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1.
PLoS One ; 18(12): e0295659, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38100455

RESUMEN

The Saccharomyces cerevisiae uses a highly glycolytic metabolism, if glucose is available, through appropriately suppressing mitochondrial functions except for some of them such as Fe/S cluster biogenesis. Puf3p, a Pumillio family protein, plays a pivotal role in modulating mitochondrial activity, especially during fermentation, by destabilizing its target mRNAs and/or by repressing their translation. Puf3p preferentially binds to 8-nt conserved binding sequences in the 3'-UTR of nuclear-encoded mitochondrial (nc-mitochondrial) mRNAs, leading to broad effects on gene expression under fermentable conditions. To further explore how Puf3p post-transcriptionally regulates nc-mitochondrial mRNAs in response to cell growth conditions, we initially focused on nc-mitochondrial mRNAs known to be enriched in monosomes in a glucose-rich environment. We unexpectedly found that one of the monosome-enriched mRNAs, CAT5/COQ7 mRNA, directly interacts with Puf3p through its non-canonical Puf3p binding sequence, which is generally less considered as a Puf3p binding site. Western blot analysis showed that Puf3p represses translation of Cat5p, regardless of culture in fermentable or respiratory medium. In vitro binding assay confirmed Puf3p's direct interaction with CAT5 mRNA via this non-canonical Puf3p-binding site. Although cat5 mutants of the non-canonical Puf3p-binding site grow normally, Cat5p expression is altered, indicating that CAT5 mRNA is a bona fide Puf3p target with additional regulatory factors acting through this sequence. Unlike other yeast PUF proteins, Puf3p uniquely regulates Cat5p by destabilizing mRNA and repressing translation, shedding new light on an unknown part of the Puf3p regulatory network. Given that pathological variants of human COQ7 lead to CoQ10 deficiency and yeast cat5Δ can be complemented by hCOQ7, our findings may also offer some insights into clinical aspects of COQ7-related disorders.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Humanos , Glucosa/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética
2.
Biosci Biotechnol Biochem ; 86(10): 1398-1404, 2022 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-35948278

RESUMEN

Saccharomyces cerevisiae has 6 synonymous tRNATrpCCA genes encoding the identical sequence, including their intronic region. They are supposed to express tRNATrpCCA in the same quality and quantity. Here, we generated single to quintuple deletion strains with all the possible combinations of the synonymous tRNATrpCCA genes to analyze whether those individual genes equally contribute cell viability and tRNA production. The quintuple deletion strains that only harbor tW(CCA)J, tW(CCA)M, or tW(CCA)P were viable but almost lethal while the other quintuple deletions showed moderately impaired growth. These growth differences were not obvious among the quadruple deletion strains, which expressed almost one third of mature tRNATrpCCA in the wild type. Therefore, no dosage compensation operates for tRNATrpCCA amount, and growth variations among the quintuple deletion strains may not simply reflect differences in tRNATrpCCA shortage. Yeast may retain the redundancy of tRNATrpCCA genes for a noncanonical function(s) beyond the supply of the tRNA to translation.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Intrones , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
RNA ; 2021 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-33674420

RESUMEN

To maintain optimal proteome, both codon choice of each mRNA and supply of aminoacyl-tRNAs are two principal factors in translation. Recent reports have revealed that the amounts of tRNAs in cells are more dynamic than we had expected. High-throughput methods such as RNA-Seq and microarrays are versatile for comprehensive detection of changes in individual tRNA amounts, but they suffer from inability to assess signal production efficiencies of individual tRNA species. Thus, they are not the perfect choice to measure absolute amounts of tRNAs. Here, we introduce a novel method for this purpose, termed Oligonucleotide-directed Three-prime Terminal Extension of RNA (OTTER), which employs fluorescence-labeling at the 3'-terminus of a tRNA by optimized reverse primer extension and an assessment step of each labeling efficiency by northern blotting. Using this method, we quantified the absolute amounts of the 34 individual and 4 pairs of isoacceptor tRNAs out of the total 42 nuclear-encoded isoacceptors in the yeast Saccharomyces cerevisiae. We found that the amounts of tRNAs in log phase yeast cells grown in a rich glucose medium range from 0.030 to 0.73 pmol/µg RNA. The tRNA amounts seem to be altered at the isoacceptor level by a few folds in response to physiological growing conditions. The data obtained by OTTER are poorly correlated with those by simple RNA-Seq, marginally with those by microarrays and by microscale thermophoresis. However, the OTTER data showed good agreement with the data obtained by 2D-gel analysis of in vivo radiolabeled RNAs. Thus, OTTER is a suitable method for quantifying absolute amounts of tRNAs at the level of isoacceptor resolution.

4.
Sci Rep ; 10(1): 19669, 2020 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-33184379

RESUMEN

eIF2α phosphorylation-mediated translational regulation is crucial for global translation repression by various stresses, including the unfolded protein response (UPR). However, translational control during UPR has not been demonstrated in yeast. This study investigated ribosome ubiquitination-mediated translational controls during UPR. Tunicamycin-induced ER stress enhanced the levels of ubiquitination of the ribosomal proteins uS10, uS3 and eS7. Not4-mediated monoubiquitination of eS7A was required for resistance to tunicamycin, whereas E3 ligase Hel2-mediated ubiquitination of uS10 was not. Ribosome profiling showed that the monoubiquitination of eS7A was crucial for translational regulation, including the upregulation of the spliced form of HAC1 (HAC1i) mRNA and the downregulation of Histidine triad NucleoTide-binding 1 (HNT1) mRNA. Downregulation of the deubiquitinating enzyme complex Upb3-Bre5 increased the levels of ubiquitinated eS7A during UPR in an Ire1-independent manner. These findings suggest that the monoubiquitination of ribosomal protein eS7A plays a crucial role in translational controls during the ER stress response in yeast.


Asunto(s)
Estrés del Retículo Endoplásmico , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Endopeptidasas/metabolismo , Regulación Fúngica de la Expresión Génica , Hidrolasas/genética , Hidrolasas/metabolismo , Biosíntesis de Proteínas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Respuesta de Proteína Desplegada
6.
Nucleic Acids Res ; 47(11): 5936-5949, 2019 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-30997502

RESUMEN

In eukaryotes and archaea, tRNA genes frequently contain introns, which are removed during maturation. However, biological roles of tRNA introns remain elusive. Here, we constructed a complete set of Saccharomyces cerevisiae strains in which the introns were removed from all the synonymous genes encoding 10 different tRNA species. All the intronless strains were viable, but the tRNAPheGAA and tRNATyrGUA intronless strains displayed slow growth, cold sensitivity and defective growth under respiratory conditions, indicating physiological importance of certain tRNA introns. Northern analyses revealed that removal of the introns from genes encoding three tRNAs reduced the amounts of the corresponding mature tRNAs, while it did not affect aminoacylation. Unexpectedly, the tRNALeuCAA intronless strain showed reduced 5.8S rRNA levels and abnormal nucleolar morphology. Because pseudouridine (Ψ) occurs at position 34 of the tRNAIleUAU anticodon in an intron-dependent manner, tRNAIleUAU in the intronless strain lost Ψ34. However, in a portion of tRNAIleUAU population, position 34 was converted into 5-carbamoylmethyluridine (ncm5U), which could reduce decoding fidelity. In summary, our results demonstrate that, while introns are dispensable for cell viability, some introns have diverse roles, such as ensuring proper growth under various conditions and controlling the appropriate anticodon modifications for accurate pairing with the codon.


Asunto(s)
Intrones , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anticodón , Northern Blotting , Codón , Genoma Fúngico , Leucina/química , Mutación , Conformación de Ácido Nucleico , Fenotipo , Plásmidos/metabolismo , Seudouridina , ARN/química , Procesamiento Postranscripcional del ARN , ARN de Hongos/metabolismo , ARN Ribosómico 5.8S/metabolismo
8.
J Plant Res ; 131(5): 803-815, 2018 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-29923137

RESUMEN

In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global-local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global-local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.


Asunto(s)
Citoesqueleto de Actina/ultraestructura , Citocinesis , Nicotiana/ultraestructura , Huso Acromático/ultraestructura , Citoesqueleto de Actina/fisiología , Tamaño de la Célula , Células Cultivadas , Centrifugación , Proteínas Fluorescentes Verdes , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Huso Acromático/fisiología , Nicotiana/fisiología
9.
Microscopy (Oxf) ; 65(3): 211-21, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26754563

RESUMEN

The pennate diatom, Bacillaria paxillifer, forms a colony in which adjacent cells glide smoothly and almost continuously, yet no obvious apparatus driving the movement, such as flagella or cilia, is observed. Thus far, neither the mechanism nor physiological significance of this movement has been well understood. Here, we report quantitative analysis of the gliding motion of B. paxillifer and morphological analysis of this diatom with light and electron microscopes. The gliding of pairs of adjacent B. paxillifer cells in a colony was cyclic with rather constant periods while the average gliding period varied from a few seconds to multiples of 10 s among colonies. The gliding was compromised reversibly by inhibitors for actin and myosin, suggesting involvement of the actomyosin system. Indeed, we observed two closely apposed actin bundles near the raphe by fluorescence-labeled phalloidin staining. Using electron microscopy, we observed filamentous structures that resemble the actin bundles seen with fluorescence microscopy, and we also found novel electron-dense structures located between the plasma membrane and these actin-like filaments. From these and other observations, we suggest that B. paxillifer also uses actin bundles and propose a putative myosin as a molecular motor in the gliding of unicellular diatoms.


Asunto(s)
Actomiosina/metabolismo , Membrana Celular/fisiología , Diatomeas/fisiología , Diatomeas/ultraestructura , Movimiento/fisiología , Citoesqueleto de Actina , Actinas/antagonistas & inhibidores , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Citocalasinas/farmacología , Diacetil/análogos & derivados , Diacetil/farmacología , Inhibidores Enzimáticos/farmacología , Microscopía Electrónica , Microscopía Fluorescente , Proteínas Motoras Moleculares/metabolismo , Miosinas/antagonistas & inhibidores , Tiazolidinas/farmacología
10.
Nucleus ; 6(5): 339-43, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26280499

RESUMEN

tRNAs, a class of non-coding RNAs essential for translation, are unique among cytosolic RNA species in that they shuttle between the nucleus and cytoplasm during their life. Although their export from the nucleus has been studied in detail, limited information on import machinery was available. Our group recently reported that Ssa2p, one of major cytosolic Hsp70s in Saccharomyces cerevisiae, acts as a crucial factor for tRNA import upon nutrient starvation. Ssa2p can bind tRNAs and a nucleoporin directly in an ATP-sensitive manner, suggesting that it acts as a nuclear import carrier for tRNAs, like importin-ß proteins. In vitro assays revealed that Ssa2p binds tRNA specifically but has preference for loosely folded tRNAs. In this Extra View, these features of Ssa2p as a new import factor is discussed with other recent findings related to nucleocytoplasmic transport of tRNAs reported from other groups.


Asunto(s)
Regulación Fúngica de la Expresión Génica , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo
11.
J Biol Chem ; 290(26): 16021-30, 2015 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-25971974

RESUMEN

The tRNA splicing endonuclease (Sen) complex is located on the mitochondrial outer membrane and splices precursor tRNAs in Saccharomyces cerevisiae. Here, we demonstrate that the Sen complex cleaves the mitochondria-localized mRNA encoding Cbp1 (cytochrome b mRNA processing 1). Endonucleolytic cleavage of this mRNA required two cis-elements: the mitochondrial targeting signal and the stem-loop 652-726-nt region. Mitochondrial localization of the Sen complex was required for cleavage of the CBP1 mRNA, and the Sen complex cleaved this mRNA directly in vitro. We propose that the Sen complex cleaves the CBP1 mRNA, which is co-translationally localized to mitochondria via its mitochondrial targeting signal.


Asunto(s)
Endorribonucleasas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimología , Secuencia de Bases , Endorribonucleasas/genética , Mitocondrias/genética , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Empalme del ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Elife ; 42015 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-25853343

RESUMEN

tRNAs are unique among various RNAs in that they shuttle between the nucleus and the cytoplasm, and their localization is regulated by nutrient conditions. Although nuclear export of tRNAs has been well documented, the import machinery is poorly understood. Here, we identified Ssa2p, a major cytoplasmic Hsp70 in Saccharomyces cerevisiae, as a tRNA-binding protein whose deletion compromises nuclear accumulation of tRNAs upon nutrient starvation. Ssa2p recognizes several structural features of tRNAs through its nucleotide-binding domain, but prefers loosely-folded tRNAs, suggesting that Ssa2p has a chaperone-like activity for RNAs. Ssa2p also binds Nup116, one of the yeast nucleoporins. Sis1p and Ydj1p, cytoplasmic co-chaperones for Ssa proteins, were also found to contribute to the tRNA import. These results unveil a novel function of the Ssa2p system as a tRNA carrier for nuclear import by a novel mode of substrate recognition. Such Ssa2p-mediated tRNA import likely contributes to quality control of cytosolic tRNAs.


Asunto(s)
Regulación Fúngica de la Expresión Génica , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular/genética , Secuencia de Bases , Sitios de Unión , Núcleo Celular/metabolismo , Citosol/metabolismo , Proteínas del Choque Térmico HSP40/genética , Proteínas HSP70 de Choque Térmico/genética , Datos de Secuencia Molecular , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Conformación de Ácido Nucleico , Fosforilación , Unión Proteica , Transporte de ARN/genética , ARN de Transferencia/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal
13.
Front Genet ; 5: 213, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25071838

RESUMEN

Introns are found in various tRNA genes in all the three kingdoms of life. Especially, archaeal and eukaryotic genomes are good sources of tRNA introns that are removed by proteinaceous splicing machinery. Most intron-containing tRNA genes both in archaea and eukaryotes possess an intron at a so-called canonical position, one nucleotide 3' to their anticodon, while recent bioinformatics have revealed unusual types of tRNA introns and their derivatives especially in archaeal genomes. Gain and loss of tRNA introns during various stages of evolution are obvious both in archaea and eukaryotes from analyses of comparative genomics. The splicing of tRNA molecules has been studied extensively from biochemical and cell biological points of view, and such analyses of eukaryotic systems provided interesting findings in the past years. Here, I summarize recent progresses in the analyses of tRNA introns and the splicing process, and try to clarify new and old questions to be solved in the next stages.

14.
EMBO Rep ; 15(6): 670-7, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24781694

RESUMEN

The membrane topology of Om45 in the yeast mitochondrial outer membrane (OM) is under debate. Here, we confirm that Om45 is anchored to the OM from the intermembrane space (IMS) by its N-terminal hydrophobic segment. We show that import of Om45 requires the presequence receptors, Tom20 and Tom22, and the import channel of Tom40. Unlike any of the known OM proteins, Om45 import requires the TIM23 complex in the inner membrane, a translocator for presequence-containing proteins, and the membrane potential (ΔΨ). Therefore, Om45 is anchored to the OM via the IMS by a novel import pathway involving the TIM23 complex.


Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Interacciones Hidrofóbicas e Hidrofílicas , Immunoblotting , Potencial de la Membrana Mitocondrial/fisiología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Octoxinol , Saccharomyces cerevisiae/metabolismo
16.
Nucleic Acids Res ; 41(6): 3901-14, 2013 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-23396276

RESUMEN

In all eukaryotes, transcribed precursor tRNAs are maturated by processing and modification processes in nucleus and are transported to the cytoplasm. The cytoplasmic export protein (Cex1p) captures mature tRNAs from the nuclear export receptor (Los1p) on the cytoplasmic side of the nuclear pore complex, and it delivers them to eukaryotic elongation factor 1α. This conserved Cex1p function is essential for the quality control of mature tRNAs to ensure accurate translation. However, the structural basis of how Cex1p recognizes tRNAs and shuttles them to the translational apparatus remains unclear. Here, we solved the 2.2 Å resolution crystal structure of Saccharomyces cerevisiae Cex1p with C-terminal 197 disordered residues truncated. Cex1p adopts an elongated architecture, consisting of N-terminal kinase-like and a C-terminal α-helical HEAT repeat domains. Structure-based biochemical analyses suggested that Cex1p binds tRNAs on its inner side, using the positively charged HEAT repeat surface and the C-terminal disordered region. The N-terminal kinase-like domain acts as a scaffold to interact with the Ran-exportin (Los1p·Gsp1p) machinery. These results provide the structural basis of Los1p·Gsp1p·Cex1p·tRNA complex formation, thus clarifying the dynamic mechanism of tRNA shuttling from exportin to the translational apparatus.


Asunto(s)
Modelos Moleculares , Proteínas de Transporte Nucleocitoplasmático/química , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Saccharomyces cerevisiae/química , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Cristalografía por Rayos X , Citoplasma/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
RNA ; 17(9): 1760-9, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21784868

RESUMEN

A part of eukaryotic tRNA genes harbor an intron at one nucleotide 3' to the anticodon, so that removal of the intron is an essential processing step for tRNA maturation. While some tRNA introns have important roles in modification of certain nucleotides, essentiality of the tRNA intron in eukaryotes has not been tested extensively. This is partly because most of the eukaryotic genomes have multiple genes encoding an isoacceptor tRNA. Here, we examined whether the intron of tRNA-Trp(CCA) genes, six copies of which are scattered on the genome of yeast, Saccharomyces cerevisiae, is essential for growth or translation of the yeast in vivo. We devised a procedure to remove all of the tRNA introns from the yeast genome iteratively with marker cassettes containing both positive and negative markers. Using this procedure, we removed all the introns from the six tRNA-Trp(CCA) genes, and found that the intronless strain grew normally and expressed tRNA-Trp(CCA) in an amount similar to that of the wild-type genes. Neither incorporation of (35)S-labeled amino acids into a TCA-insoluble fraction nor the major protein pattern on SDS-PAGE/2D gel were affected by complete removal of the intron, while expression levels of some proteins were marginally affected. Therefore, the tRNA-Trp(CCA) intron is dispensable for growth and bulk translation of the yeast. This raises the possibility that some mechanism other than selective pressure from translational efficiency maintains the tRNA intron on the yeast genome.


Asunto(s)
Intrones , Biosíntesis de Proteínas , ARN de Hongos/genética , ARN de Transferencia de Triptófano/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/genética , Anticodón , Secuencia de Bases , Cromosomas Fúngicos , Electroforesis en Gel de Poliacrilamida , Genes Fúngicos , Datos de Secuencia Molecular , Plásmidos , Eliminación de Secuencia
18.
Mol Biol Cell ; 21(21): 3722-34, 2010 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-20844078

RESUMEN

The unfolded protein response (UPR) is an essential signal transduction to cope with protein-folding stress in the endoplasmic reticulum. In the yeast UPR, the unconventional splicing of HAC1 mRNA is a key step. Translation of HAC1 pre-mRNA (HAC1(u) mRNA) is attenuated on polysomes and restarted only after splicing upon the UPR. However, the precise mechanism of this restart remained unclear. Here we show that yeast tRNA ligase (Rlg1p/Trl1p) acting on HAC1 ligation has an unexpected role in HAC1 translation. An RLG1 homologue from Arabidopsis thaliana (AtRLG1) substitutes for yeast RLG1 in tRNA splicing but not in the UPR. Surprisingly, AtRlg1p ligates HAC1 exons, but the spliced mRNA (HAC1(i) mRNA) is not translated efficiently. In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1(i) mRNA. Furthermore, the HAC1 5' UTR itself enables yeast Rlg1p to regulate translation of the following ORF. RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1(u) mRNA. These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , Polinucleótido Ligasas/metabolismo , Precursores del ARN/metabolismo , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Respuesta de Proteína Desplegada , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/biosíntesis , Citoplasma/metabolismo , Regulación Fúngica de la Expresión Génica , Intrones , Hidrolasas Diéster Fosfóricas/genética , Polinucleótido 5'-Hidroxil-Quinasa/genética , Polinucleótido Ligasas/genética , Precursores del ARN/genética , Empalme del ARN , Proteínas Represoras/biosíntesis , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética
19.
Cell ; 142(3): 362-3, 2010 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-20691896

RESUMEN

Although mitochondrial biogenesis requires the import of specific RNAs, the pathways and cellular machineries involved are only poorly understood. Wang et al. (2010) now find that polynucleotide phosphorylase in the intermembrane space of mammalian mitochondria facilitates import of several RNAs into the mitochondrial matrix.

20.
J Biol Chem ; 284(46): 31635-46, 2009 Nov 13.
Artículo en Inglés | MEDLINE | ID: mdl-19767391

RESUMEN

Mitochondrial protein traffic requires precise recognition of the mitochondrial targeting signals by the import receptors on the mitochondrial surface including a general import receptor Tom20 and a receptor for presequence-less proteins, Tom70. Here we took a proteome-wide approach of mitochondrial protein import in vitro to find a set of presequence-containing precursor proteins for recognition by Tom70. The presequences of the Tom70-dependent precursor proteins were recognized by Tom20, whereas their mature parts exhibited Tom70-dependent import when attached to the presequence of Tom70-independent precursor proteins. The mature parts of the Tom70-dependent precursor proteins have the propensity to aggregate, and the presence of the receptor domain of Tom70 prevents their aggregate formation. Therefore Tom70 plays the role of a docking site for not only cytosolic chaperones but also aggregate-prone substrates to maintain their solubility for efficient transfer to downstream components of the mitochondrial import machineries.


Asunto(s)
Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Precursores de Proteínas/metabolismo , Proteoma/análisis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Electroforesis en Gel Bidimensional , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Chaperonas Moleculares , Precursores de Proteínas/genética , ARN de Hongos/genética , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
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