RESUMEN
Gene editing using CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) is under development as a therapeutic tool for the modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas9 systems from Streptococcus pyogenes and Staphylococcus aureus, alternative CRISPR systems have been identified from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of CRISPR/Cas enzymes. The Cas12e enzymes from non-pathogenic Deltaproteobacteria (CasX1, DpeCas12e) and Planctomycetes (CasX2, PlmCas12e) are smaller than Cas9, have a selective protospacer adjacent motif (PAM), and deliver a staggered cleavage cut with a 5-7 nucleotide overhang. We investigated the impact of guide RNA spacer length and alternative PAM sequences on cleavage activity to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes the CCR5 coreceptor used by human immunodeficiency virus-type 1 (HIV-1) to infect target cells. A 32 base-pair deletion in CCR5 (CCR5-[Formula: see text]32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR/Cas. We determined that CCR5 cleavage activity varied with the target site, spacer length, and the fourth nucleotide in the previously described PAM sequence, TTCN. Our analyses demonstrated a PAM preference for purines (adenine, guanine) over pyrimidines (thymidine, cytosine) in the fourth position of the CasX2 PAM. This improved understanding of CasX2 cleavage requirements facilitates the development of therapeutic strategies to recreate the CCR5-[Formula: see text]32 mutation in haematopoietic stem cells.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Humanos , Mutación , ARN/genética , Nucleótidos , Receptores CCR5/genéticaRESUMEN
Essential messenger RNA (mRNA) export factors execute critical steps to mediate directional transport through nuclear pore complexes (NPCs). At cytoplasmic NPC filaments, the ATPase activity of DEAD-box protein Dbp5 is activated by inositol hexakisphosphate (IP(6))-bound Gle1 to mediate remodeling of mRNA-protein (mRNP) complexes. Whether a single Dbp5 executes multiple remodeling events and how Dbp5 is recycled are unknown. Evidence suggests that Dbp5 binding to Nup159 is required for controlling interactions with Gle1 and the mRNP. Using in vitro reconstitution assays, we found here that Nup159 is specifically required for ADP release from Dbp5. Moreover, Gle1-IP(6) stimulates ATP binding, thus priming Dbp5 for RNA loading. In vivo, a dbp5-R256D/R259D mutant with reduced ADP binding bypasses the need for Nup159 interaction. However, NPC spatial control is important, as a dbp5-R256D/R259D nup42Δ double mutant is temperature-sensitive for mRNA export. Further analysis reveals that remodeling requires a conformational shift to the Dbp5-ADP form. ADP release factors for DEAD-box proteins have not been reported previously and reflect a new paradigm for regulation. We propose a model wherein Nup159 and Gle1-IP(6) regulate Dbp5 cycles by controlling its nucleotide-bound state, allowing multiple cycles of mRNP remodeling by a single Dbp5 at the NPC.
Asunto(s)
Núcleo Celular/metabolismo , ARN Helicasas DEAD-box , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Nucleótidos/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Mutación , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Unión Proteica , Conformación Proteica , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Nuclear export of messenger RNA (mRNA) occurs by translocation of mRNA/protein complexes (mRNPs) through nuclear pore complexes (NPCs). The DEAD-box protein Dbp5 mediates export by triggering removal of mRNP proteins in a spatially controlled manner. This requires Dbp5 interaction with Nup159 in NPC cytoplasmic filaments and activation of Dbp5's ATPase activity by Gle1 bound to inositol hexakisphosphate (IP(6)). However, the precise sequence of events within this mechanism has not been fully defined. Here we analyze dbp5 mutants that alter ATP binding, ATP hydrolysis, or RNA binding. We found that ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs. Interestingly, mutants defective for RNA binding are dominant-negative (DN) for mRNA export in yeast and human cells. We show that the DN phenotype stems from competition with wild-type Dbp5 for Gle1 at NPCs. The Dbp5-Gle1 interaction is limiting for export and, importantly, can be independent of Nup159. Fluorescence recovery after photobleaching experiments in yeast show a very dynamic association between Dbp5 and NPCs, averaging <1 sec, similar to reported NPC translocation rates for mRNPs. This work reveals critical steps in the Gle1-IP(6)/Dbp5/Nup159 cycle, and suggests that the number of remodeling events mediated by a single Dbp5 is limited.
Asunto(s)
Núcleo Celular/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Adenosina Trifosfato/metabolismo , Línea Celular Tumoral , Células HeLa , Humanos , Hidrólisis , Mutación , Proteínas de Complejo Poro Nuclear/metabolismo , Fenotipo , Unión Proteica/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Proper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously-Brr6, which is closely related to Brl1, and Apq12-function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Biogénesis de Organelos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adaptación Fisiológica/efectos de los fármacos , Alcohol Bencilo/farmacología , Epistasis Genética/efectos de los fármacos , Homeostasis/efectos de los fármacos , Lípidos de la Membrana/metabolismo , Mutación/genética , Membrana Nuclear/efectos de los fármacos , Membrana Nuclear/metabolismo , Transporte de ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Esteroles/metabolismo , ViscosidadRESUMEN
CRISPR/Cas is under development as a therapeutic tool for the cleavage, excision, and/or modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9), alternative CRISPR systems have been identified using metagenomic datasets from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of gene editors. The Cas12e (CasX1, CasX2) endonucleases from non-pathogenic Deltaproteobacteria (DpeCas12e) and Planctomycetes (PlmCas12e) are more compact than SpCas9, have a more selective protospacer adjacent motif (PAM) requirement, and deliver a staggered cleavage cut with 5-7 base overhangs. We investigated varying guide RNA (spacer) lengths and alternative PAM sequences to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes one of two chemokine coreceptors required by HIV-1 to infect target cells, and a mutation of CCR5 (delta-32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR, TALENs, and ZFNs. We determined that CCR5 cleavage activity varied with the target site, guide RNA length, and the terminal nucleotide in the PAM sequence. Our analyses demonstrated a PlmCas12e PAM preference for purines (A, G) over pyrimidines (T, C) in the fourth position of the CasX2 PAM (TTCN). These analyses have contributed to a better understanding of CasX2 cleavage requirements and will position us more favorably to develop a therapeutic that creates the delta-32 mutation in the CCR5 gene in hematopoietic stem cells.
RESUMEN
Cells of Saccharomyces cerevisiae lacking Apq12, a nuclear envelope (NE)-endoplasmic reticulum (ER) integral membrane protein, are defective in assembly of nuclear pore complexes (NPCs), possibly because of defects in regulating membrane fluidity. We identified BRR6, which encodes an essential integral membrane protein of the NE-ER, as a dosage suppressor of apq12 Delta. Cells carrying the temperature-sensitive brr6-1 allele have been shown to have defects in nucleoporin localization, mRNA metabolism and nuclear transport. Electron microscopy revealed that brr6-1 cells have gross NE abnormalities and proliferation of the ER. brr6-1 cells were hypersensitive to compounds that affect membrane biophysical properties and to inhibitors of lipid biosynthetic pathways, and displayed strong genetic interactions with genes encoding non-essential lipid biosynthetic enzymes. Strikingly, brr6-1 cells accumulated, in or near the NE, elevated levels of the two classes of neutral lipids, steryl esters and triacylglycerols, and over-accumulated sterols when they were provided exogenously. Although neutral lipid synthesis is dispensable in wild-type cells, viability of brr6-1 cells was fully dependent on neutral lipid production. These data indicate that Brr6 has an essential function in regulating lipid homeostasis in the NE-ER, thereby impacting NPC formation and nucleocytoplasmic transport.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mutantes/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular/genética , Retículo Endoplásmico , Metabolismo de los Lípidos/genética , Fluidez de la Membrana , Proteínas de la Membrana/genética , Proteínas Mutantes/genética , Membrana Nuclear/genética , Poro Nuclear/genética , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Esteroles/metabolismo , Temperatura , Triglicéridos/metabolismoRESUMEN
Although the structure and function of components of the nuclear pore complex (NPC) have been the focus of many studies, relatively little is known about NPC biogenesis. In this study, we report that Apq12 is required for efficient NPC biogenesis in Saccharomyces cerevisiae. Apq12 is an integral membrane protein of the nuclear envelope (NE) and endoplasmic reticulum. Cells lacking Apq12 are cold sensitive for growth, and a subset of their nucleoporins (Nups), those that are primarily components of the cytoplasmic fibrils of the NPC, mislocalize to the cytoplasm. APQ12 deletion also causes defects in NE morphology. In the absence of Apq12, most NPCs appear to be associated with the inner but not the outer nuclear membrane. Low levels of benzyl alcohol, which increases membrane fluidity, prevented Nup mislocalization and restored the proper localization of Nups that had accumulated in cytoplasmic foci upon a shift to lower temperature. Thus, Apq12p connects nuclear pore biogenesis to the dynamics of the NE.
Asunto(s)
Proteínas de la Membrana/metabolismo , Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Alcohol Bencilo/metabolismo , División Celular/fisiología , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/genética , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Gene editing approaches using CRISPR/Cas9 are being developed as a means for targeting the integrated HIV-1 provirus. Enthusiasm for the use of gene editing as an anti-HIV-1 therapeutic has been tempered by concerns about the specificity and efficacy of this approach. Guide RNAs (gRNAs) that target conserved sequences across a wide range of genetically diverse HIV-1 isolates will have greater clinical utility. However, on-target efficacy should be considered in the context of off-target cleavage events as these may comprise an essential safety parameter for CRISPR-based therapeutics. We analyzed a panel of Streptococcus pyogenes Cas9 (SpCas9) gRNAs directed to the 5' and 3' long terminal repeat (LTR) regions of HIV-1. We used in vitro cleavage assays with genetically diverse HIV-1 LTR sequences to determine gRNA activity across HIV-1 clades. Lipid-based transfection of gRNA/Cas9 ribonucleoproteins was used to assess targeting of the integrated HIV-1 proviral sequence in cells (in vivo). For both the in vitro and in vivo experiments, we observed increased efficiency of sequence disruption through the simultaneous use of two distinct gRNAs. Next, CIRCLE-Seq was utilized to identify off-target cleavage events using genomic DNA from cells with integrated HIV-1 proviral DNA. We identified a gRNA targeting the U3 region of the LTR (termed SpCas9-127HBX2) with broad cleavage efficiency against sequences from genetically diverse HIV-1 strains. Based on these results, we propose a workflow for identification and development of anti-HIV CRISPR therapeutics.
Asunto(s)
Infecciones por VIH , VIH-1 , Sistemas CRISPR-Cas , Edición Génica , Infecciones por VIH/genética , VIH-1/genética , Humanos , ARN Guía de Kinetoplastida/genéticaRESUMEN
Coordination of the multiple steps of mRNA biogenesis helps to ensure proper regulation of gene expression. The Saccharomyces cerevisiae DEAD-box protein Rat8p/Dbp5p is an essential mRNA export factor that functions at the nuclear pore complex (NPC) where it is thought to remodel mRNA/protein complexes during mRNA export. Rat8p also functions in translation termination and has been implicated in functioning during early transcription. We conducted a synthetic genetic array analysis (SGA) using a strain harboring the temperature-sensitive rat8-2 allele. Although RAT8 had been shown to interact genetically with >15 other genes, we identified >40 additional genes whose disruption in a rat8-2 background causes synthetic lethality or dramatically reduced growth. Included were five that encode components of P-bodies, sites of cytoplasmic mRNA turnover and storage. Wild-type Rat8p localizes to NPCs and diffusely throughout the cell but rat8-2p localized to cytoplasmic granules at nonpermissive temperature that are distinct from P-bodies. In some genetic backgrounds, these granules also contain poly(A)-binding protein, Pab1p, and additional mRNA export factors. Although these foci are distinct from P-bodies, the two merge under heat-stress conditions. We suggest that these granules reflect defective mRNP remodeling during mRNA export and during cytoplasmic mRNA metabolism.
Asunto(s)
ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Genes Fúngicos , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Alelos , Citoplasma/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Sintéticos , Poro Nuclear/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Polirribosomas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Nuclear pore complexes (NPCs) play an essential role in RNA export. Nucleoporins required for mRNA export in Saccharomyces cerevisiae are found in the Nup84p and Nup82p subcomplexes of the NPC. The Nup82p subcomplex contains Nup82p, Rat7p/Nup159p, Nsp1p, Gle1p/Rss1p, and Rip1p/Nup42p and is found only on the cytoplasmic face of NPCs. Both Rat7p and Gle1p contain binding sites for Rat8p/Dbp5p, an essential DEAD box protein and putative RNA helicase. Rip1p interacts directly with Gle1p and is the only protein known to be essential for mRNA export after heat shock but not under normal growth conditions. We report that in cells lacking Rip1p, both Gle1p and Rat8p dissociate from NPCs following heat shock at 42 degrees C. Rat8p but not Gle1p was retained at NPCs if rip1Delta cells were first shifted to 37 degrees C and then to 42 degrees C, and this was correlated with preserving mRNA export in heat-shocked rip1Delta cells. Export following ethanol shock was less dependent on the presence of Rip1p. Exposure to 10% ethanol led to dissociation of Rat8p from NPCs in both wild-type and rip1Delta cells. Following this treatment, Rat8p was primarily nuclear in wild-type cells but primarily cytoplasmic in rip1Delta cells. We also determined that efficient export of heat shock mRNA after heat shock depends upon a novel 6-amino-acid element within Rat8p. This motif is not required under normal growth conditions or following ethanol shock. These studies suggest that the molecular mechanism responsible for the defect in export of heat shock mRNAs in heat-shocked rip1Delta cells is dissociation of Rat8p from NPCs. These studies also suggest that both nuclear pores and Rat8p have features not required for mRNA export in growing cells but which enhance the ability of mRNAs to be exported following heat shock.
Asunto(s)
Calor , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Helicasas/metabolismo , ARN Mensajero/metabolismo , Antiinfecciosos Locales/farmacología , Proteínas Portadoras/metabolismo , ARN Helicasas DEAD-box , Etanol/farmacología , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Complejo Poro Nuclear , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ProteínaRESUMEN
The regulated transport of proteins across the nuclear envelope occurs through nuclear pore complexes (NPCs), which are composed of >30 different protein subunits termed nucleoporins. While some nucleoporins are glycosylated, little about the role of glycosylation in NPC activity is understood. We have identified loss-of-function alleles of ALG12, encoding a mannosyltransferase, as suppressors of a temperature-sensitive mutation in the gene encoding the FXFG-nucleoporin NUP1. We observe that nup1Delta cells import nucleophilic proteins more efficiently when ALG12 is absent, suggesting that glycosylation may influence nuclear transport. Conditional nup1 and nup82 mutations are partially suppressed by the glycosylation inhibitor tunicamycin, while nic96 and nup116 alleles are hypersensitive to tunicamycin treatment, further implicating glycosylation in NPC function. Because Pom152p is a glycosylated, transmembrane nucleoporin, we examined genetic interactions between pom152 mutants and nup1Delta. A nup1 deletion is lethal in combination with pom152Delta, as well as with truncations of the N-terminal and transmembrane regions of Pom152p. However, truncations of the N-glycosylated, lumenal domain of Pom152p and pom152 mutants lacking N-linked glycosylation sites are viable in combination with nup1Delta, suppress nup1Delta temperature sensitivity, and partially suppress the nuclear protein import defects associated with the deletion of NUP1. These data provide compelling evidence for a role for glycosylation in influencing NPC function.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Poro Nuclear/fisiología , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Glicosilación , Manosiltransferasas/genética , Manosiltransferasas/metabolismo , Glicoproteínas de Membrana/genética , Mutación , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de Secuencia , TemperaturaRESUMEN
Heat shock leads to accumulation of polyadenylated RNA in nuclei of Saccharomyces cerevisiae cells, transcriptional induction of heat shock genes, and efficient export of polyadenylated heat shock mRNAs. These studies were conducted to examine the requirements for export of mRNA following heat shock. We used in situ hybridization to detect SSA4 mRNA (encoding Hsp70) and flow cytometry to measure the amount of Ssa4p-green fluorescent protein (GFP) produced following heat shock. Npl3p and Yra1p are mRNA-binding proteins recruited to nascent mRNAs and are essential for proper mRNA biogenesis and export. Heat shock mRNA was exported efficiently in temperature-sensitive npl3, yra1, and npl3 yra1 mutant strains. Nevertheless, Yra1p was recruited to heat shock mRNA, as were Nab2p and Npl3p. Interestingly, Yra1p was not recruited to heat shock mRNA in yra1-1 cells, suggesting that Npl3p is required for recruitment of Yra1p. The THO complex, which functions in transcription elongation and in recruitment of Yra1p, was not required for heat shock mRNA export, although normal mRNA export is impaired in growing cells lacking THO complex proteins. Taken together, these studies indicate that export following heat shock depends upon fewer factors than does mRNA export in growing cells. Furthermore, even though some mRNA-binding proteins are dispensable for efficient export of heat shock mRNA, those that are present in nuclei of heat shocked cells were recruited to heat shock mRNA.
Asunto(s)
Respuesta al Choque Térmico , Mutación/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte de ARN , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/fisiología , Núcleo Celular/metabolismo , Eliminación de Gen , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico/efectos de los fármacos , Ribonucleoproteínas Nucleares Heterogéneas , Calor , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares , Transporte de ARN/efectos de los fármacos , ARN de Hongos/efectos de los fármacos , ARN de Hongos/metabolismo , ARN Mensajero/efectos de los fármacos , Proteínas de Unión al ARN , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Eukaryotic gene expression requires the export of mRNA from the nucleus to the cytoplasm. The DEAD box protein Dbp5p is an essential export factor conserved from yeast to man. A fraction of Dbp5p forms a complex with nucleoporins of the cytoplasmic filaments of the nuclear pore complex. Gfd1p was identified originally as a multicopy suppressor of the rat8-2 ts allele of DBP5. Here we reported that Dbp5p and Gfd1p interact with Zds1p, a protein previously identified as a multicopy suppressor in several yeast genetic screens. By using the two-hybrid system, we showed that Zds1p interacts in vivo with both Gfd1p and Dbp5p. In vitro binding experiments revealed that Gfd1p and Dbp5p bind directly to the C-terminal part of Zds1p. In addition, ZDS1 interacted genetically with mutant alleles of genes encoding key factors in mRNA export, including DBP5 and MEX67. Furthermore, deletion of ZDS1 or of both ZDS1 and the closely related ZDS2 exacerbated the poly(A)+ export defects shown by dbp5-2 and mex67-5 mutants. We proposed that Zds1p associates with the complex formed by Dbp5p, Gfd1p, and nucleoporins at the cytosolic fibrils of the nuclear pore complex and is required for optimal mRNA export.
Asunto(s)
ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Bases , Citosol/metabolismo , Cartilla de ADN , Genoma Fúngico , Datos de Secuencia Molecular , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In eukaryotic cells, pre-mRNAs undergo extensive processing in the nucleus prior to export. Processing is subject to a quality-control mechanism that retains improperly processed transcripts at or near sites of transcription. A poly(A) tail added by the normal 3'-processing machinery is necessary but not sufficient for export. Retention depends on the exosome. In this study, we identify the poly(A)-binding protein, Pab1, and the poly(A) nuclease, PAN, as important factors that couple 3' processing to export. Pab1 contains a nonessential leucine-rich nuclear export signal and shuttles between the nucleus and the cytoplasm. It can exit the nucleus either as cargo of exportin 1 or bound to mRNA. Pab1 is essential but several bypass suppressors have been identified. Deletion of PAB1 from these bypass suppressor strains results in exosome-dependent retention at sites of transcription. Retention is also seen in cells lacking PAN, which Pab1 is thought to recruit and which may be responsible for the final step of mRNA biogenesis, trimming of the poly(A) tail to the length found on newly exported mRNAs. The studies presented here suggest that proper loading of Pab1 onto mRNAs and final trimming of the tail allows release from transcription sites and couples pre-mRNA processing to export.