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1.
Cell ; 162(5): 940-1, 2015 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-26317461

RESUMEN

The RNA exosome degrades many different RNAs. Thoms et al. now fill an important gap in our understanding of how the exosome recognizes distinct subsets of target RNAs.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Exosomas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ribosómicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Humanos
2.
RNA ; 30(4): 354-366, 2024 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-38307611

RESUMEN

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.


Asunto(s)
Mucorales , Proteínas de Saccharomyces cerevisiae , Humanos , ARN Ligasa (ATP)/genética , ARN Ligasa (ATP)/metabolismo , Saccharomyces cerevisiae/genética , ARN de Transferencia/química , Proteínas de Saccharomyces cerevisiae/genética , Precursores del ARN/metabolismo , Empalme del ARN , Mucorales/genética , Mucorales/metabolismo
3.
RNA ; 2024 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-39332835

RESUMEN

Eukaryotic genomes typically encode one member of the DXO/Dxo1/Rai1 family of enzymes, which can hydrolyze the 5' ends of RNAs with a variety of structures that deviate from the canonical 7mGpppN. In contrast, the Saccharomyces genome encodes two family members and the second copy, Dxo1, is a distributive 5' exoribonuclease that is required for the final maturation of the 5' end of 25S rRNA from a 25S' precursor. Here we show that this 25S rRNA maturation function is not conserved across kingdoms, but arose in the budding yeasts. Interestingly, the origin of 25S processing capacity coincides with the duplication of this gene and this capacity is absent in the nonduplicated genes. Strikingly, two different clades of budding yeasts have undergone parallel evolution: Both duplicated their DXO/Dxo1/Rai1 gene, and in both cases one copy gained the 25S processing function. This was accompanied by many parallel sequence changes, a remarkable case of reproducible neofunctionalization.

4.
J Biol Chem ; 300(8): 107571, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39009343

RESUMEN

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.


Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma , Mutación Missense , Biosíntesis de Proteínas , Humanos , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Masculino , Femenino , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Alelos , Exosomas/metabolismo , Exosomas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Nucleic Acids Res ; 51(19): 10606-10618, 2023 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-37742077

RESUMEN

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.


Asunto(s)
Aminoacil-ARNt Sintetasas , Estrés Proteotóxico , Humanos , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacilación , Mutación , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/metabolismo , Treonina-ARNt Ligasa/genética
6.
Biochemistry ; 63(1): 159-170, 2024 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-38085597

RESUMEN

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.


Asunto(s)
Exosomas , Proteínas de Saccharomyces cerevisiae , Exosomas/genética , Exosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , ARN Helicasas DEAD-box/química , ARN Helicasas/química , ADN Helicasas/metabolismo
7.
J Biol Chem ; 299(9): 105138, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37544645

RESUMEN

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.


Asunto(s)
Endorribonucleasas , Eucariontes , Precursores del ARN , Empalme del ARN , ARN de Transferencia , Humanos , Intrones/genética , Conformación de Ácido Nucleico , Precursores del ARN/química , Precursores del ARN/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Especificidad por Sustrato , Eucariontes/enzimología , Endorribonucleasas/química , Endorribonucleasas/metabolismo
8.
Antimicrob Agents Chemother ; : e0000224, 2024 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-38629858

RESUMEN

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.

9.
RNA ; 28(5): 657-667, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35140172

RESUMEN

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.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Exonucleasas/genética , Exonucleasas/metabolismo , Exorribonucleasas/metabolismo , Proteínas Nucleares/genética , ARN/genética , ARN/metabolismo , ARN Ribosómico , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcriptoma/genética
10.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33649230

RESUMEN

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.


Asunto(s)
Endorribonucleasas , Empalme del ARN/genética , Estabilidad del ARN/genética , ARN de Hongos , ARN Mensajero , ARN de Transferencia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
RNA ; 27(9): 1046-1067, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34162742

RESUMEN

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.


Asunto(s)
Exorribonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Mutación Missense , ARN de Hongos/genética , Proteínas de Unión al ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Enanismo/enzimología , Enanismo/genética , Enanismo/patología , Exorribonucleasas/química , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Facies , Expresión Génica , Glicina/química , Glicina/metabolismo , Pérdida Auditiva/enzimología , Pérdida Auditiva/genética , Pérdida Auditiva/patología , Humanos , Modelos Biológicos , Modelos Moleculares , Conformación Proteica , ARN de Hongos/química , ARN de Hongos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Retinitis Pigmentosa/enzimología , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/patología , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Síndrome
12.
Am J Med Genet A ; 191(7): 1923-1928, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37024942

RESUMEN

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.


Asunto(s)
Enfermedades Cerebelosas , Microcefalia , Malformaciones del Sistema Nervioso , Atrofias Olivopontocerebelosas , Humanos , Femenino , Lactante , Microcefalia/genética , Enfermedades Cerebelosas/diagnóstico , Enfermedades Cerebelosas/genética , Atrofias Olivopontocerebelosas/genética , Mutación , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Proteínas de Unión al ARN/genética
13.
Hum Mol Genet ; 29(13): 2218-2239, 2020 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-32504085

RESUMEN

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.


Asunto(s)
Antígenos de Neoplasias/genética , Cerebelo/anomalías , Discapacidades del Desarrollo/genética , Enanismo/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Malformaciones del Sistema Nervioso/genética , Proteínas de Unión al ARN/genética , Animales , Cerebelo/patología , Discapacidades del Desarrollo/patología , Enanismo/patología , Mutación del Sistema de Lectura/genética , Homocigoto , Humanos , Mutación Missense/genética , Malformaciones del Sistema Nervioso/patología , Linaje , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo
14.
RNA ; 26(10): 1464-1480, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32631843

RESUMEN

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.


Asunto(s)
Empalme Alternativo/genética , Proteoma/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Evolución Molecular , Duplicación de Gen/genética , Genoma/genética , Isoformas de Proteínas/genética
15.
PLoS Genet ; 15(1): e1007944, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30695063

RESUMEN

Heme-containing peroxidases are important components of innate immunity. Many of them functionally associate with NADPH oxidase (NOX)/dual oxidase (DUOX) enzymes by using the hydrogen peroxide they generate in downstream reactions. Caenorhabditis elegans encodes for several heme peroxidases, and in a previous study we identified the ShkT-containing peroxidase, SKPO-1, as necessary for pathogen resistance. Here, we demonstrated that another peroxidase, HPX-2 (Heme-PeroXidase 2), is required for resistance against some, but not all pathogens. Tissue specific RNA interference (RNAi) revealed that HPX-2 functionally localizes to the hypodermis of the worm. In congruence with this observation, hpx-2 mutant animals possessed a weaker cuticle structure, indicated by higher permeability to a DNA dye, but exhibited no obvious morphological defects. In addition, fluorescent labeling of HPX-2 revealed its expression in the pharynx, an organ in which BLI-3 is also present. Interestingly, loss of HPX-2 increased intestinal colonization of E. faecalis, suggesting its role in the pharynx may limit intestinal colonization. Moreover, disruption of a catalytic residue in the peroxidase domain of HPX-2 resulted in decreased survival on E. faecalis, indicating its peroxidase activity is required for pathogen resistance. Finally, RNA-seq analysis of an hpx-2 mutant revealed changes in genes encoding for cuticle structural components under the non-pathogenic conditions. Under pathogenic conditions, genes involved in infection response were differentially regulated to a greater degree, likely due to increased microbial burden. In conclusion, the characterization of the heme-peroxidase, HPX-2, revealed that it contributes to C. elegans pathogen resistance through a role in generating cuticle material in the hypodermis and pharynx.


Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Inmunidad Innata/genética , Oxidorreductasas/genética , Peroxidasa/genética , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/microbiología , Enterococcus faecalis/patogenicidad , Hemo/genética , Peróxido de Hidrógeno/química , Oxidación-Reducción , Faringe/enzimología , Faringe/microbiología , Interferencia de ARN , Homología de Secuencia de Aminoácido
16.
Proc Natl Acad Sci U S A ; 115(29): E6808-E6816, 2018 07 17.
Artículo en Inglés | MEDLINE | ID: mdl-29967155

RESUMEN

Eukaryotes maintain fidelity of gene expression by preferential degradation of aberrant mRNAs that arise by errors in RNA processing reactions. In Saccharomyces cerevisiae, Ski7 plays an important role in this mRNA quality control by mediating mRNA degradation by the RNA exosome. Ski7 was initially thought to be restricted to Saccharomyces cerevisiae and close relatives because the SKI7 gene and its paralog HBS1 arose by whole genome duplication (WGD) in a recent ancestor. We have recently shown that the preduplication gene was alternatively spliced and that Ski7 function predates WGD. Here, we use transcriptome analysis of diverse eukaryotes to show that diverse eukaryotes use alternative splicing of SKI7/HBS1 to encode two proteins. Although alternative splicing affects the same intrinsically disordered region of the protein, the pattern of splice site usage varies. This alternative splicing event arose in an early eukaryote that is a common ancestor of plants, animals, and fungi. Remarkably, through changes in alternative splicing and gene duplication, the Ski7 protein has diversified such that different species express one of four distinct Ski7-like proteins. We also show experimentally that the Saccharomyces cerevisiae SKI7 gene has undergone multiple changes that are incompatible with the Hbs1 function and may also have undergone additional changes to optimize mRNA quality control. The combination of transcriptome analysis in diverse eukaryotes and genetic analysis in yeast clarifies the mechanism by which a Ski7-like protein is expressed across eukaryotes and provides a unique view of changes in alternative splicing patterns of one gene over long evolutionary time.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Empalme Alternativo , Evolución Molecular , Duplicación de Gen , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
Infect Immun ; 88(5)2020 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-32094252

RESUMEN

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.


Asunto(s)
Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Interacciones Huésped-Patógeno/fisiología , Nutrientes/metabolismo , Factores de Transcripción/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Animales , Candida albicans/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/metabolismo , Femenino , Regulación Fúngica de la Expresión Génica/fisiología , Células HT29 , Interacciones Huésped-Patógeno/genética , Humanos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos ICR , Mutación/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nutrientes/genética , Factores de Transcripción/genética
18.
Nucleic Acids Res ; 43(3): 1848-58, 2015 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-25589546

RESUMEN

The RNA exosome is one of the main 3' to 5' exoribonucleases in eukaryotic cells. Although it is responsible for degradation or processing of a wide variety of substrate RNAs, it is very specific and distinguishes between substrate and non-substrate RNAs as well as between substrates that need to be 3' processed and those that need to be completely degraded. This specificity does not appear to be determined by the exosome itself but rather by about a dozen other proteins. Four of these exosome cofactors have enzymatic activity, namely, the nuclear RNA-dependent ATPase Mtr4, its cytoplasmic paralog Ski2 and the nuclear non-canonical poly(A) polymerases, Trf4 and Trf5. Mtr4 and either Trf4 or Trf5 assemble into a TRAMP complex. However, how these enzymes assemble into a TRAMP complex and the functional consequences of TRAMP complex assembly remain unknown. Here, we identify an important interaction site between Mtr4 and Trf5, and show that disrupting the Mtr4/Trf interaction disrupts specific TRAMP and exosome functions, including snoRNA processing.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Péptidos/fisiología , Polinucleotido Adenililtransferasa/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , ARN Nucleolar Pequeño/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Unión Proteica , Proteínas de Saccharomyces cerevisiae/química , Técnicas del Sistema de Dos Híbridos
19.
RNA ; 20(7): 1057-67, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24860016

RESUMEN

RNA degradation plays important roles for maintaining temporal control and fidelity of gene expression, as well as processing of transcripts. In Saccharomyces cerevisiae the RNA exosome is a major 3'-to-5' exoribonuclease and also has an endonuclease domain of unknown function. Here we report a physiological role for the exosome in response to a stimulus. We show that inactivating the exoribonuclease active site of Rrp44 up-regulates the iron uptake regulon. This up-regulation is caused by increased levels of reactive oxygen species (ROS) in the mutant. Elevated ROS also causes hypersensitivity to H2O2, which can be reduced by the addition of iron to H2O2 stressed cells. Finally, we show that the previously characterized slow growth phenotype of rrp44-exo(-) is largely ameliorated during fermentative growth. While the molecular functions of Rrp44 and the RNA exosome have been extensively characterized, our studies characterize how this molecular function affects the physiology of the organism.


Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma/fisiología , Hierro/metabolismo , Estrés Oxidativo/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae , Resistencia a Medicamentos/genética , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Peróxido de Hidrógeno/farmacología , Redes y Vías Metabólicas/efectos de los fármacos , Redes y Vías Metabólicas/genética , Organismos Modificados Genéticamente , Estabilidad del ARN/efectos de los fármacos , Estabilidad del ARN/genética , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Nucleic Acids Res ; 42(22): 13861-72, 2014 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-25414331

RESUMEN

Mtr4 is a conserved Ski2-like RNA helicase and a subunit of the TRAMP complex that activates exosome-mediated 3'-5' turnover in nuclear RNA surveillance and processing pathways. Prominent features of the Mtr4 structure include a four-domain ring-like helicase core and a large arch domain that spans the core. The 'ratchet helix' is positioned to interact with RNA substrates as they move through the helicase. However, the contribution of the ratchet helix in Mtr4 activity is poorly understood. Here we show that strict conservation along the ratchet helix is particularly extensive for Ski2-like RNA helicases compared to related helicases. Mutation of residues along the ratchet helix alters in vitro activity in Mtr4 and TRAMP and causes slow growth phenotypes in vivo. We also identify a residue on the ratchet helix that influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain has minimal effect on Mtr4 unwinding activity. We now show that combining the arch deletion with ratchet helix mutations abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates.


Asunto(s)
ARN Helicasas DEAD-box/química , ARN/química , Proteínas de Saccharomyces cerevisiae/química , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Modelos Moleculares , Mutación , Poli A/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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