RESUMO
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 copurification 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 the 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.
RESUMO
The ribosome is the central hub for protein synthesis and the target of many antibiotics. Although the majority of ribosome-targeting antibiotics inhibit protein synthesis and are bacteriostatic, aminoglycosides promote protein mistranslation and are bactericidal. Understanding the resistance mechanisms of bacteria against aminoglycosides is not only vital for improving the efficacy of this critically important group of antibiotics but also crucial for studying the molecular basis of translational fidelity. In this work, we analyzed Salmonella mutants evolved in the presence of the aminoglycoside streptomycin (Str) and identified a novel gene rimP to be involved in Str resistance. RimP is a ribosome assembly factor critical for the maturation of the 30S small subunit that binds Str. Deficiency in RimP increases resistance against Str and facilitates the development of even higher resistance. Deleting rimP decreases mistranslation and cellular uptake of Str and further impairs flagellar motility. Our work thus highlights a previously unknown mechanism of aminoglycoside resistance via defective ribosome assembly.
RESUMO
Some eukaryotic pre-tRNAs contain an intron that is removed by a dedicated set of enzymes. Intron-containing pre-tRNAs are cleaved by tRNA splicing endonuclease, followed by ligation of the two exons and release of the intron. Fungi use a "heal and seal" pathway that requires three distinct catalytic domains of the tRNA ligase enzyme, Trl1. In contrast, humans use a "direct ligation" pathway carried out by RTCB, an enzyme completely unrelated to Trl1. Because of these mechanistic differences, Trl1 has been proposed as a promising drug target for fungal infections. To validate Trl1 as a broad-spectrum drug target, we show that fungi from three different phyla contain Trl1 orthologs with all three domains. This includes the major invasive human fungal pathogens, and these proteins can each functionally replace yeast Trl1. In contrast, species from the order Mucorales, including the pathogens Rhizopus arrhizus and Mucor circinelloides, have an atypical Trl1 that contains the sealing domain but lacks both healing domains. Although these species contain fewer tRNA introns than other pathogenic fungi, they still require splicing to decode three of the 61 sense codons. These sealing-only Trl1 orthologs can functionally complement defects in the corresponding domain of yeast Trl1 and use a conserved catalytic lysine residue. We conclude that Mucorales use a sealing-only enzyme together with unidentified nonorthologous healing enzymes for their heal and seal pathway. This implies that drugs that target the sealing activity are more likely to be broader-spectrum antifungals than drugs that target the healing domains.
Assuntos
Mucorales , Proteínas de Saccharomyces cerevisiae , Humanos , RNA Ligase (ATP)/genética , RNA Ligase (ATP)/metabolismo , Saccharomyces cerevisiae/genética , RNA de Transferência/química , Proteínas de Saccharomyces cerevisiae/genética , Precursores de RNA/metabolismo , Splicing de RNA , Mucorales/genética , Mucorales/metabolismoRESUMO
The ribosome is the central hub for protein synthesis and the target of many antibiotics. Whereas the majority of ribosome-targeting antibiotics inhibit protein synthesis and are bacteriostatic, aminoglycosides promote protein mistranslation and are bactericidal. Understanding the resistance mechanisms of bacteria against aminoglycosides is not only vital for improving the efficacy of this critically important group of antibiotics but also crucial for studying the molecular basis of translational fidelity. In this work, we analyzed Salmonella mutants evolved in the presence of the aminoglycoside streptomycin (Str) and identified a novel gene rimP to be involved in Str resistance. RimP is a ribosome assembly factor critical for the maturation of the 30S small subunit that binds Str. Deficiency in RimP increases resistance against Str and facilitates the development of even higher resistance. Deleting rimP decreases mistranslation and cellular uptake of Str, and further impairs flagellar motility. Our work thus highlights a previously unknown mechanism of aminoglycoside resistance via defective ribosome assembly.
RESUMO
Mtr4 is an essential RNA helicase involved in nuclear RNA processing and degradation and is a member of the Ski2-like helicase family. Ski2-like helicases share a common core architecture that includes two RecA-like domains, a winged helix, and a helical bundle (HB) domain. In Mtr4, a short C-terminal tail immediately follows the HB domain and is positioned at the interface of the RecA-like domains. The tail ends with a SLYΦ sequence motif that is highly conserved in a subset of Ski2-like helicases. Here, we show that this sequence is critical for Mtr4 function. Mutations in the C-terminus result in decreased RNA unwinding activity. Mtr4 is a key activator of the RNA exosome complex, and mutations in the SLYΦ motif produce a slow growth phenotype when combined with a partial exosome defect in S. cerevisiae, suggesting an important role of the C-terminus of Mtr4 and the RNA exosome. We further demonstrate that C-terminal mutations impair RNA degradation activity by the major RNA exosome nuclease Rrp44 in vitro. These data demonstrate a role for the Mtr4 C-terminus in regulating helicase activity and coordinating Mtr4-exosome interactions.
Assuntos
Exossomos , Proteínas de Saccharomyces cerevisiae , Exossomos/genética , Exossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/química , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , RNA Helicases DEAD-box/química , RNA Helicases/química , DNA Helicases/metabolismoRESUMO
IMPORTANCE: Although inflammatory bowel diseases are on the rise, what factors influence IBD risk and severity, and the underlying mechanisms remain to be fully understood. Although host genetics, microbiome, and environmental factors have all been shown to correlate with the development of IBD, cause and effect are difficult to disentangle in this context. For example, AIEC is a known pathobiont found in IBD patients, but it remains unclear if gut inflammation during IBD facilitates colonization with AIEC, or if AIEC colonization makes the host more susceptible to pro-inflammatory stimuli. It is critical to understand the mechanisms that contribute to AIEC infections in a susceptible host in order to develop successful therapeutics. Here, we show that the larval zebrafish model recapitulates key features of AIEC infections in other animal models and can be utilized to address these gaps in knowledge.
Assuntos
Colite , Doença de Crohn , Enterocolite , Infecções por Escherichia coli , Doenças Inflamatórias Intestinais , Humanos , Animais , Peixe-Zebra , Colite/induzido quimicamente , Doença de Crohn/complicações , Escherichia coli/genética , Mucosa Intestinal , Enterocolite/complicaçõesRESUMO
Some eukaryotic pre-tRNAs contain an intron that is removed by a dedicated set of enzymes. Intron-containing pre-tRNAs are cleaved by tRNA splicing endonuclease (TSEN), followed by ligation of the two exons and release of the intron. Fungi use a "heal and seal" pathway that requires three distinct catalytic domains of the tRNA ligase enzyme, Trl1. In contrast, humans use a "direct ligation" pathway carried out by RTCB, an enzyme completely unrelated to Trl1. Because of these mechanistic differences, Trl1 has been proposed as a promising drug target for fungal infections. To validate Trl1 as a broad-spectrum drug target, we show that fungi from three different phyla contain Trl1 orthologs with all three domains. This includes the major invasive human fungal pathogens, and these proteins each can functionally replace yeast Trl1. In contrast, species from the order Mucorales, including the pathogens Rhizopus arrhizus and Mucor circinelloides, contain an atypical Trl1 that contains the sealing domain, but lack both healing domains. Although these species contain fewer tRNA introns than other pathogenic fungi, they still require splicing to decode three of the 61 sense codons. These sealing-only Trl1 orthologs can functionally complement defects in the corresponding domain of yeast Trl1 and use a conserved catalytic lysine residue. We conclude that Mucorales use a sealing-only enzyme together with unidentified non-orthologous healing enzymes for their heal and seal pathway. This implies that drugs that target the sealing activity are more likely to be broader-spectrum antifungals than drugs that target the healing domains.
RESUMO
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.
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Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that ligate amino acids to tRNAs, and often require editing to ensure accurate protein synthesis. Recessive mutations in aaRSs cause various neurological disorders in humans, yet the underlying mechanism remains poorly understood. Pathogenic aaRS mutations frequently cause protein destabilization and aminoacylation deficiency. In this study, we report that combined aminoacylation and editing defects cause severe proteotoxicity. We show that the ths1-C268A mutation in yeast threonyl-tRNA synthetase (ThrRS) abolishes editing and causes heat sensitivity. Surprisingly, experimental evolution of the mutant results in intragenic mutations that restore heat resistance but not editing. ths1-C268A destabilizes ThrRS and decreases overall Thr-tRNAThr synthesis, while the suppressor mutations in the evolved strains improve aminoacylation. We further show that deficiency in either ThrRS aminoacylation or editing is insufficient to cause heat sensitivity, and that ths1-C268A impairs ribosome-associated quality control. Our results suggest that aminoacylation deficiency predisposes cells to proteotoxic stress.
Assuntos
Aminoacil-tRNA Sintetases , Estresse Proteotóxico , Humanos , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Aminoacilação , Mutação , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Treonina-tRNA Ligase/genéticaRESUMO
Through its role in intron cleavage, tRNA splicing endonuclease (TSEN) plays a critical function in the maturation of intron-containing pre-tRNAs. The catalytic mechanism and core requirement for this process is conserved between archaea and eukaryotes, but for decades, it has been known that eukaryotic TSENs have evolved additional modes of RNA recognition, which have remained poorly understood. Recent research identified new roles for eukaryotic TSEN, including processing or degradation of additional RNA substrates, and determined the first structures of pre-tRNA-bound human TSEN complexes. These recent discoveries have changed our understanding of how the eukaryotic TSEN targets and recognizes substrates. Here, we review these recent discoveries, their implications, and the new questions raised by these findings.
Assuntos
Endorribonucleases , Eucariotos , Precursores de RNA , Splicing de RNA , RNA de Transferência , Humanos , Íntrons/genética , Conformação de Ácido Nucleico , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Especificidade por Substrato , Eucariotos/enzimologia , Endorribonucleases/química , Endorribonucleases/metabolismoRESUMO
Pontocerebellar hypoplasia (PCH) is a heterogeneous group of rare neurodegenerative disorders characterized by a wide phenotypic range including severe motor and cognitive impairments, microcephaly, distinctive facial features, and other features according to the type. Several classes of PCH1 have been linked to mutations in the evolutionarily conserved RNA exosome complex that consists of nine subunits (EXOSC1 to EXOSC9) and facilitates the degradation and processing of cytoplasmic and nuclear RNA from the 3' end. Only a single individual with an EXOSC1 mutation was reported with clinical features of PCH type 1 (PCH1F). Here, we report a 3-month-old female with PCH and additional clinical features not previously reported to be associated with PCH1, including dilated cardiomyopathy. On assessment, failure to thrive, microcephaly, distinctive facial features, and bluish sclera, were noted. Whole-exome sequencing was performed and revealed a novel homozygous missense variant c.547C > T (p.Arg183Trp) in the EXOSC1 gene. Functional studies in a budding yeast model that expresses the human EXOSC1 variant Arg183Trp show a slow-growth phenotype, whereas the previously identified PCH1F allele EXOSC1-Ser35Leu is lethal, indicating impaired exosome function for both of these variants. The protein levels of both EXOSC1 variants are reduced compared with wild-type when expressed in budding yeast. Herein, we ascertain the second case of PCH associated with a EXOSC1 variant that causes defects in RNA exosome function and provide a model organism system to distinguish between benign and pathogenic variants in EXOSC1.
Assuntos
Doenças Cerebelares , Microcefalia , Malformações do Sistema Nervoso , Atrofias Olivopontocerebelares , Humanos , Feminino , Lactente , Microcefalia/genética , Doenças Cerebelares/diagnóstico , Doenças Cerebelares/genética , Atrofias Olivopontocerebelares/genética , Mutação , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Proteínas de Ligação a RNA/genéticaRESUMO
tRNA splicing endonuclease (TSEN) has a well-characterized role in transfer RNA (tRNA) splicing but also other functions. For yeast TSEN, these other functions include degradation of a subset of mRNAs that encode mitochondrial proteins and an unknown essential function. In this study, we use yeast genetics to characterize the unknown tRNA-independent function(s) of TSEN. Using a high-copy suppressor screen, we found that sen2 mutants can be suppressed by overexpression of SEN54. This effect was seen both for tRNA-dependent and tRNA-independent functions indicating that SEN54 is a general suppressor of sen2, likely through structural stabilization. A spontaneous suppressor screen identified mutations in the intron-debranching enzyme, Dbr1, as tRNA splicing-independent suppressors. Transcriptome analysis showed that sen2 mutation activates the Gcn4 stress response. These Gcn4 target transcripts decreased considerably in the sen2 dbr1 double mutant. We propose that Dbr1 and TSEN may compete for a shared substrate, which TSEN normally processes into an essential RNA, while Dbr1 initiates its degradation. These data provide further insight into the essential function(s) of TSEN. Importantly, single amino acid mutations in TSEN cause the generally fatal neuronal disease pontocerebellar hypoplasia (PCH). The mechanism by which defects in TSEN cause this disease is unknown, and our results reveal new possible mechanisms.
Assuntos
Precursores de RNA , Saccharomyces cerevisiae , Íntrons , Precursores de RNA/genética , Saccharomyces cerevisiae/metabolismo , Splicing de RNA , RNA de Transferência/genética , MutaçãoRESUMO
The Dxo1/Rai1/DXO family of decapping and exonuclease enzymes can catalyze the in vitro removal of chemically diverse 5' ends from RNA. Specifically, these enzymes act poorly on RNAs with a canonical 7mGpppN cap, but instead prefer RNAs with a triphosphate, monophosphate, hydroxyl, or nonconventional cap. In each case, these enzymes generate an RNA with a 5' monophosphate, which is then thought to be further degraded by Rat1/Xrn1 5' exoribonucleases. For most Dxo1/Rai1/DXO family members, it is not known which of these activities is most important in vivo. Here we describe the in vivo function of the poorly characterized cytoplasmic family member, yeast Dxo1. Using RNA-seq of 5' monophosphate ends, we show that Dxo1 can act as a distributive exonuclease, removing a few nucleotides from endonuclease or decapping products. We also show that Dxo1 is required for the final 5' end processing of 25S rRNA, and that this is the primary role of Dxo1. While Dxo1/Rai1/DXO members were expected to act upstream of Rat1/Xrn1, this order is reversed in 25S rRNA processing, with Dxo1 acting downstream from Rat1. Such a hand-off from a processive to a distributive exonuclease may be a general phenomenon in the precise maturation of RNA ends.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Exonucleases/genética , Exonucleases/metabolismo , Exorribonucleases/metabolismo , Proteínas Nucleares/genética , RNA/genética , RNA/metabolismo , RNA Ribossômico , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcriptoma/genéticaRESUMO
RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4 The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies.
Assuntos
Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Mutação de Sentido Incorreto , RNA Fúngico/genética , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Nanismo/enzimologia , Nanismo/genética , Nanismo/patologia , Exorribonucleases/química , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/química , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Fácies , Expressão Gênica , Glicina/química , Glicina/metabolismo , Perda Auditiva/enzimologia , Perda Auditiva/genética , Perda Auditiva/patologia , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , RNA Fúngico/química , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Retinose Pigmentar/enzimologia , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , SíndromeRESUMO
Eukaryotes share a conserved messenger RNA (mRNA) decay pathway in which bulk mRNA is degraded by exoribonucleases. In addition, it has become clear that more specialized mRNA decay pathways are initiated by endonucleolytic cleavage at particular sites. The transfer RNA (tRNA) splicing endonuclease (TSEN) has been studied for its ability to remove introns from pre-tRNAs. More recently it has been shown that single amino acid mutations in TSEN cause pontocerebellar hypoplasia. Other recent studies indicate that TSEN has other functions, but the nature of these functions has remained obscure. Here we show that yeast TSEN cleaves a specific subset of mRNAs that encode mitochondrial proteins, and that the cleavage sites are in part determined by their sequence. This provides an explanation for the counterintuitive mitochondrial localization of yeast TSEN. To identify these mRNA target sites, we developed a "comPARE" (comparative parallel analysis of RNA ends) bioinformatic approach that should be easily implemented and widely applicable to the study of endoribonucleases. The similarity of tRNA endonuclease-initiated decay to regulated IRE1-dependent decay of mRNA suggests that mRNA specificity by colocalization may be an important determinant for the degradation of localized mRNAs in a variety of eukaryotic cells.
Assuntos
Endorribonucleases , Splicing de RNA/genética , Estabilidade de RNA/genética , RNA Fúngico , RNA Mensageiro , RNA de Transferência , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Endorribonucleases/genética , Endorribonucleases/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Faithful degradation of mRNAs is a critical step in gene expression, and eukaryotes share a major conserved mRNA decay pathway. In this major pathway, the two rate-determining steps in mRNA degradation are the initial gradual removal of the poly(A) tail, followed by removal of the cap structure. Removal of the cap structure is carried out by the decapping enzyme, containing the Dcp2 catalytic subunit. Although the mechanism and regulation of mRNA decay is well understood, the consequences of defects in mRNA degradation are less clear. Dcp2 has been reported as either essential or nonessential. Here, we clarify that Dcp2 is not absolutely required for spore germination and extremely slow growth, but in practical terms it is impossible to continuously culture dcp2∆ under laboratory conditions without suppressors arising. We show that null mutations in at least three different genes are each sufficient to restore growth to a dcp2∆, of which kap123∆ and tl(gag)g∆ appear the most specific. We show that kap123∆ and tl(gag)g∆ suppress dcp2 by mechanisms that are different from each other and from previously isolated dcp2 suppressors. The suppression mechanism for tL(GAG)G is determined by the unique GAG anticodon of this tRNA, and thus likely by translation of some CUC or CUU codons. Unlike previously reported suppressors of decapping defects, these suppressors do not detectably restore decapping or mRNA decay to normal rates, but instead allow survival while only modestly affecting RNA homeostasis. These results provide important new insight into the importance of decapping, resolve previously conflicting publications about the essentiality of DCP2, provide the first phenotype for a tl(gag)g mutant, and show that multiple distinct mechanisms can bypass Dcp2 requirement.
Assuntos
Endorribonucleases/metabolismo , Estabilidade de RNA , RNA de Transferência de Leucina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , beta Carioferinas/metabolismo , Endorribonucleases/genética , Mutação com Perda de Função , RNA de Transferência de Leucina/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Esporos Fúngicos/genética , beta Carioferinas/genéticaRESUMO
Many eukaryotes use RNA processing, including alternative splicing, to express multiple gene products from the same gene. The budding yeast Saccharomyces cerevisiae has been successfully used to study the mechanism of splicing and the splicing machinery, but alternative splicing in yeast is relatively rare and has not been extensively studied. Alternative splicing of SKI7/HBS1 is widely conserved, but yeast and a few other eukaryotes have replaced this one alternatively spliced gene with a pair of duplicated, unspliced genes as part of a whole genome doubling (WGD). We show that other examples of alternative splicing known to have functional consequences are widely conserved within Saccharomycotina. A common mechanism by which alternative splicing has disappeared is by replacement of an alternatively spliced gene with duplicate unspliced genes. This loss of alternative splicing does not always take place soon after duplication, but can take place after sufficient time has elapsed for speciation. Saccharomycetaceae that diverged before WGD use alternative splicing more frequently than S. cerevisiae, suggesting that WGD is a major reason for infrequent alternative splicing in yeast. We anticipate that WGDs in other lineages may have had the same effect. Having observed that two functionally distinct splice-isoforms are often replaced by duplicated genes allowed us to reverse the reasoning. We thereby identify several splice isoforms that are likely to produce two functionally distinct proteins because we find them replaced by duplicated genes in related species. We also identify some alternative splicing events that are not conserved in closely related species and unlikely to produce functionally distinct proteins.
Assuntos
Processamento Alternativo/genética , Proteoma/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Evolução Molecular , Duplicação Gênica/genética , Genoma/genética , Isoformas de Proteínas/genéticaRESUMO
The RNA exosome is an essential ribonuclease complex required for processing and/or degradation of both coding and non-coding RNAs. We identified five patients with biallelic variants in EXOSC5, which encodes a structural subunit of the RNA exosome. The clinical features of these patients include failure to thrive, short stature, feeding difficulties, developmental delays that affect motor skills, hypotonia and esotropia. Brain MRI revealed cerebellar hypoplasia and ventriculomegaly. While we ascertained five patients, three patients with distinct variants of EXOSC5 were studied in detail. The first patient had a deletion involving exons 5-6 of EXOSC5 and a missense variant, p.Thr114Ile, that were inherited in trans, the second patient was homozygous for p.Leu206His and the third patient had paternal isodisomy for chromosome 19 and was homozygous for p.Met148Thr. The additional two patients ascertained are siblings who had an early frameshift mutation in EXOSC5 and the p.Thr114Ile missense variant that were inherited in trans. We employed three complementary approaches to explore the requirement for EXOSC5 in brain development and assess consequences of pathogenic EXOSC5 variants. Loss of function for exosc5 in zebrafish results in shortened and curved tails/bodies, reduced eye/head size and edema. We modeled pathogenic EXOSC5 variants in both budding yeast and mammalian cells. Some of these variants cause defects in RNA exosome function as well as altered interactions with other RNA exosome subunits. These findings expand the number of genes encoding RNA exosome subunits linked to human disease while also suggesting that disease mechanism varies depending on the specific pathogenic variant.
Assuntos
Antígenos de Neoplasias/genética , Cerebelo/anormalidades , Deficiências do Desenvolvimento/genética , Nanismo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Malformações do Sistema Nervoso/genética , Proteínas de Ligação a RNA/genética , Animais , Cerebelo/patologia , Deficiências do Desenvolvimento/patologia , Nanismo/patologia , Mutação da Fase de Leitura/genética , Homozigoto , Humanos , Mutação de Sentido Incorreto/genética , Malformações do Sistema Nervoso/patologia , Linhagem , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Nutrient acquisition is a central challenge for all organisms. For the fungal pathogen Candida albicans, utilization of amino acids has been shown to be critical for survival, immune evasion, and escape, while the importance of catabolism of host-derived proteins and peptides in vivo is less well understood. Stp1 and Stp2 are paralogous transcription factors (TFs) regulated by the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing system and have been proposed to have distinct, if uncertain, roles in protein and amino acid utilization. We show here that Stp1 is required for proper utilization of peptides but has no effect on amino acid catabolism. In contrast, Stp2 is critical for utilization of both carbon sources. Commensurate with this observation, we found that Stp1 controls a very limited set of genes, while Stp2 has a much more extensive regulon that is partly dependent on the Ssy1 amino acid sensor (amino acid uptake and catabolism) and partly Ssy1 independent (genes associated with filamentous growth, including the regulators UME6 and SFL2). The ssy1Δ/Δ and stp2Δ/Δ mutants showed reduced fitness in a gastrointestinal (GI) colonization model, yet induced greater damage to epithelial cells and macrophages in a manner that was highly dependent on the growth status of the fungal cells. Surprisingly, the stp1Δ/Δ mutant was better able to colonize the gut but the mutation had no effect on host cell damage. Thus, proper protein and amino acid utilization are both required for normal host interaction and are controlled by an interrelated network that includes Stp1 and Stp2.