Biology subjects

Ghalei, H.

Publications and source records attributed to Ghalei, H..

10 recordsLinked to original sources

bioRxiv2026

Systematic comparison of pathogenic variants of the RNA exosome gene EXOSC3/RRP40 in Saccharomyces cerevisiae reveals variant-specific functional consequences

The RNA exosome is an essential, evolutionarily conserved ribonuclease complex that processes and degrades many classes of RNA. The complex is composed of three structural cap subunits (EXOSC1-3/Csl4, Rrp4, Rrp40; H. sapiens/S. cerevisiae), six structural core subunits (EXOSC4-9/Rrp41,Rrp46,Mtr3,Rrp42,Rrp43,Rrp45), and a catalytic ribonuclease (DIS3 or DIS3L/Dis3). Cofactors that associate with the RNA exosome confer specificity to target specific RNAs for processing and/or decay. Missense mutations in genes encoding structural subunits of the RNA exosome have been linked to neurological diseases. Notably, several pathogenic mutations have been identified in EXOSC3 that are associated with pontocerebellar hypoplasia type 1b (PCH1b). These pathogenic alleles cause a broad spectrum of clinical severity, suggesting variant-specific functional consequences. Given the high degree of conservation between the human and budding yeast RNA exosome complexes, we performed a systematic analysis of eight pathogenic EXOSC3 variants modeled in budding yeast Rrp40. We find that two Rrp40 variants cause growth defects, show distinct negative genetic interactions with RNA exosome cofactor mutants, and impair RNA processing in budding yeast. One of these variants, EXOSC3-Y109N/Rrp40-Y64N, had not been previously characterized in any mechanistic studies. Computational stability predictions and immunoblot analyses indicate that most EXOSC3/Rrp40 variants display reduced steady-state protein levels, but decreased protein levels do not strictly correlate with phenotype or disease severity, suggesting that individual variants disrupt RNA exosome function through distinct mechanisms. Collectively, our studies suggest that pathogenic EXOSC3 variants alter RNA exosome function through distinct mechanisms and provide insight into the specific molecular defects that could underlie pathology.

genetics↗

bioRxiv2025

EXOSC3 S1-domain variants implicated in PCH1b alter RNA exosome cap subunit abundance and thermal stability disrupting rRNA processing and targeting of AU-rich mRNA

Missense variants in EXOSC3, an RNA exosome subunit, have been identified in patients with PCH1b. We investigated three missense variants in the S1 domain of EXOSC3, including one variant of uncertain significance (VUS) and two pathogenic variants (hence S1 variants). EXOSC3 S1 variant cell lines were generated using CRISPR-Cas9 resulting in widespread proteome changes including decreases in some RNA exosome subunits paired with increases in the catalytic subunit DIS3. Thermal stability, analyzed by PISA, revealed extensive destabilization of RNA exosome cap subunits and the cap-associated exonuclease EXOSC10. Functionally, S1 variants altered rRNA processing with corresponding protein compensation observed in rRNA processing proteins outside the RNA exosome. Exogenous overexpression of EXOSC3 rescues many molecular defects caused by S1 variants suggesting that protein destabilization and turnover strongly contribute to molecular defects. Overall, our findings define the mechanisms through which cells respond to EXOSC3 S1 variant disruption of RNA processing homeostasis.

systems biology↗

bioRxiv2024

Differential roles of putative arginine fingers of AAA+ ATPases Rvb1 and Rvb2

The evolutionarily conserved AAA+ ATPases Rvb1 and Rvb2 proteins form a heteromeric complex (Rvb1/2) required for assembly or remodeling of macromolecular complexes in essential cellular processes ranging from chromatin remodeling to ribosome biogenesis. Rvb1 and Rvb2 have a high degree of sequence and structural similarity, and both contain the classical features of ATPases of their clade, including an N-terminal AAA+ subdomain with the Walker A motif, an insertion domain that typically interacts with various binding partners, and a C-terminal AAA+ subdomain containing a Walker B motif, the Sensor I and II motifs, and an arginine finger. In this study, we find that despite the high degree of structural similarity, Rvb1 and Rvb2 have distinct active sites that impact their activities and regulation within the Rvb1/2 complex. Using a combination of biochemical and genetic approaches, we show that replacing the homologous arginine fingers of Rvb1 and Rvb2 with different amino acids not only has distinct effects on the catalytic activity of the complex, but also impacts cell growth, and the Rvb1/2 interactions with binding partners. Using molecular dynamics simulations, we find that changes near the active site of Rvb1 and Rvb2 cause long-range effects on the protein dynamics in the insertion domain, suggesting a molecular basis for how enzymatic activity within the catalytic site of ATP hydrolysis can be relayed to other domains of the Rvb1/2 complex to modulate its function. Further, we show the impact that the arginine finger variants have on snoRNP biogenesis and validate the findings from molecular dynamics simulations using a targeted genetic screen. Together, our results reveal new aspects of the regulation of the Rvb1/2 complex by identifying a relay of long-range molecular communication from the ATPase active site of the complex to the binding site of cofactors. Most importantly, our findings suggest that despite high similarity and cooperation within the same protein complex, the two proteins have evolved with unique properties critical for the regulation and function of the Rvb1/2 complex. SignificanceAAA ATPases constitute a large family of proteins involved in various essential cellular functions in living organisms in all kingdoms of life. Members of this family typically form homo or hetero multimers that convert the energy from ATP hydrolysis to mechanical work. How the conserved features of AAA ATPases relay the energy from ATP hydrolysis to other functional domains of the complex remains largely unknown. Here, using arginine finger variants of Rvb1 and Rvb2, two evolutionarily conserved closely related AAA+ ATPases that form a heterohexameric complex, we reveal how individual protomers in a heteromeric complex can uniquely contribute to the overall function of the complex and how changes in the ATP binding site can be relayed to distal functional domains.

biochemistry↗

bioRxiv2023

Comparative analyses of disease-linked missense mutations in the RNA exosome modeled in budding yeast reveal distinct functional consequences in translation

The RNA exosome is a multi-subunit, evolutionarily conserved ribonuclease complex that is essential for processing, decay and surveillance of many cellular RNAs. Missense mutations in genes encoding the structural subunits of the RNA exosome complex cause a diverse range of diseases, collectively known as RNA exosomopathies, often involving neurological and developmental defects. The varied symptoms suggest that different mutations lead to distinct in vivo consequences. To investigate these functional consequences and distinguish whether they are unique to each RNA exosomopathy mutation, we generated a collection of in vivo models by introducing pathogenic missense mutations in orthologous S. cerevisiae genes. Comparative RNA-seq analysis assessing broad transcriptomic changes in each mutant model revealed that three yeast mutant models, rrp4-G226D, rrp40-W195R and rrp46-L191H, which model mutations in the genes encoding EXOSC2, EXOSC3 and EXOSC5, respectively, had the largest transcriptomic differences. While some transcriptomic changes, particularly in transcripts related to ribosome biogenesis, were shared among mutant models, each mutation also induced unique transcriptomic changes. Thus, our data suggests that while there are some shared consequences, there are also distinct differences in RNA exosome function by each variant. Assessment of ribosome biogenesis and translation defects in the three models revealed distinct differences in polysome profiles. Collectively, our results provide the first comparative analyses of RNA exosomopathy mutant models and suggest that different RNA exosome gene mutations result in in vivo consequences that are both unique and shared across each variant, providing further insight into the biology underlying each distinct pathology.

molecular biology↗

bioRxiv2023

Sinefungin, a natural nucleoside analog of S-adenosyl methionine, impairs the pathogenicity of Candida albicans

Candida albicans, an opportunistic fungal human pathogen, is a major threat to the healthcare system due to both infections in immunocompromised individuals and the emergence of antifungal resistance. Fungal infection caused by C. albicans, candidiasis, is a life-threatening condition in immunocompromised patients and the current treatments are mostly restricted to polyenes, azoles, and echinocandins. Use of these antifungals is limited by toxicity, drug-drug interactions, and the emergence of resistance, underscoring the importance of identifying novel therapeutic targets and the need for new treatment approaches. C. albicans can undergo a morphological transition from yeast to hyphae and this transition is central to C. albicans virulence. Here, we determine the impact of sinefungin, a natural nucleoside analog of S-adenosyl methionine, on the virulence of C. albicans strain SC5314 by evaluating treatment effects on the morphological transition, human epithelial cell adhesion, and biofilm formation. Our data indicate that sinefungin impairs pathogenic traits of C. albicans including hyphal lengthening, biofilm formation and the adhesion to the human epithelial cell lines, without adversely affecting human cells, therefore highlighting sinefungin as a potential avenue for therapeutic intervention. We determine that the formation of N6-methyladenosine (m6A) is particularly disturbed by sinefungin. More broadly, this study underscores the importance of considering the post-transcriptional control mechanisms of pathogenicity when designing therapeutic solutions to fungal infection.

biochemistry↗

bioRxiv2023

The RNA helicase DDX1 associates with the nuclear RNA exosome and modulates R-loops

The RNA exosome is a ribonuclease complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays roles in regulating gene expression and protecting the genome, including modulating the accumulation of RNA-DNA hybrids (R-loops). The function of the RNA exosome is facilitated by cofactors, such as the RNA helicase MTR4, which binds/remodels RNAs. Recently, missense mutations in RNA exosome subunit genes have been linked to neurological diseases. One possibility to explain why missense mutations in genes encoding RNA exosome subunits lead to neurological diseases is that the complex may interact with cell- or tissue-specific cofactors that are impacted by these changes. To begin addressing this question, we performed immunoprecipitation of the RNA exosome subunit, EXOSC3, in a neuronal cell line (N2A) followed by proteomic analyses to identify novel interactors. We identified the putative RNA helicase, DDX1, as an interactor. DDX1 plays roles in double-strand break repair, rRNA processing, and R-loop modulation. To explore the functional connections between EXOSC3 and DDX1, we examined the interaction following double-strand breaks, and analyzed changes in R-loops in N2A cells depleted for EXOSC3 or DDX1 by DNA/RNA immunoprecipitation followed by sequencing (DRIP-Seq). We find that EXOSC3 interaction with DDX1 is decreased in the presence of DNA damage and that loss of EXOSC3 or DDX1 alters R-loops. These results suggest EXOSC3 and DDX1 interact during events of cellular homeostasis and potentially suppress unscrupulous expression of genes promoting neuronal projection.

biochemistry↗

bioRxiv2022

in vivo RNA structural probing of guanine and uracil nucleotides in yeast

RNAs have critical catalytic or regulatory functions in the cell and play significant roles in many steps of gene expression regulation. RNA structure can be essential for its cellular function. Therefore, methods to investigate the structure of RNA in vivo are of great importance for understanding the role of cellular RNAs. RNA structural probing is an indirect method to probe the three-dimensional structure of RNA by analyzing the reactivity of different nucleotides to chemical modifications. The chemical modifications can target either the RNA backbone or the Watson-Crick face of nucleotides. The selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) can probe the ribose sugar in all unpaired RNA nucleotides. In contrast, Dimethyl sulfate (DMS) alkylates adenine and cytosine and reports on base pairing context but is not reactive to guanine (G) or uracil (U). Recently, new compounds were used to modify Gs and Us in the plant model system Oryza sativa and in the prokaryotic organisms Bacillus subtilis and Echerichia coli, as well as human cells. To complement the scope of RNA structural probing by chemical modifications in the model organism yeast, we analyzed the effectiveness of guanine modification by a family of aldehyde derivatives, the glyoxal family, in Saccharomyces cerevisiae and Candida albicans. We also explored the effectiveness of uracil modification by carbodiimide N-cyclohexyl-N-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMCT) in vivo. We show that among the glyoxal family, phenylglyoxal (PGO) is the best guanine probe for structural probing in S. cerevisiae and C. albicans. We also demonstrate uracil modification by CMCT in S. cerevisiae in vivo. Further, we show that PGO treatment does not affect the processing of different RNA species in the cell and is not toxic for the cells under the conditions we have established for RNA structural probing. Our results provide the conditions for in vivo probing the reactivity of guanine and uracil in RNA structures in yeast and offer a valuable tool for studying RNA structure and function in two widely used yeast model systems.

biochemistry↗

bioRxiv2021

Ribosomal RNA 2'-O-methylations regulate translation by impacting ribosome dynamics

Protein synthesis by ribosomes is critically important for gene expression in all cells. The ribosomal RNAs (rRNAs) are marked by numerous chemical modifications. An abundant group of rRNA modifications, present in all domains of life, is 2-O-methylation guided by box C/D small nucleolar RNAs (snoRNAs) which are part of small ribonucleoprotein complexes (snoRNPs). Although 2-O-methylations are required for proper production of ribosomes, the mechanisms by which these modifications contribute to translation have remained elusive. Here, we show that a change in box C/D snoRNP biogenesis in actively growing yeast cells results in the production of hypo 2-O-methylated ribosomes with distinct translational properties. Using RiboMeth-Seq for the quantitative analysis of 2-O methylations, we identify site-specific perturbations of the rRNA 2-O-methylation pattern and uncover sites that are not required for ribosome production under normal conditions. Characterization of the hypo 2-O-methylated ribosomes reveals significant translational fidelity defects including frameshifting and near-cognate start codon selection. Using rRNA structural probing, we show that hypo 2-O-methylation affects the inherent dynamics of the ribosomal subunits and impacts the binding of translation factor eIF1 thereby causing translational defects. Our data reveal an unforeseen spectrum of 2-O-methylation heterogeneity in yeast rRNA and suggest a significant role for rRNA 2-O-methylation in regulating cellular translation by controlling ribosome dynamics and ligand binding.

biochemistry↗

bioRxiv2019

A kinase-dependent checkpoint prevents escape of immature ribosomes into the translating pool

Premature release of nascent ribosomes into the translating pool must be prevented, as these do not support viability and may be prone to mistakes. Here we show that the kinase Rio1, the nuclease Nob1, and its binding partner Pno1 cooperate to establish a checkpoint that prevents the escape of immature ribosomes into polysomes. Nob1 blocks mRNA recruitment, and rRNA cleavage is required for its dissociation from nascent 40S subunits, thereby setting up a checkpoint for maturation. Rio1 releases Nob1 and Pno1 from pre-40S ribosomes to discharge nascent 40S into the translating pool. Weakly binding Nob1 and Pno1 mutants can bypass the requirement for Rio1, and Pno1 mutants rescue cell viability. In these strains, immature ribosomes escape into the translating pool, where they cause fidelity defects and perturb protein homeostasis. Thus, the Rio1-Nob1-Pno1 network establishes a checkpoint that safeguards against the release of immature ribosomes into the translating pool.

molecular biology↗

bioRxiv2019

Subunit joining exposes nascent pre-40S rRNA for processing and quality control

During their maturation, nascent 40S subunits enter a translation-like quality control cycle, where they are joined by mature 60S subunits to form 80S-like ribosomes. While these assembly intermediates are essential for maturation and quality control, how they form, and how their structure promotes quality control remains unknown. To address these questions, we determined the structure of an 80S-like ribosome assembly intermediate to an overall resolution of 3.4 [A]. The structure, validated by biochemical data, resolves a large body of previously paradoxical data and illustrates how assembly and translation factors cooperate to promote the formation of an interface that lacks many mature subunit contacts but is stabilized by the universally conserved Dim1. We also show how Tsr1 enables this interface by blocking the canonical binding of eIF5B to 40S subunits, while maintaining its binding to 60S. The structure also shows how this interface leads to unfolding of the platform, which allows for temporal regulation of the ATPase Fap7, thus linking 40S maturation to quality-control during ribosome assembly.

biochemistry↗