RESUMEN
Control of the differential abundance or activity of tRNAs can be important determinants of gene regulation. RNA polymerase (RNAP) III synthesizes all tRNAs in eukaryotes and it derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of the target of rapamycin (TOR) that acts to integrate transcriptional output and protein synthetic demand toward metabolic economy. Studies in budding yeast have indicated that the global tRNA gene activation that occurs with derepression of RNAP III via maf1-deletion is accompanied by a paradoxical loss of tRNA-mediated nonsense suppressor activity, manifested as an antisuppression phenotype, by an unknown mechanism. We show that maf1-antisuppression also occurs in the fission yeast S. pombe amidst general activation of RNAP III. We used tRNA-HydroSeq to document that little changes occurred in the relative levels of different tRNAs in maf1Δ cells. By contrast, the efficiency of N2,N2-dimethyl G26 (m(2)2G26) modification on certain tRNAs was decreased in response to maf1-deletion and associated with antisuppression, and was validated by other methods. Over-expression of Trm1, which produces m(2)2G26, reversed maf1-antisuppression. A model that emerges is that competition by increased tRNA levels in maf1Δ cells leads to m(2)2G26 hypomodification due to limiting Trm1, reducing the activity of suppressor-tRNASerUCA and accounting for antisuppression. Consistent with this, we show that RNAP III mutations associated with hypomyelinating leukodystrophy decrease tRNA transcription, increase m(2)2G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m(2)2G26 modification and that this response is conserved among highly divergent yeasts and human cells.
Asunto(s)
ARN Polimerasa III/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , ARNt Metiltransferasas/metabolismo , Secuencia de Aminoácidos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Células HEK293/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Mutación , ARN Polimerasa III/genética , ARN de Transferencia/biosíntesis , ARN de Transferencia de Serina/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Sirolimus/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , ARNt Metiltransferasas/genéticaRESUMEN
PUF proteins are potent repressors that serve important roles in stem cell maintenance, neurological processes, and embryonic development. These functions are driven by PUF protein recognition of specific binding sites within the 3' untranslated regions of target mRNAs. In this study, we investigated mechanisms of repression by the founding PUF, Drosophila Pumilio, and its human orthologs. Here, we evaluated a previously proposed model wherein the Pumilio RNA binding domain (RBD) binds Argonaute, which in turn blocks the translational activity of the eukaryotic elongation factor 1A. Surprisingly, we found that Argonautes are not necessary for repression elicited by Drosophila and human PUFs in vivo. A second model proposed that the RBD of Pumilio represses by recruiting deadenylases to shorten the mRNA's polyadenosine tail. Indeed, the RBD binds to the Pop2 deadenylase and accelerates deadenylation; however, this activity is not crucial for regulation. Rather, we determined that the poly(A) is necessary for repression by the RBD. Our results reveal that poly(A)-dependent repression by the RBD requires the poly(A) binding protein, pAbp. Furthermore, we show that repression by the human PUM2 RBD requires the pAbp ortholog, PABPC1. Pumilio associates with pAbp but does not disrupt binding of pAbp to the mRNA. Taken together, our data support a model wherein the Pumilio RBD antagonizes the ability of pAbp to promote translation. Thus, the conserved function of the PUF RBD is to bind specific mRNAs, antagonize pAbp function, and promote deadenylation.
Asunto(s)
Poli A/metabolismo , Poliadenilación/fisiología , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Argonautas/química , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sitios de Unión , Proteínas de Drosophila/metabolismo , Exorribonucleasas/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Motivos de Nucleótidos , Factor 1 de Elongación Peptídica/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Alineación de Secuencia , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
The degradation of messenger RNA is a critical node of gene regulation. A major pathway of mRNA decay is initiated by shortening of the poly(A) tail, followed by removal of the 5' cap structure (decapping) and subsequent degradation. Decapping is an important determinate in the destruction of many transcripts. Detailed kinetic analysis of in vivo decapping rates is necessary to understand how this step is regulated. Importantly, the product of decapping is recalcitrant for investigation, in part due to its transient nature. As such, little in vivo kinetic information is available. Here we report the development of an assay that measures decapping of mRNAs by combining splinted ligation and quantitative RT-PCR (qSL-RT-PCR). We apply this method to determine the decapping rate constant for a natural mRNA in vivo for the first time. The qSL-RT-PCR assay may be adapted for use on any mRNA, providing a new tool to study regulation of mRNA decay.
Asunto(s)
Caperuzas de ARN/análisis , Caperuzas de ARN/metabolismo , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ribosómicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Endorribonucleasas/metabolismo , Poli A/genética , Caperuzas de ARN/genética , ARN de Hongos/genética , ARN de Hongos/metabolismo , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
The N(6)-(isopentenyl)adenosine (i(6)A) modification of some tRNAs at position A37 is found in all kingdoms and facilitates codon-specific mRNA decoding, but occurs in different subsets of tRNAs in different species. Here we examine yeasts' tRNA isopentenyltransferases (i.e., dimethylallyltransferase, DMATase, members of the Δ(2)-isopentenylpyrophosphate transferase, IPPT superfamily) encoded by tit1(+) in Schizosaccharomyces pombe and MOD5 in Saccharomyces cerevisiae, whose homologs are Escherichia coli miaA, the human tumor suppressor TRIT1, and the Caenorhabditis elegans life-span gene product GRO-1. A major determinant of miaA activity is known to be the single-stranded tRNA sequence, A36A37A38, in a stem-loop. tRNA(Trp)(CCA) from either yeast is a Tit1p substrate, but neither is a Mod5p substrate despite the presence of A36A37A38. We show that Tit1p accommodates a broader range of substrates than Mod5p. tRNA(Trp)(CCA) is distinct from Mod5p substrates, which we sort into two classes based on the presence of G at position 34 and other elements. A single substitution of C34 to G converts tRNA(Trp)(CCA) to a Mod5p substrate in vitro and in vivo, consistent with amino acid contacts to G34 in existing Mod5p-tRNA(Cys)(GCA) crystal structures. Mutation of Mod5p in its G34 recognition loop region debilitates it differentially for its G34 (class I) substrates. Multiple alignments reveal that the G34 recognition loop sequence of Mod5p differs significantly from Tit1p, which more resembles human TRIT1 and other DMATases. We show that TRIT1 can also modify tRNA(Trp)(CCA) consistent with broad recognition similar to Tit1p. This study illustrates previously unappreciated molecular plasticity and biological diversity of the tRNA-isopentenyltransferase system of eukaryotes.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Anticodón/genética , ARN de Transferencia/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Schizosaccharomyces/enzimología , Transferasas Alquil y Aril/genética , Secuencia de Bases , Humanos , Mutación , Conformación de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
The conserved nuclear RNA-binding factor known as La protein arose in an ancient eukaryote, phylogenetically associated with another eukaryotic hallmark, synthesis of tRNA by RNA polymerase III (RNAP III). Because 3'-oligo(U) is the sequence-specific signal for transcription termination by RNAP III as well as the high affinity binding site for La, the latter is linked to the intranuclear posttranscriptional processing of eukaryotic precursor-tRNAs. The pre-tRNA processing pathway must accommodate a variety of substrates that are destined for both common steps as well as tRNA-specific events. The order of intranuclear pre-tRNA processing steps is mediated in part by three activities derived from interaction with La protein: 3'-end protection from untimely decay by 3' exonucleases, nuclear retention and chaperone activity that helps prevent pre-tRNA misfolding and mischanneling into offline pathways. A focus of this perspective will be on differences between yeast and mammals in the subcellular partitioning of pre-tRNA intermediates and differential interactions with La. We review how this is most relevant to pre-tRNA splicing which occurs in the cytoplasm of yeasts but in nuclei of higher eukaryotes. Also divergent is La architecture, comprised of three RNA-binding domains in organisms in all examined branches of the eukaryal tree except yeast, which have lost the C-terminal RNA recognition motif-2α (RRM2α) domain. We also review emerging data that suggest mammalian La interacts with nuclear pre-tRNA splicing intermediates and may impact this branch of the tRNA maturation pathway. Finally, because La is involved in intranuclear tRNA biogenesis we review relevant aspects of tRNA-associated neurodegenerative diseases. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.
Asunto(s)
Autoantígenos/genética , Células Eucariotas/metabolismo , ARN de Transferencia/metabolismo , Ribonucleoproteínas/genética , Levaduras/genética , Secuencia de Aminoácidos , Animales , Autoantígenos/química , Autoantígenos/metabolismo , Sitios de Unión , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Humanos , Modelos Moleculares , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Oligorribonucleótidos/genética , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN/genética , ARN/metabolismo , ARN Polimerasa III/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , Motivo de Reconocimiento de ARN , Empalme del ARN/fisiología , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN de Transferencia/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/deficiencia , Ribonucleoproteínas/metabolismo , Especificidad de la Especie , Fracciones Subcelulares/metabolismo , Nucleótidos de Uracilo/genética , Levaduras/metabolismo , Antígeno SS-BRESUMEN
Human La antigen (Sjögren's syndrome antigen B [SSB]) is an abundant multifunctional RNA-binding protein. In the nucleoplasm, La binds to and protects from 3' exonucleases, the ends of precursor tRNAs, and other transcripts synthesized by RNA polymerase III and facilitates their maturation, while a nucleolar isoform has been implicated in rRNA biogenesis by multiple independent lines of evidence. We showed previously that conditional La knockout (La cKO) from mouse cortex neurons results in defective tRNA processing, although the pathway(s) involved in neuronal loss thereafter was unknown. Here, we demonstrate that La is stably associated with a spliced pre-tRNA intermediate. Microscopic evidence of aberrant nuclear accumulation of 5.8S rRNA in La cKO is supported by a 10-fold increase in a pre-5.8S rRNA intermediate. To identify pathways involved in subsequent neurodegeneration and loss of brain mass in the cKO cortex, we employed mRNA sequencing (mRNA-Seq), immunohistochemistry, and other approaches. This revealed robust enrichment of immune and astrocyte reactivity in La cKO cortex. Immunohistochemistry, including temporal analyses, demonstrated neurodegeneration, followed by astrocyte invasion associated with immune response and decreasing cKO cortex size over time. Thus, deletion of La from postmitotic neurons results in defective pre-tRNA and pre-rRNA processing and progressive neurodegeneration with loss of cortical brain mass.
Asunto(s)
Astrocitos/patología , Autoantígenos/genética , Gliosis/genética , Neuronas/patología , Precursores del ARN/metabolismo , ARN Ribosómico 5.8S/metabolismo , ARN de Transferencia/metabolismo , Ribonucleoproteínas/genética , Animales , Astrocitos/metabolismo , Autoantígenos/fisiología , Muerte Celular/genética , Células Cultivadas , Femenino , Gliosis/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Neuronas/metabolismo , Ribonucleoproteínas/fisiología , Antígeno SS-BRESUMEN
The blood-brain barrier is essential for the proper homeostasis and function of the CNS, but its mechanism of function is poorly understood. Perivascular cells surrounding brain blood vessels are thought to be important for blood-brain barrier establishment, but their roles are not well defined. Here, we describe a novel perivascular cell population closely associated with blood vessels on the zebrafish brain. Based on similarities in their morphology, location, and scavenger behavior, these cells appear to be the zebrafish equivalent of cells variably characterized as Fluorescent Granular Perithelial cells (FGPs), perivascular macrophages, or 'Mato Cells' in mammals. Despite their macrophage-like morphology and perivascular location, zebrafish FGPs appear molecularly most similar to lymphatic endothelium, and our imaging studies suggest that these cells emerge by differentiation from endothelium of the optic choroidal vascular plexus. Our findings provide the first report of a perivascular cell population in the brain derived from vascular endothelium.
Asunto(s)
Vasos Sanguíneos/citología , Barrera Hematoencefálica/citología , Encéfalo/citología , Células Endoteliales/citología , Pez Cebra , Animales , Diferenciación CelularRESUMEN
Paradoxically, both anticancer immunosurveillance and tumor progression have been associated with intact autophagy, which is regulated by the target of rapamycin (Tor1). Here, we describe the potential impact on the design of cancer therapeutics of a newly described highly conserved post-transcriptional mechanism whereby Tor regulates autophagy.
Asunto(s)
ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Anticodón/química , Anticodón/genética , Anticodón/metabolismo , Codón/química , Codón/genética , Codón/metabolismo , Daño del ADN , Biosíntesis de Proteínas/genética , Procesamiento Postranscripcional del ARN , ARN Mensajero/química , ARN Mensajero/genética , ARN de Transferencia/química , ARN de Transferencia/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ARNt Metiltransferasas/química , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismoRESUMEN
Regulation of autophagy is required to maintain cellular equilibrium and prevent disease. While extensive study of post-translational mechanisms has yielded important insights into autophagy induction, less is known about post-transcriptional mechanisms that could potentiate homeostatic control. In our study, we showed that the RNA-binding protein, Dhh1 in Saccharomyces cerevisiae and Vad1 in the pathogenic yeast Cryptococcus neoformans is involved in recruitment and degradation of key autophagy mRNAs. In addition, phosphorylation of the decapping protein Dcp2 by the target of rapamycin (TOR), facilitates decapping and degradation of autophagy-related mRNAs, resulting in repression of autophagy under nutrient-replete conditions. The post-transcriptional regulatory process is conserved in both mouse and human cells and plays a role in autophagy-related modulation of the inflammasome product IL1B. These results were then applied to provide mechanistic insight into autoimmunity of a patient with a PIK3CD/p110δ gain-of-function mutation. These results thus identify an important new post-transcriptional mechanism of autophagy regulation that is highly conserved between yeast and mammals.
Asunto(s)
Autofagia/genética , ARN Helicasas DEAD-box/genética , Estabilidad del ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Animales , Femenino , HumanosRESUMEN
Autophagy is an essential eukaryotic pathway requiring tight regulation to maintain homeostasis and preclude disease. Using yeast and mammalian cells, we report a conserved mechanism of autophagy regulation by RNA helicase RCK family members in association with the decapping enzyme Dcp2. Under nutrient-replete conditions, Dcp2 undergoes TOR-dependent phosphorylation and associates with RCK members to form a complex with autophagy-related (ATG) mRNA transcripts, leading to decapping, degradation and autophagy suppression. Simultaneous with the induction of ATG mRNA synthesis, starvation reverses the process, facilitating ATG mRNA accumulation and autophagy induction. This conserved post-transcriptional mechanism modulates fungal virulence and the mammalian inflammasome, the latter providing mechanistic insight into autoimmunity reported in a patient with a PIK3CD/p110δ gain-of-function mutation. We propose a dynamic model wherein RCK family members, in conjunction with Dcp2, function in controlling ATG mRNA stability to govern autophagy, which in turn modulates vital cellular processes affecting inflammation and microbial pathogenesis.
Asunto(s)
Autofagia/genética , ARN Helicasas DEAD-box/genética , Estabilidad del ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Animales , Autoinmunidad/genética , Línea Celular Tumoral , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , ARN Helicasas DEAD-box/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Femenino , Regulación Fúngica de la Expresión Génica , Células HeLa , Humanos , Immunoblotting , Inflamasomas/genética , Inflamasomas/metabolismo , Ratones Endogámicos C57BL , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Eukaryotic cells utilize macroautophagy (hereafter autophagy) to recycle cellular materials during nutrient stress. Target of rapamycin (Tor) is a central regulator of this process, acting by post-translational mechanisms, phosphorylating preformed autophagy-related (Atg) proteins to repress autophagy during log-phase growth. We recently reported an additional role for post-transcriptional regulation of autophagy, whereby the mRNA decapping protein, Dcp2, undergoes Tor-dependent phosphorylation, resulting in increased ATG mRNA decapping and degradation under nutrient-rich, repressing conditions. Dephosphorylation of Dcp2 during starvation is associated with dissociation of the decapping-ATG mRNA complex, with resultant stabilization of, and accumulation of, ATG transcripts, leading to induction of autophagy. Regulation of mRNA degradation occurs in concert with known mRNA synthetic inductive mechanisms to potentiate overall transcriptional regulation. This mRNA degradative pathway thus constitutes a type of transcriptional 'futile cycle' where under nutrient-rich conditions transcript is constantly being generated and degraded. As nutrient levels decline, steady state mRNA levels are increased by both inhibition of degradation as well as increased de novo synthesis. A role for this regulatory process in fungal virulence was further demonstrated by showing that overexpression of the Dcp2-associated mRNA-binding protein Vad1 in the AIDS-associated pathogen Cryptococcus neoformans results in constitutive repression of autophagy even under starvation conditions as well as attenuated virulence in a mouse model. In summary, Tor-dependent post-transcriptional regulation of autophagy plays a key role in the facilitation of microbial pathogenesis.
RESUMEN
La antigen (Sjögren's syndrome antigen B) is a phosphoprotein associated with nascent precursor tRNAs and other RNAs, and it is targeted by autoantibodies in patients with Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Increased levels of La are associated with leukemias and other cancers, and various viruses usurp La to promote their replication. Yeast cells (Saccharomyces cerevisiae and Schizosaccharomyces pombe) genetically depleted of La grow and proliferate, whereas deletion from mice causes early embryonic lethality, raising the question of whether La is required by mammalian cells generally or only to surpass a developmental stage. We developed a conditional La allele and used it in mice that express Cre recombinase in either B cell progenitors or the forebrain. B cell Mb1(Cre) La-deleted mice produce no B cells. Consistent with αCamKII Cre, which induces deletion in hippocampal CA1 cells in the third postnatal week and later throughout the neocortex, brains develop normally in La-deleted mice until â¼5 weeks and then lose a large amount of forebrain cells and mass, with evidence of altered pre-tRNA processing. The data indicate that La is required not only in proliferating cells but also in nondividing postmitotic cells. Thus, La is essential in different cell types and required for normal development of various tissue types.
Asunto(s)
Autoantígenos/inmunología , Linfocitos B/inmunología , Lóbulo Frontal/inmunología , Neuronas/inmunología , Ribonucleoproteínas/inmunología , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Linfocitos B/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/inmunología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Lóbulo Frontal/metabolismo , Lóbulo Frontal/patología , Hipocampo/inmunología , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Inmunohistoquímica , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuronas/metabolismo , ARN/genética , ARN/inmunología , ARN/metabolismo , Precursores del ARN/genética , Precursores del ARN/inmunología , Precursores del ARN/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/inmunología , ARN de Transferencia/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Síndrome de Sjögren/genética , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/metabolismo , Factores de Tiempo , Antígeno SS-BRESUMEN
tRNA isopentenyltransferases (Tit1) modify tRNA position 37, adjacent to the anticodon, to N6-isopentenyladenosine (i6A37) in all cells, yet the tRNA subsets selected for modification vary among species, and their relevance to phenotypes is unknown. We examined the function of i6A37 in Schizosaccharomyces pombe tit1+ and tit1-Δ cells by using a ß-galactosidase codon-swap reporter whose catalytic activity is sensitive to accurate decoding of codon 503. i6A37 increased the activity of tRNACys at a cognate codon and that of tRNATyr at a near-cognate codon, suggesting that i6A37 promotes decoding activity generally and increases fidelity at cognate codons while decreasing fidelity at noncognate codons. S. pombe cells lacking tit1+ exhibit slow growth in glycerol or rapamycin. While existing data link wobble base U34 modifications to translation of functionally related mRNAs, whether this might extend to the anticodon-adjacent position 37 was unknown. Indeed, we found a biased presence of i6A37-cognate codons in high-abundance mRNAs for ribosome subunits and energy metabolism, congruent with the observed phenotypes and the idea that i6A37 promotes translational efficiency. Polysome profiles confirmed the decreased translational efficiency of mRNAs in tit1-Δ cells. Because subsets of i6A37-tRNAs differ among species, as do their cognate codon-sensitive mRNAs, these genomic variables may underlie associated phenotypic differences.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Isopenteniladenosina/genética , ARN de Hongos/genética , ARN Mensajero/genética , ARN de Transferencia/genética , Schizosaccharomyces/genética , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Codón/genética , Codón/metabolismo , Eliminación de Gen , Isopenteniladenosina/metabolismo , Biosíntesis de Proteínas , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Schizosaccharomyces/enzimología , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismoRESUMEN
PUF proteins are eukaryotic RNA-binding proteins that repress specific mRNAs. The mechanisms and corepressors involved in PUF repression remain to be fully identified. Here, we investigated the mode of repression by Saccharomyces cerevisiae Puf5p and Puf4p and found that Puf5p specifically requires Eap1p to repress mRNAs, whereas Puf4p does not. Surprisingly, we observed that Eap1p, which is a member of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP) class of translational inhibitors, does not inhibit the efficient polyribosome association of a Puf5p target mRNA. Rather, we found that Eap1p accelerates mRNA degradation by promoting decapping, and the ability of Eap1p to interact with eIF4E facilitates this activity. Deletion of EAP1 dramatically reduces decapping, resulting in accumulation of deadenylated, capped mRNA. In support of this phenotype, Eap1p associates both with Puf5p and the Dhh1p decapping factor. Furthermore, recruitment of Eap1p to downregulated mRNA is mediated by Puf5p. On the basis of these results, we propose that Puf5p promotes decapping by recruiting Eap1p and associated decapping factors to mRNAs. The implication of these findings is that a 4E-BP can repress protein expression by promoting specific mRNA degradation steps in addition to or in lieu of inhibiting translation initiation.
Asunto(s)
Factor 4E Eucariótico de Iniciación/metabolismo , Caperuzas de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia Abajo , Polirribosomas/metabolismo , Unión ProteicaRESUMEN
Human RFamide-related peptide-1 (hRFRP-1, MPHSFANLPLRF-NH(2)) binds to neuropeptide FF receptor 2 (NPFF(2)R) to dramatically diminish cardiovascular performance. hRFRP-1 and its signaling pathway may provide targets to address cardiac dysfunction. Here, structure-activity relationship, transcript, Ca(2+) transient, and phospholabeling data indicate the presence of a hRFRP-1 pathway in cardiomyocytes. Alanyl-substituted and N-terminal truncated analogues identified that R(11) was essential for activity, hRFRP-1((8-12)) mimicked hRFRP-1, and [A(11)]hRFRP-1((8-12)) antagonized the effect of hRFRP-1 in cellular and integrated cardiac performance. RFRP and NPFF(2)R transcripts were amplified from cardiomyocytes and heart. Maintenance of the Ca(2+) transient when hRFRP-1 impaired myocyte shortening indicated the myofilament was its primary downstream target. Enhanced myofilament protein phosphorylation detected after hRFRP-1 treatment but absent in [A(11)]hRFRP-1((8-12))-treated cells was consistent with this result. Protein kinase C (PKC) but not PKA inhibitor diminished the influence of hRFRP-1 on the Ca(2+) transient. Molecules targeting this pathway may help address cardiovascular disease.