The putative RNA helicase DDX1 associates with the nuclear RNA exosome and modulates RNA/DNA hybrids (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. 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.

A Biallelic Variant of the RNA Exosome Gene, EXOSC4, Associated with Neurodevelopmental Defects Impairs RNA Exosome Function and Translation.

The RNA exosome is an evolutionarily conserved complex required for both precise RNA processing and decay. Pathogenic variants in EXOSC genes, which encode structural subunits of this complex, are linked to several autosomal recessive disorders. Here, we describe a missense allele of the EXOSC4 gene that causes a collection of clinical features in two affected siblings. This missense variant (NM_019037.3: exon3:c.560T>C), changes a leucine residue within a conserved region of EXOSC4 to proline (p.Leu187Pro). The two affected individuals show prenatal growth restriction, failure to thrive, global developmental delay, intracerebral and basal ganglia calcifications, and kidney failure. Homozygosity for the damaging variant was identified by exome sequencing with Sanger sequencing to confirm segregation. To explore the functional consequences of this amino acid change, we modeled EXOSC4-L187P in the corresponding budding yeast protein, Rrp41 (Rrp41-L187P). Cells that express Rrp41-L187P as the sole copy of the essential Rrp41 protein show growth defects. Steady-state levels of both Rrp41-L187P and EXOSC4-L187P are decreased compared to controls and EXOSC4-L187P shows decreased co-purification with other RNA exosome subunits. RNA exosome target transcripts accumulate in rrp41-L187P cells, including the 7S precursor of 5.8S rRNA. Polysome profiles show a decrease in actively translating ribosomes in rrp41-L187P cells as compared to control cells with incorporation of 7S pre-rRNA into polysomes. This work adds EXOSC4 to the structural subunits of the RNA exosome that have been linked to human disease and defines foundational molecular defects that could contribute to the adverse phenotypes caused by EXOSC pathogenic variants.

The ATPase Fap7 Tests the Ability to Carry Out Translocation-like Conformational Changes and Releases Dim1 during 40S Ribosome Maturation.

Late in their maturation, nascent small (40S) ribosomal subunits bind 60S subunits to produce 80S-like ribosomes. Because of the analogy of this translation-like cycle to actual translation, and because 80S-like ribosomes do not produce any protein, it has been suggested that this represents a quality control mechanism for subunit functionality. Here we use genetic and biochemical experiments to show that the essential ATPase Fap7 promotes formation of the rotated state, a key intermediate in translocation, thereby releasing the essential assembly factor Dim1 from pre-40S subunits. Bypassing this quality control step produces defects in reading frame maintenance. These results show how progress in the maturation cascade is linked to a test for a key functionality of 40S ribosomes: their ability to translocate the mRNA⋅tRNA pair. Furthermore, our data demonstrate for the first time that the translation-like cycle is a quality control mechanism that ensures the fidelity of the cellular ribosome pool.

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

SignificanceThe presence of RNA chemical modifications has long been known, but their precise molecular consequences remain unknown. 2'-O-methylation is an abundant modification that exists in RNA in all domains of life. Ribosomal RNA (rRNA) represents a functionally important RNA that is heavily modified by 2'-O-methylations. Although abundant at functionally important regions of the rRNA, the contribution of 2'-O-methylations to ribosome activities is unknown. By establishing a method to disturb rRNA 2'-O-methylation patterns, we show that rRNA 2'-O-methylations affect the function and fidelity of the ribosome and change the balance between different ribosome conformational states. Our work links 2'-O-methylation to ribosome dynamics and defines a set of critical rRNA 2'-O-methylations required for ribosome biogenesis and others that are dispensable.

Studies of mutations of assembly factor Hit1 in budding yeast suggest translation defects as the molecular basis for PEHO syndrome.

Regulation of protein synthesis is critical for control of gene expression in all cells. Ribosomes are ribonucleoprotein machines responsible for translating cellular proteins. Defects in ribosome production, function, or regulation are detrimental to the cell and cause human diseases, such as progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy (PEHO) syndrome. PEHO syndrome is a devastating neurodevelopmental disorder caused by mutations in the ZNHIT3 gene, which encodes an evolutionarily conserved nuclear protein. The precise mechanisms by which ZNHIT3 mutations lead to PEHO syndrome are currently unclear. Studies of the human zinc finger HIT-type containing protein 3 homolog in budding yeast (Hit1) revealed that this protein is critical for formation of small nucleolar ribonucleoprotein complexes that are required for rRNA processing and 2'-O-methylation. Here, we use budding yeast as a model system to reveal the basis for the molecular pathogenesis of PEHO syndrome. We show that missense mutations modeling those found in PEHO syndrome patients cause a decrease in steady-state Hit1 protein levels, a significant reduction of box C/D snoRNA levels, and subsequent defects in rRNA processing and altered cellular translation. Using RiboMethSeq analysis of rRNAs isolated from actively translating ribosomes, we reveal site-specific changes in the rRNA modification pattern of PEHO syndrome mutant yeast cells. Our data suggest that PEHO syndrome is a ribosomopathy and reveal potential new aspects of the molecular basis of this disease in translation dysregulation.

An open interface in the pre-80S ribosome coordinated by ribosome assembly factors Tsr1 and Dim1 enables temporal regulation of Fap7.

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 Å. 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 methyltransferase 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.

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

[This corrects the article DOI: 10.1371/journal.pbio.3000329.].

Maturation of small nucleolar RNAs: from production to function.

Small Nucleolar RNAs (snoRNAs) are an abundant group of non-coding RNAs with well-defined roles in ribosomal RNA processing, folding and chemical modification. Besides their classic roles in ribosome biogenesis, snoRNAs are also implicated in several other cellular activities including regulation of splicing, transcription, RNA editing, cellular trafficking, and miRNA-like functions. Mature snoRNAs must undergo a series of processing steps tightly regulated by transiently associating factors and coordinated with other cellular processes including transcription and splicing. In addition to their mature forms, snoRNAs can contribute to gene expression regulation through their derivatives and degradation products. Here, we review the current knowledge on mechanisms of snoRNA maturation, including the different pathways of processing, and the regulatory mechanisms that control snoRNA levels and complex assembly. We also discuss the significance of studying snoRNA maturation, highlight the gaps in the current knowledge and suggest directions for future research in this area.

Practical advice for mentoring and supporting faculty colleagues in STEM fields: Views from mentor and mentee perspectives.

In 2020, the American Society of Biochemistry and Molecular Biology (ASBMB) Women in Biochemistry and Molecular Biology Committee introduced the ASBMB Leadership Awards to recognize individuals with a strong commitment to advancing the careers of women in biochemistry and molecular biology along with demonstrated excellence in research, discovery, and/or service. This innovative award recognizes efforts to mentor and support trainees and colleagues at all levels. Such a leadership award provides the opportunity to focus briefly on the important role of mentoring within the STEM disciplines. The goal of this commentary, which brings together perspectives from a senior scientist and recent recipient of the ASBMB Mid-Career Leadership Award as well as two junior faculty, is to highlight approaches for purposeful support of colleagues, with an emphasis on going beyond formal mentoring committees. The commentary primarily focuses on mentoring within the academic arena of extramural funding and publication, highlighting the reality that multiple mentors with diverse expertise and perspectives are critical to support success within STEM careers.

Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization.

Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome assembly, alternative splicing, and mRNA export. Notably, serine-/arginine- (SR)-rich RBPs are densely phosphorylated compared with the remainder of the proteome. Previously, we showed that dephosphorylation of the splicing factor SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. However, the large-scale functional and structural impact of these modifications on RBPs remains unclear. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass-spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. We determined that phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high-molecular-weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine/arginine protein kinase 2 (SRPK2) in vitro decreased high-molecular-weight SRSF2 species formation. Furthermore, upon pharmacological inhibition of SRPKs in mammalian cells, we observed SRSF2 cytoplasmic mislocalization and increased formation of cytoplasmic granules as well as cytoplasmic tubular structures that associated with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.

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 because 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. Weak-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.

A conserved Bcd1 interaction essential for box C/D snoRNP biogenesis.

Precise modification and processing of rRNAs are required for the production of ribosomes and accurate translation of proteins. Small nucleolar ribonucleoproteins (snoRNPs) guide the folding, modification, and processing of rRNAs and are thus critical for all eukaryotic cells. Bcd1, an essential zinc finger HIT protein functionally conserved in eukaryotes, has been implicated as an early regulator for biogenesis of box C/D snoRNPs and controls steady-state levels of box C/D snoRNAs through an unknown mechanism. Using a combination of genetic and biochemical approaches, here we found a conserved N-terminal motif in Bcd1 from Saccharomyces cerevisiae that is required for interactions with box C/D snoRNAs and the core snoRNP protein, Snu13. We show that both the Bcd1-snoRNA and Bcd1-Snu13 interactions are critical for snoRNP assembly and ribosome biogenesis. Our results provide mechanistic insight into Bcd1 interactions that likely control the early steps of snoRNP maturation and contribute to the essential role of this protein in maintaining the steady-state levels of snoRNAs in the cell.

Entrapment of DNA in an intersubunit tunnel system of a single-stranded DNA-binding protein.

Instead of a classical single-stranded deoxyribonuleic acid (DNA)-binding protein (SSB), some hyperthermophilic crenarchaea harbor a non-canonical SSB termed ThermoDBP. Two related but poorly characterized groups of proteins, which share the ThermoDBP N-terminal DNA-binding domain, have a broader phylogenetic distribution and co-exist with ThermoDBPs and/or other SSBs. We have investigated the nucleic acid binding properties and crystal structures of representatives of these groups of ThermoDBP-related proteins (ThermoDBP-RPs) 1 and 2. ThermoDBP-RP 1 and 2 oligomerize by different mechanisms and only ThermoDBP-RP2 exhibits strong single-stranded DNA affinity in vitro. A crystal structure of ThermoDBP-RP2 in complex with DNA reveals how the NTD common to ThermoDBPs and ThermoDBP-RPs can contact the nucleic acid in a manner that allows a symmetric homotetrameric protein complex to bind single-stranded DNA molecules asymmetrically. While single-stranded DNA wraps around the surface or binds along channels of previously investigated SSBs, it traverses an internal, intersubunit tunnel system of a ThermoDBP-RP2 tetramer. Our results indicate that some archaea have acquired special SSBs for genome maintenance in particularly challenging environments.

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.

Structural basis for the dual U4 and U4atac snRNA-binding specificity of spliceosomal protein hPrp31.

Human proteins 15.5K and hPrp31 are components of the major spliceosomal U4 snRNP and of the minor spliceosomal U4atac snRNP. The two proteins bind to related 5'-stem loops (5'SLs) of the U4 and U4atac snRNAs in a strictly sequential fashion. The primary binding 15.5K protein binds at K-turns that exhibit identical sequences in the two snRNAs. However, RNA sequences contacted by the secondary binding hPrp31 differ in U4 and U4atac snRNAs, and the mechanism by which hPrp31 achieves its dual specificity is presently unknown. We show by crystal structure analysis that the capping pentaloops of the U4 and U4atac 5'SLs adopt different structures in the ternary hPrp31-15.5K-snRNA complexes. In U4atac snRNA, a noncanonical base pair forms across the pentaloop, based on which the RNA establishes more intimate interactions with hPrp31 compared with U4 snRNA. Stacking of hPrp31-His270 on the noncanonical base pair at the base of the U4atac pentaloop recapitulates intramolecular stabilizing principles known from the UUCG and GNRA families of RNA tetraloops. Rational mutagenesis corroborated the importance of the noncanonical base pair and the U4atac-specific hPrp31-RNA interactions for complex stability. The more extensive hPrp31-U4atac snRNA interactions are in line with a higher stability of the U4atac compared with the U4-based ternary complex seen in gel-shift assays, which may explain how U4atac snRNA can compete with the more abundant U4 snRNA for the same protein partners in vivo.

RNA structural probing of guanine and uracil nucleotides in yeast.

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 structure probing is an indirect method to asess the three-dimensional structure of RNA by analyzing the reactivity of different nucleotides to chemical modifications. Dimethyl sulfate (DMS) is a well-established compound that reports on base pairing context of adenine (A) and cytidine (C) in-vitro and in-vivo, but is not reactive to guanine (G) or uracil (U). Recently, new compounds were used to modify Gs and Us in plant, bacteria, and human cells. To complement the scope of RNA structural probing by chemical modifications in the model organism yeast, we analyze the effectiveness of guanine modification by the glyoxal family in Saccharomyces cerevisiae and Candida albicans. We show that within glyoxal family of compounds, phenylglyoxal (PGO) is the best guanine probe for structural probing in S. cerevisiae and C. albicans. 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. We also explore the effectiveness of uracil modification by Cyclohexyl-3-(2-Morpholinoethyl) Carbodiimide metho-p-Toluenesulfonate (CMCT) in vivo and demonstrate that uracils can be modified by CMCT in S. cerevisiae in vivo. Our results provide the conditions for in vivo probing the reactivity of guanine and uracil nucleotides in RNA structures in yeast and offer a valuable tool for studying RNA structure and function in two widely used yeast model systems.

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.

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 an evolutionarily conserved exoribonuclease complex that consists of a 3-subunit cap, a 6-subunit barrel-shaped core, and a catalytic base subunit. Missense mutations in genes encoding structural subunits of the RNA exosome cause a growing family of diseases with diverse pathologies, collectively termed RNA exosomopathies. The disease symptoms vary and can manifest as neurological defects or developmental disorders. The diversity of the RNA exosomopathy pathologies suggests that the different missense mutations in structural genes result in 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 using budding yeast by introducing pathogenic missense mutations in orthologous S. cerevisiae genes. We then performed a comparative RNA-seq analysis to assess broad transcriptomic changes in each mutant model. Three of the mutant models rrp4-G226D, rrp40-W195R and rrp46-L191H, which model mutations in the genes encoding structural subunits of the RNA exosome, EXOSC2, EXOSC3 and EXOSC5 showed the largest transcriptomic differences. Further analyses revealed shared increased transcripts enriched in translation or ribosomal RNA modification/processing pathways across the three mutant models. Studies of the impact of the mutations on translation revealed shared defects in ribosome biogenesis but distinct impacts on translation. Collectively, our results provide the first comparative analysis of several RNA exosomopathy mutant models and suggest that different RNA exosomopathy mutations result in in vivo consequences that are both unique and shared across each variant, providing more insight into the biology underlying each distinct pathology.

A Biallelic Variant of the RNA Exosome Gene EXOSC4 Causes Translational Defects Associated with a Neurodevelopmental Disorder.

The RNA exosome is an evolutionarily conserved complex required for both precise RNA processing and decay. Mutations in EXOSC genes encoding structural subunits of the complex are linked to several autosomal recessive disorders. Here, we describe a missense allele of the EXOSC4 gene, which causes a collection of clinical features in two affected siblings. This missense mutation (NM_019037.3: exon3:c.560T>C), changes a leucine residue within a highly conserved region of EXOSC4 to proline (p.Leu187Pro). The two affected individuals presented with prenatal growth restriction, failure to thrive, global developmental delay, intracerebral and basal ganglia calcifications, and kidney failure. Homozygosity for the damaging variant was identified through exome sequencing and Sanger sequencing confirmed segregation. To explore the functional consequences of this amino acid change, we modeled EXOSC4-L187P in the corresponding budding yeast protein, Rrp41 (Rrp41-L187P). Cells that express Rrp41-L187P as the sole copy of the essential Rrp41 protein show significant growth defects. The steady-state level of both the Rrp41-L187P and the EXOSC4-L187P proteins is significantly decreased compared to control Rrp41/EXOSC4. Consistent with this observation, targets of the RNA exosome accumulate in rrp41-L187P cells, including the 7S precursor of 5.8S rRNA. Polysome profiles show a significant decrease in translation in rrp41-L187P cells as compared to control cells with apparent incorporation of 7S pre-rRNA into polysomes. Taken together, this work adds the EXOSC4 subunit of the RNA exosome to the structural subunits of this complex that have been linked to human disease and defines foundational molecular defects that could contribute to the adverse growth phenotypes caused by this novel EXOSC4 pathogenic variant.