RESUMEN
RN7SL1 (RNA component of signal recognition particle 7SL1), a component of the signal recognition particle, is a non-coding RNA possessing a small ORF (smORF). However, whether it is translated into peptides is unknown. Here, we generated the RN7SL1-Green Fluorescent Protein (GFP) gene, in which the smORF of RN7SL1 was replaced by GFP, introduced it into 293T cells, and observed cells emitting GFP fluorescence. Furthermore, RNA-seq of GFP-positive cells revealed that they were in an oncogenic state, suggesting that RN7SL1 smORF may be translated under special conditions.
Asunto(s)
Péptidos , Partícula de Reconocimiento de Señal , Partícula de Reconocimiento de Señal/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Péptidos/metabolismoRESUMEN
The signal recognition particle (SRP) is a critical component in protein sorting pathways in all domains of life. Human SRP contains six proteins bound to the 7S RNA and their structures and functions have been mostly elucidated. The SRP68/72 dimer is the largest SRP component and is essential for SRP function. Although the structures of the SRP68/72 RNA binding and dimerization domains have been previously reported, the structure and function of large portions of the SRP68/72 dimer remain unknown. Here, we analyse full-length SRP68/72 using cryo-EM and report that SRP68/72 depend on each other for stability and form an extended dimerization domain. This newly observed dimerization domain is both a protein- and RNA-binding domain. Comparative analysis with current structural models suggests that this dimerization domain undergoes dramatic translocation upon SRP docking onto SRP receptor and eventually comes close to the Alu domain. We propose that the SRP68/72 dimerization domain functions by binding and detaching the Alu domain and SRP9/14 from the ribosomal surface, thus releasing elongation arrest upon docking onto the ER membrane.
Asunto(s)
Microscopía por Crioelectrón , Modelos Moleculares , Multimerización de Proteína , Partícula de Reconocimiento de Señal , Humanos , Sitios de Unión , Unión Proteica , Dominios Proteicos , ARN/química , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/ultraestructura , Partícula de Reconocimiento de Señal/química , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
Peptide nucleic acid (PNA) based antisense strategy is a promising therapeutic approach to specifically inhibit target gene expression. However, unlike protein coding genes, identification of an ideal PNA binding site for non-coding RNA is not straightforward. Here, we compare the inhibitory activities of PNA molecules that bind a non-coding 4.5S RNA called SRP RNA, a key component of the bacterial signal recognition particle (SRP). A 9-mer PNA (PNA9) complementary to the tetraloop region of the RNA was more potent in inhibiting its interaction with the SRP protein, compared to an 8-mer PNA (PNA8) targeting a stem-loop. PNA9, which contained a homo-pyrimidine sequence could form a triplex with the complementary stretch of RNA inâ vitro as confirmed using a fluorescent derivative of PNA9 (F-PNA13). The RNA-PNA complex formation resulted in inhibition of SRP function with PNA9 and F-PNA13, but not PNA8 highlighting the importance of target site selection. Surprisingly, F-PNA13 which was more potent in inhibiting SRP function inâ vitro, showed weaker antibacterial activity compared to PNA9 likely due to poor cell penetration of the longer PNA. Our results underscore the importance of suitable target site selection and optimum PNA length to develop better antisense molecules against non-coding RNA.
Asunto(s)
Ácidos Nucleicos de Péptidos , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/farmacología , Ácidos Nucleicos de Péptidos/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Sitios de Unión , ARN no Traducido/genética , ARN no Traducido/química , ARN no Traducido/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Partícula de Reconocimiento de Señal/química , Partícula de Reconocimiento de Señal/genética , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Secuencia de Bases , Conformación de Ácido NucleicoRESUMEN
SRP14 is a crucial protein subunit of the signal recognition particle (SRP), a ribonucleoprotein complex essential for co-translational translocation to the endoplasmic reticulum. During our investigation of SRP14 expression across diverse cell lines, we observe variations in its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with some cells exhibiting slower migration and others migrating faster. However, the cause of this phenomenon remains elusive. Our research rules out alternative splicing as the cause and, instead, identifies the presence of a P124A mutation in SRP14 (SRP14 P124A) among the faster-migrating variants, while the slower-migrating variants lack this mutation. Subsequent ectopic expression of wild-type SRP14 P124 or SRP14 WT and SRP14 P124A in various cell lines confirms that the P124A mutation indeed leads to faster migration of SRP14. Further mutagenesis analysis shows that the P117A and A121P mutations within the alanine-rich domain at the C-terminus of SRP14 are responsible for migration alterations on SDS-PAGE, whereas mutations outside this domain, such as P39A, Y27F, and T45A, have no such effect. Furthermore, the ectopic expression of SRP14 WT and SRP14 P124A yields similar outcomes in terms of SRP RNA stability, cell morphology, and cell growth, indicating that SRP14 P124A represents a natural variant of SRP14 and retains comparable functionality. In conclusion, the substitution of proline for alanine in the alanine-rich tail of SRP14 results in faster migration on SDS-PAGE, but has little effect on its function.
Asunto(s)
Alanina , Partícula de Reconocimiento de Señal , Partícula de Reconocimiento de Señal/genética , Partícula de Reconocimiento de Señal/metabolismo , Mutación , Mutagénesis , Electroforesis en Gel de Poliacrilamida , Alanina/genéticaRESUMEN
The DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation that belongs to the DDX3 subfamily. The purified Ded1 protein is an ATP-dependent RNA-binding protein and an RNA-dependent ATPase, but it was previously found to lack substrate specificity and enzymatic regulation. Here we demonstrate through yeast genetics, yeast extract pull-down experiments, in situ localization, and in vitro biochemical approaches that Ded1 is associated with, and regulated by, the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 in the presence of SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results provide a new understanding of the role of Ded1 during translation.
Asunto(s)
ARN Helicasas DEAD-box , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Partícula de Reconocimiento de Señal , Partícula de Reconocimiento de Señal/metabolismo , Partícula de Reconocimiento de Señal/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Transporte de ProteínasRESUMEN
The signal recognition particle is essential for targeting transmembrane and secreted proteins to the endoplasmic reticulum. Remarkably, because they work together in the cytoplasm, the SRP and ribosomes are assembled in the same biomolecular condensate: the nucleolus. How important is the nucleolus for SRP assembly is not known. Using quantitative proteomics, we have investigated the interactomes of SRP components. We reveal that SRP proteins are associated with scores of nucleolar proteins important for ribosome biogenesis and nucleolar structure. Having monitored the subcellular distribution of SRP proteins upon controlled nucleolar disruption, we conclude that an intact organelle is required for their proper localization. Lastly, we have detected two SRP proteins in Cajal bodies, which indicates that previously undocumented steps of SRP assembly may occur in these bodies. This work highlights the importance of a structurally and functionally intact nucleolus for efficient SRP production and suggests that the biogenesis of SRP and ribosomes may be coordinated in the nucleolus by common assembly factors.
Asunto(s)
Nucléolo Celular , Proteómica , Ribosomas , Partícula de Reconocimiento de Señal , Partícula de Reconocimiento de Señal/metabolismo , Nucléolo Celular/metabolismo , Ribosomas/metabolismo , Humanos , Proteómica/métodos , Proteínas Nucleares/metabolismo , Cuerpos Enrollados/metabolismo , Células HeLa , Retículo Endoplásmico/metabolismoRESUMEN
The unfolded protein response (UPR) relieves endoplasmic reticulum (ER) stress through multiple strategies, including reducing protein synthesis, increasing protein folding capabilities, and enhancing misfolded protein degradation. After a multi-omics analysis, we find that signal recognition particle 14 (SRP14), an essential component of the SRP, is markedly reduced in cells undergoing ER stress. Further experiments indicate that SRP14 reduction requires PRKR-like ER kinase (PERK)-mediated eukaryotic translation initiation factor 2α (eIF2α) phosphorylation but is independent of ATF4 or ATF3 transcription factors. The decrease of SRP14 correlates with reduced translocation of fusion proteins and endogenous cathepsin D. Enforced expression of an SRP14 variant with elongation arrest capability prevents the reduced translocation of cathepsin D in stressed cells, whereas an SRP14 mutant without the activity does not. Finally, overexpression of SRP14 augments the UPR and aggravates ER-stress-induced cell death. These data suggest that translocational attenuation mediated by the PERK-SRP14 axis is a protective measure for the UPR to mitigate ER stress.
Asunto(s)
Estrés del Retículo Endoplásmico , Respuesta de Proteína Desplegada , eIF-2 Quinasa , eIF-2 Quinasa/metabolismo , Humanos , Fosforilación , Transporte de Proteínas , Partícula de Reconocimiento de Señal/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Células HEK293 , Células HeLaRESUMEN
Although RNA molecules are synthesized via transcription, little is known about the general impact of cotranscriptional folding in vivo. We present different computational approaches for the simulation of changing structure ensembles during transcription, including interpretations with respect to experimental data from literature. Specifically, we analyze different mutations of the E. coli SRP RNA, which has been studied comparatively well in previous literature, yet the details of which specific metastable structures form as well as when they form are still under debate. Here, we combine thermodynamic and kinetic, deterministic, and stochastic models with automated and visual inspection of those systems to derive the most likely scenario of which substructures form at which point during transcription. The simulations do not only provide explanations for present experimental observations but also suggest previously unnoticed conformations that may be verified through future experimental studies.
Asunto(s)
Escherichia coli , Conformación de Ácido Nucleico , Pliegue del ARN , ARN Bacteriano , Termodinámica , Transcripción Genética , ARN Bacteriano/química , ARN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Partícula de Reconocimiento de Señal/química , Partícula de Reconocimiento de Señal/metabolismo , Partícula de Reconocimiento de Señal/genética , Cinética , Biología Computacional/métodos , Mutación , Modelos MolecularesRESUMEN
Many insulin gene variants alter the protein sequence and result in monogenic diabetes due to insulin insufficiency. However, the molecular mechanisms of various disease-causing mutations are unknown. Insulin is synthesized as preproinsulin containing a signal peptide (SP). SPs of secreted proteins are recognized by the signal recognition particle (SRP) or by another factor in a SRP-independent pathway. If preproinsulin uses SRP-dependent or independent pathways is still debatable. We demonstrate by the use of site-specific photocrosslinking that the SRP subunit, SRP54, interacts with the preproinsulin SP. Moreover, SRP54 depletion leads to the decrease of insulin mRNA and protein expression, supporting the involvement of the RAPP protein quality control in insulin biogenesis. RAPP regulates the quality of secretory proteins through degradation of their mRNA. We tested five disease-causing mutations in the preproinsulin SP on recognition by SRP and on their effects on mRNA and protein levels. We demonstrate that the effects of mutations are associated with their position in the SP and their severity. The data support diverse molecular mechanisms involved in the pathogenesis of these mutations. We show for the first time the involvement of the RAPP protein quality control pathway in insulin biogenesis that is implicated in the development of neonatal diabetes caused by the Leu13Arg mutation.
Asunto(s)
Insulina , Precursores de Proteínas , Estabilidad del ARN , Partícula de Reconocimiento de Señal , Humanos , Recién Nacido , Diabetes Mellitus , Insulina/genética , Insulina/metabolismo , Precursores de Proteínas/metabolismo , Señales de Clasificación de Proteína/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
Signal recognition particles (SRPs) are essential for regulating intracellular protein transport and secretion. Patients with tumors with high SRP9 expression tend to have a poorer overall survival. However, to the best of our knowledge, no reports have described the relationship between SRP9 localization and prognosis in pancreatic cancer. Thus, the present study aimed to investigate this relationship. Immunohistochemical staining for SRP9 using excised specimens from pancreatic cancer surgery cases without preoperative chemotherapy or radiotherapy showed that SRP9 was preferentially expressed in the nucleus of the cancerous regions in some cases, which was hardly detected in other cases, indicating that SRP9 was transported to the nucleus in the former cases. To compare the prognosis of patients with SRP9 nuclear translocation, patients were divided into two groups: Those with a nuclear translocation rate of >50% and those with a nuclear translocation rate of ≤50%. The nuclear translocation rate of >50% group had a significantly better recurrencefree survival than the nuclear translocation rate of ≤50% group (P=0.037). Subsequent in vitro experiments were conducted; notably, the nuclear translocation rate of SRP9 was reduced under amino aciddeficient conditions, suggesting that multiple factors are involved in this phenomenon. To further study the function of SRP9 nuclear translocation, in vitro experiments were performed by introducing SRP9 splicing variants (v1 and v2) and their deletion mutants lacking Cterminal regions into MiaPaCa pancreatic cancer cells. The results demonstrated that both splicing variants showed nuclear translocation regardless of the Cterminal deletions, suggesting the role of the Nterminal regions. Given that SRP9 is an RNAbinding protein, the study of RNA immunoprecipitation revealed that signaling pathways involved in cancer progression and protein translation were downregulated in nucleartranslocated v1 and v2. Undoubtedly, further studies of the nuclear translocation of SRP9 will open an avenue to optimize the precise evaluation and therapeutic control of pancreatic cancer.
Asunto(s)
Núcleo Celular , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Pronóstico , Masculino , Femenino , Núcleo Celular/metabolismo , Persona de Mediana Edad , Anciano , Línea Celular Tumoral , Partícula de Reconocimiento de Señal/metabolismo , Partícula de Reconocimiento de Señal/genética , Transporte Activo de Núcleo Celular , Factores de Empalme Serina-Arginina/metabolismo , Factores de Empalme Serina-Arginina/genética , Adulto , Regulación Neoplásica de la Expresión GénicaRESUMEN
Translation of messenger ribonucleic acids (mRNAs) encoding integral membrane proteins or secreted proteins occurs on the surface of the endoplasmic reticulum (ER). When a nascent signal peptide is synthesized from the mRNAs, the ribosome-nascent chain complex (RNC) is recognized by the signal recognition particle (SRP) and then transported to the surface of the ER. The appropriate targeting of the RNC-SRP complex to the ER is monitored by a quality control pathway, a nuclear cap-binding complex (CBC)-ensured translational repression of RNC-SRP (CENTRE). In this study, using ribosome profiling of CBC-associated and eukaryotic translation initiation factor 4E-associated mRNAs, we reveal that, at the transcriptomic level, CENTRE is in charge of the translational repression of the CBC-RNC-SRP until the complex is specifically transported to the ER. We also find that CENTRE inhibits the nonsense-mediated mRNA decay (NMD) of mRNAs within the CBC-RNC-SRP. The NMD occurs only after the CBC-RNC-SRP is targeted to the ER and after eukaryotic translation initiation factor 4E replaces CBC. Our data indicate dual surveillance for properly targeting mRNAs encoding integral membrane or secretory proteins to the ER. CENTRE blocks gene expression at the translation level before the CBC-RNC-SRP delivery to the ER, and NMD monitors mRNA quality after its delivery to the ER.
Asunto(s)
Retículo Endoplásmico , Degradación de ARNm Mediada por Codón sin Sentido , ARN Mensajero , Partícula de Reconocimiento de Señal , Retículo Endoplásmico/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genética , Humanos , Partícula de Reconocimiento de Señal/metabolismo , Partícula de Reconocimiento de Señal/genética , Señales de Clasificación de Proteína/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Células HeLa , Ribosomas/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Biosíntesis de ProteínasRESUMEN
The basal structure of the bacterial flagellum includes a membrane embedded MS-ring (formed by multiple copies of FliF) and a cytoplasmic C-ring (composed of proteins FliG, FliM and FliN). The SRP-type GTPase FlhF is required for directing the initial flagellar protein FliF to the cell pole, but the mechanisms are unclear. Here, we show that FlhF anchors developing flagellar structures to the polar landmark protein HubP/FimV, thereby restricting their formation to the cell pole. Specifically, the GTPase domain of FlhF interacts with HubP, while a structured domain at the N-terminus of FlhF binds to FliG. FlhF-bound FliG subsequently engages with the MS-ring protein FliF. Thus, the interaction of FlhF with HubP and FliG recruits a FliF-FliG complex to the cell pole. In addition, the modulation of FlhF activity by the MinD-type ATPase FlhG controls the interaction of FliG with FliM-FliN, thereby regulating the progression of flagellar assembly at the pole.
Asunto(s)
Proteínas Bacterianas , Flagelos , Flagelos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Unión Proteica , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Partícula de Reconocimiento de Señal/metabolismo , Partícula de Reconocimiento de Señal/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de la MembranaRESUMEN
The signal recognition particle (SRP) system delivers approximately 30% of the proteome to the endoplasmic reticulum (ER) membrane. SRP receptor alpha (SRα) binds to SRP for targeting nascent secreted proteins to the ER membrane in eukaryotic cells. In this study, the SRα homologous gene was identified in the sea cucumber, Apostichopus japonicus (AjSRα). AjSRα codes for 641 amino acids and has 54.94% identity with its mammalian homologs. Like mammalian SRα, it is expected to contain the SRP-alpha N domain, SRP54_N domain, and SRP54 domain. In addition, AjSRα is ubiquitously expressed in adult tissues and exhibits a sexually dimorphic expression pattern, with significantly higher expression in ovaries compared to testes. As a maternal factor, AjSRα can be continuously detected during embryonic development. Importantly, we first attempted to investigate its function by using lentiviral vectors for delivering SRα gene-specific shRNA, and we revealed that lentiviral vectors do not induce an upregulation of immune-related enzymes in sea cucumbers. However, compared to the dsRNA-based RNA interference (RNAi) method, lentivirus-mediated RNAi caused dynamic changes in gene expression at a later time. This study supplied the technical support for studying the functional mechanism of SRα in sea cucumbers.