of RPG regulation and may serve as a paradigm for unravelling RPG regulation in multicellular eukaryotes.

molecular biology↗

bioRxiv2019

Fork Pausing Complex Engages Topoisomerases at the Replisome

Replication forks temporarily or terminally pause at hundreds of hard-to-replicate regions around the genome. A conserved pair of budding yeast replisome components Tof1-Csm3 (fission yeast Swi1-Swi3 and human TIMELESS-TIPIN) acts as a molecular brake and promotes fork slowdown at proteinaceous replication fork barriers (RFBs), while the accessory helicase Rrm3 assists the replisome in removing protein obstacles. Here we show that Tof1-Csm3 complex promotes fork pausing independently of Rrm3 helicase by recruiting topoisomerase I (Top1) to the replisome. Topoisomerase II (Top2) partially compensates for the pausing decrease in cells when Top1 is lost from the replisome. The C-terminus of Tof1 is specifically required for Top1 recruitment to the replisome and fork pausing but not for DNA replication checkpoint (DRC) activation. We propose that forks pause at proteinaceous RFBs through a sTOP mechanism ( slowing down with TOPoisomerases I-II), which we show also contributes to protecting cells from topoisomerase-blocking agents.

molecular biology↗

bioRxiv2019

Quantitative profiling of 1-Carbon Metabolites, Amino Acids and Precursors, and Plasmalogens in human plasma using Ultra-High-Pressure Liquid Chromatography coupled with Tandem Mass Spectrometry and robotic compound extraction

Amino acids (AAs) and one-carbon (1-C) compounds are involved in a range of key metabolic pathways, and mediate numerous health and disease processes in the human body. Previous assays have quantified a limited selection of these compounds. Here, we describe an analytical method for the simultaneous quantification of 37 1-C metabolites, amino acids, precursors, and plasmalogens using reversed-phase ultra-high pressure liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Compound extraction from human plasma was tested manually before being robotically automated. The final analytical panel was validated on human plasma samples. Our automated and multiplexed method holds promise for application to large cohort studies.

biochemistry↗

bioRxiv2019

A ribosome assembly stress response regulates transcription to maintain proteome homeostasis

Ribosome biogenesis is a complex and energy-demanding process requiring tight coordination of ribosomal RNA (rRNA) and ribosomal protein (RP) production. Alteration of any step in this process may impact growth by leading to proteotoxic stress. Although the transcription factor Hsf1 has emerged as a central regulator of proteostasis, how its activity is coordinated with ribosome biogenesis is unknown. Here we show that arrest of ribosome biogenesis in the budding yeast S. cerevisiae triggers rapid activation of a highly specific stress pathway that coordinately up-regulates Hsf1 target genes and down-regulates RP genes. Activation of Hsf1 target genes requires neo-synthesis of RPs, which accumulate in an insoluble fraction, leading to sequestration of the RP transcriptional activator Ifh1. Our data suggest that levels of newly-synthetized RPs, imported into the nucleus but not yet assembled into ribosomes, work to continuously balance Hsf1 and Ifh1 activity, thus guarding against proteotoxic stress during ribosome assembly.

molecular biology↗

bioRxiv2018

Sfp1 regulates transcriptional networks driving cell growth and division through multiple promoter binding modes

Understanding how transcriptional programs help to coordinate cell growth and division is an important unresolved problem. Here we report that the nutrient- and stress-regulated transcription factor Sfp1 is rate-limiting for expression of several large classes of genes involved in yeast cell growth, including ribosomal protein, ribosome biogenesis, and snoRNA genes. Remarkably, the spectrum of Sfp1 transcription effects is concordant with a combination of chromatin immunoprecipitation and chromatin endogenous cleavage binding analyses, which together provide evidence for two distinct modes of Sfp1 promoter binding, one requiring a co-factor and the other a specific DNA-recognition motif. In addition to growth-related genes, Sfp1 binds to and regulates the promoters of cell cycle \"START\" regulon genes, including the key G1/S cyclins CLN1 and CLN2. Our findings suggest that Sfp1 acts as a master regulator of cell growth and cell size by coordinating the expression of genes implicated in mass accumulation and cell division.

molecular biology↗

bioRxiv2018

Sequence-directed action of RSC remodeler and pioneer factors positions +1 nucleosome to facilitate transcription

Accessible chromatin is important for RNA polymerase II recruitment and transcription initiation at eukaryotic promoters. We investigated the mechanistic links between promoter DNA sequence, nucleosome positioning and transcription. Our results indicate that precise positioning of the transcription start site-associated +1 nucleosome in yeast is critical for efficient TBP binding, and is driven by two key factors, the essential chromatin remodeler RSC and a small set of ubiquitous pioneer transcription factors. We find no evidence for recruitment of RSC by pioneer factors, but show instead that the strength and directionality of RSC action on nucleosomes depends upon the arrangement of two specific DNA motifs that promote its binding and nucleosome displacement activity at promoters. Thus, despite their widespread co-localization, RSC and pioneer factors predominantly act independently to generate accessible chromatin. Our results provide insight into how promoter DNA sequence instructs trans-acting factors to control nucleosome architecture and stimulate transcription initiation.

molecular biology↗

bioRxiv2017

Nasal and aerosol shedding of infectious influenza virus - a community based study

Little is known about the amount and infectiousness of influenza virus shed into exhaled breath. This contributes to uncertainty about the importance of airborne influenza transmission. We screened 355 symptomatic volunteers with acute respiratory illness and report 142 cases with confirmed influenza infection who provided 218 paired nasopharyngeal (NP) and 30-minute breath samples (coarse >5 m and fine <5 m fractions) on days 1 to 3 post symptom onset. We assessed viral RNA copy number for all samples and cultured NP swabs and fine aerosols. We recovered infectious virus from 52 (39%) of the fine aerosols and 150 (89%) of the NP swabs with valid cultures. The geometric mean RNA copy numbers were 3.8x104/30-min fine, 1.2x104/30-min coarse aerosol sample, and 8.2x108 per NP swab. Fine and coarse aerosol viral RNA was positively associated with body mass index (fine p<0.05, coarse p<0.10) and number of coughs (fine p<0.001, coarse p<0.01) and negatively associated with increasing days since symptom onset (fine p<0.05 to p<0.01, coarse p<0.10) in adjusted models. Fine aerosol viral RNA was also positively associated with having influenza vaccination for both the current and prior season (p<0.01). NP swab viral RNA was positively associated with upper respiratory symptoms (p<0.01) and negatively associated with age (p<0.01) but was not significantly associated with fine or coarse aerosol viral RNA or their predictors. Sneezing was rare, and sneezing and coughing were not necessary for infectious aerosol generation. Our observations suggest that influenza infection in the upper and lower airways are compartmentalized and independent.\n\nSignificanceLack of human data on influenza virus aerosol shedding fuels debate over the importance of airborne transmission. We provide overwhelming evidence that humans generate infectious aerosols and quantitative data to improve mathematical models of transmission and public health interventions. We show that sneezing is rare and not important for, and that coughing is not required for influenza virus aerosolization. Our findings, that upper and lower airway infection are independent and that fine particle exhaled aerosols reflect infection in the lung, open a new pathway for understanding the human biology of influenza infection and transmission. Our observation of an association between repeated vaccination and increased viral aerosol generation demonstrated the power of our method, but needs confirmation.

epidemiology↗