RESUMEN
Gene expression is a fundamental cellular process that ensures the transfer of information encoded in a gene into the final functional product [...].
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Expresión GénicaRESUMEN
Ribosomal protein uL15 (RPL27a) carries a specific modification, hydroxylation, at the His39 residue, which neighbors the CCA terminus of the E-site-bound tRNA at the mammalian ribosome. Under hypoxia, the level of hydroxylation of this protein decreases. We transiently transfected HEK293T cells with constructs expressing wild-type uL15 or mutated uL15 (His39Ala) incapable of hydroxylation, and demonstrated that ribosomes containing both proteins are competent in translation. By applying RNA-seq to the total cellular and polysome-associated mRNAs, we identified differentially expressed genes (DEGs) in cells containing exogenous uL15 or its mutant form. Analyzing mRNA features of up- and down-regulated DEGs, we found an increase in the level of more abundant mRNAs and shorter CDSs in cells with uL15 mutant for both translated and total cellular mRNAs. The level of longer and rarer mRNAs, on the contrary, decreased. Our data show how ribosome heterogeneity can change the composition of the translatome and transcriptome, depending on the properties of the translated mRNAs.
Asunto(s)
Biosíntesis de Proteínas , Proteínas Ribosómicas , Humanos , Animales , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Hidroxilación , Células HEK293 , Mutación , Mamíferos/metabolismoRESUMEN
In eukaryotic ribosomes, the conserved protein uS19, formerly known as S15, extends with its C-terminal tail to the decoding site. The cross-linking of uS19 to the A site codon has been detected using synthetic mRNAs bearing 4-thiouridine (s4U) residues. Here, we showed that the A-site tRNA prevents this cross-linking and that the P site codon does not contact uS19. Next, we focused on determining uS19-mRNA interactions in vivo by applying the photoactivatable-ribonucleoside enhancing cross-linking and immunoprecipitation method to a stable HEK293 cell line producing FLAG-tagged uS19 and grown in a medium containing s4U. We found that when translation was stopped by cycloheximide, uS19 was efficiently cross-linked to mRNA regions with a high frequency of Glu, Lys and, more rarely, Arg codons. The results indicate that the complexes, in which the A site codon is not involved in the formation of the mRNA-tRNA duplex, are present among the cycloheximide-arrested 80S complexes, which implies pausing of elongating ribosomes at the above mRNA regions. Thus, our findings demonstrate that the human ribosomal protein uS19 interacts with mRNAs during translation elongation and highlight the regions of mRNAs where ribosome pausing occurs, bringing new structural and functional insights into eukaryotic translation in vivo.
Asunto(s)
ARN Mensajero/química , Proteínas Ribosómicas/química , Ribosomas/química , Codón , Eucariontes/genética , Células HEK293 , Humanos , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN de Transferencia/química , ARN de Transferencia/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Tiouridina/químicaRESUMEN
A number of mutations in the RPS20 gene encoding the ribosomal protein uS10 have been found to be associated with a predisposition to hereditary non-polyposis colorectal carcinoma (CRC). We transfected HEK293T cells with constructs carrying the uS10 minigene with mutations identical to those mentioned above and examined the effects of the produced proteins on the cellular transcriptome. We showed that uS10 with mutations p.V50SfsX23 or p.L61EfsX11 cannot be incorporated into 40S ribosomal subunits, while the protein with the missense mutation p.V54L functionally replaces the respective endogenous protein in the 40S subunit assembly and the translation process. The comparison of RNA-seq data obtained from cells producing aberrant forms of uS10 with data for those producing the wild-type protein revealed overlapping sets of upregulated and downregulated differently expressed genes (DEGs) related to several pathways. Among the limited number of upregulated DEGs, there were genes directly associated with the progression of CRC, e.g., PPM1D and PIGN. Our findings indicate that the accumulation of the mutant forms of uS10 triggers a cascade of cellular events, similar to that which is triggered when the cell responds to a large number of erroneous proteins, suggesting that this may increase the risk of cancer.
Asunto(s)
Neoplasias Colorrectales , Proteínas Ribosómicas , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Susceptibilidad a Enfermedades , Células HEK293 , Humanos , Mutación , Proteínas Ribosómicas/genética , TranscriptomaRESUMEN
The RNA cytosine C5 methyltransferase NSUN2 has a variety of RNA substrates and plays an important role in mRNA metabolism. NSUN2 binds to specific sequences enriched in exosomal mRNAs, suggesting its possible involvement in the sorting of mRNAs into exosomes. We applied the photoactivatable.4-thiouridine-enhanced cross-linking and immunoprecipitation assay involving high-throughput RNA sequencing (RNA-seq) to HEK293T cells to determine NSUN2 mRNA targets. NSUN2 cross-linking sites were found in more than one hundred relatively abundant mRNAs with a high GC content and a pronounced secondary structure. Then, utilizing RNA-seq for the total and polysome-associated mRNA from HEK293T cells with and without the knockdown of NSUN2, we identified differentially expressed genes, as well as genes with altered translational efficiency (GATEs). It turned out that the up-regulated GATE mRNAs were much shorter on average than the down-regulated ones, and their GC content was higher; moreover, they contained motifs with C residues located in GC-rich environments. Our findings reveal the specific features of mRNAs that make them potential targets for NSUN2 and expand our understanding of the role of NSUN2 in controlling translation and, possibly, in mRNA sorting into exosomes implemented through the methylation of cytosine residues.
Asunto(s)
Metiltransferasas , ARN Mensajero/metabolismo , Células HEK293 , Humanos , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , ARN Mensajero/químicaRESUMEN
The features of previously unexplored labile complexes of human 40S ribosomal subunits with RNAs, whose formation is manifested in the cross-linking of aldehyde derivatives of RNAs to the ribosomal protein uS3 through its peptide 55-64 located outside the mRNA channel, were studied by EPR spectroscopy methods. Analysis of subatomic 40S subunit models showed that a likely site for labile RNA binding is a cluster of positively charged amino acid residues between the mRNA entry site and uS3 peptide 55-64. This is consistent with our finding that the 3'-terminal mRNA fragment hanging outside the 40S subunit prevents the cross-linking of an RNA derivative to this peptide. To detect labile complexes of 40S subunits with RNA by DEER/PELDOR spectroscopy, an undecaribonucleotide derivative with nitroxide spin labels at terminal nucleotides was utilized. We demonstrated that the 40S subunit channel occupancy with mRNA does not affect the RNA derivative binding and that uS3 peptide 55-64 is not involved in binding interactions. Replacing the RNA derivative with a DNA one revealed the importance of ribose 2'-OH groups for the complex formation. Using the single-label RNA derivatives, the distance between the mRNA entry site and the loosely bound RNA site on the 40S subunit was estimated.
Asunto(s)
Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Unión Proteica , ARN Mensajero/química , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/químicaRESUMEN
The protein eL38 is one of the smallest proteins of the mammalian ribosome, which is a component of its large (60S) subunit. The haploinsufficiency of eL38 in mice leads to the Tail-short mutant phenotype characterized by defects in the development of the axial skeleton caused by the poor translation of mRNA subsets of Hox genes. Using the ribosome profiling assay applied to HEK293 cells knocked down of eL38, we examined the effects of the lack of eL38 in 60S subunits on gene expression at the level of translation. A four-fold decrease in the cell content of eL38 was shown to result in significant changes in the translational efficiencies of 150 genes. Among the genes, whose expression at the level of translation was enhanced, there were mainly those associated with basic metabolic processes; namely, translation, protein folding, chromosome organization, splicing, and others. The set of genes with reduced translation efficiencies contained those that are mostly involved in the processes related to the regulation of transcription, including the activation of Hox genes. Thus, we demonstrated that eL38 insufficiency significantly affects the expression of certain genes at the translational level. Our findings facilitate understanding the possible causes of some anomalies in eL38-deficient animals.
Asunto(s)
Regulación de la Expresión Génica/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Expresión Génica/genética , Células HEK293 , Humanos , Biosíntesis de Proteínas , ARN Mensajero/genética , Subunidades Ribosómicas Grandes de Eucariotas/genética , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ribosomas/metabolismo , Transcriptoma/genéticaRESUMEN
Protein uL5 (formerly called L11) is an integral component of the large (60S) subunit of the human ribosome, and its deficiency in cells leads to the impaired biogenesis of 60S subunits. Using RNA interference, we reduced the level of uL5 in HEK293T cells by three times, which caused an almost proportional decrease in the content of the fraction corresponding to 80S ribosomes, without a noticeable diminution in the level of polysomes. By RNA sequencing of uL5-deficient and control cell samples, which were those of total mRNA and mRNA from the polysome fraction, we identified hundreds of differentially expressed genes (DEGs) at the transcriptome and translatome levels and revealed dozens of genes with altered translational efficiency (GATEs). Transcriptionally up-regulated DEGs were mainly associated with rRNA processing, pre-mRNA splicing, translation and DNA repair, while down-regulated DEGs were genes of membrane proteins; the type of regulation depended on the GC content in the 3' untranslated regions of DEG mRNAs. The belonging of GATEs to up-regulated and down-regulated ones was determined by the coding sequence length of their mRNAs. Our findings suggest that the effects observed in uL5-deficient cells result from an insufficiency of translationally active ribosomes caused by a deficiency of 60S subunits.
Asunto(s)
Regulación de la Expresión Génica/genética , Proteínas Ribosómicas/deficiencia , Proteínas Ribosómicas/metabolismo , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Células HEK293 , Humanos , Biosíntesis de Proteínas/fisiología , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , Proteínas Ribosómicas/genética , Ribosomas/metabolismo , Transcripción Genética/fisiología , Transcriptoma/genéticaRESUMEN
The model mRNA (MR), 11-mer RNA containing two nitroxide spin labels at the 5'- and 3'-terminal nucleotides and prone to form a stable homodimer (MR)2, was used for Electron Paramagnetic Resonance study of structural rearrangements in mRNA occurring upon its binding to human 80S ribosomes. The formation of two different types of ribosomal complexes with MR was observed. First, there were stable complexes where MR was fixed in the ribosomal mRNA-binding channel by the codon-anticodon interaction(s) with cognate tRNA(s). Second, we for the first time detected complexes assembled without tRNA due to the binding of MR most likely to an exposed peptide of ribosomal protein uS3 away from the mRNA channel. The analysis of interspin distances allowed the conclusion that 80S ribosomes facilitate dissociation of the duplex (MR)2: the equilibrium between the duplex and the single-stranded MR shifts to MR due to its efficient binding with ribosomes. Furthermore, we observed a significant influence of tRNA bound at the ribosomal exit (E) and/or aminoacyl (A) sites on the stability of ribosomal complexes. Our findings showed that a part of mRNA bound in the ribosome channel, which is not involved in codon-anticodon interactions, has more degrees of freedom than that interacting with tRNAs.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Anticodón/metabolismo , Sitios de Unión , Codón/metabolismo , Humanos , Conformación de Ácido Nucleico , Unión Proteica , ARN Mensajero/química , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Marcadores de SpinRESUMEN
Ribosomal proteins are involved in many cellular processes through interactions with various RNAs. Here, applying the photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation approach to HEK293 cells overproducing ribosomal protein (rp) eS1, we determined the products of RNU5A-1 and RNU11 genes encoding U5 and U11 snRNAs as the RNA partners of ribosome-unbound rp eS1. U11 pre-snRNA-associated rp eS1 was revealed in the cytoplasm and nucleus where rp eS1-bound U11/U12 di-snRNP was also found. Utilizing recombinant rp eS1 and 4-thiouridine-containing U11 snRNA transcript, we identified an N-terminal peptide contacting the U-rich sequence in the Sm site-containing RNA region. We also showed that the rp eS1 binding site on U11 snRNA is located in the cleft between stem-loops I and III and that its structure mimics the respective site on the 18S rRNA. It was found that cell depletion of rp eS1 leads to a decrease in the splicing efficiency of minor introns and to an increase in the level of U11 pre-snRNA with the unprocessed 3' terminus. Our findings demonstrate the engagement of human rp eS1 in events related to the U11 snRNA processing and to minor-class splicing. Contacts of rp eS1 with U5 snRNA in the minor pre-catalytic spliceosome are discussed.
Asunto(s)
Biosíntesis de Proteínas , Empalme del ARN , ARN Ribosómico 18S/genética , ARN Nuclear Pequeño/química , Ribonucleoproteínas Nucleares Pequeñas/genética , Proteínas Ribosómicas/química , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Intrones , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , ARN Ribosómico 18S/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismo , Tiouridina/metabolismoRESUMEN
Translation termination in eukaryotes is mediated by release factors: eRF1, which is responsible for stop codon recognition and peptidyl-tRNA hydrolysis, and GTPase eRF3, which stimulates peptide release. Here, we have utilized ribose-specific probes to investigate accessibility of rRNA backbone in complexes formed by association of mRNA- and tRNA-bound human ribosomes with eRF1â¢eRF3â¢GMPPNP, eRF1â¢eRF3â¢GTP, or eRF1 alone as compared with complexes where the A site is vacant or occupied by tRNA. Our data show which rRNA ribose moieties are protected from attack by the probes in the complexes with release factors and reveal the rRNA regions increasing their accessibility to the probes after the factors bind. These regions in 28S rRNA are helices 43 and 44 in the GTPase associated center, the apical loop of helix 71, and helices 89, 92, and 94 as well as 18S rRNA helices 18 and 34. Additionally, the obtained data suggest that eRF3 neither interacts with the rRNA ribose-phosphate backbone nor dissociates from the complex after GTP hydrolysis. Taken together, our findings provide new information on architecture of the eRF1 binding site on mammalian ribosome at various translation termination steps and on conformational rearrangements induced by binding of the release factors.
Asunto(s)
Terminación de la Cadena Péptídica Traduccional , Factores de Terminación de Péptidos/metabolismo , ARN Mensajero/química , ARN Ribosómico 18S/química , ARN Ribosómico 28S/química , Aminoacil-ARN de Transferencia/química , Sitios de Unión , Codón de Terminación , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Conformación de Ácido Nucleico , Factores de Terminación de Péptidos/genética , Placenta/química , Embarazo , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , ARN Ribosómico 28S/genética , ARN Ribosómico 28S/metabolismo , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Human ribosomal protein eS26 is an indispensable component of the small (40S) ribosomal subunit and, along with other ribosomal proteins, is involved in interaction with mRNAs during translation. Here, we explored the behavior of the exogenous ribosomal protein eS26 modified at the C-terminus in the events related to translation in human cells using a doxycycline-inducible HEK293-derived cell line enabling the stable production of C-terminal FLAG-tagged eS26 (eS26FLAG). The production of eS26FLAG in cells was accompanied by a decrease in the endogenous eS26 content although its mRNA level did not change. Exogenous eS26FLAG was able to replace endogenous eS26 in 40S ribosomal subunits, without affecting the assembly and translational activity of 80S ribosomes. However, eS26FLAG-containing ribosome fractions from the respective polysome profile displayed a reduced content of nucleophosmin, a multifunctional protein, which, as is known, is involved in the formation and nuclear export of ribosomal subunits. In general, our data showed that although the appearance of the FLAG tag at the C-terminus of eS26 does not affect translation, it interferes with nucleophosmin incorporation into the 40S subunit, pointing out the importance of the C-terminus integrity of eS26 for nucleophosmin binding. In addition, with the recombinant protein, we demonstrated the binding of nucleophosmin to both isolated eS26 and 40S subunits in the presence of HeLa nuclear extract that phosphorylated the recombinant nucleophosmin. These findings suggest that for nuclear export, nucleophosmin could directly bind to pre-40S subunits in the mRNA exit site region where the C-terminus of eS26 is located.
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Proteínas Nucleares/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/metabolismo , Sitios de Unión , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Nucleofosmina , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Ribosomas/química , Ribosomas/genética , TransfecciónRESUMEN
Nanoscale distance measurements by pulse dipolar Electron paramagnetic resonance (EPR) spectroscopy allow new insights into the structure and dynamics of complex biopolymers. EPR detection requires site directed spin labeling (SDSL) of biomolecule(s), which remained challenging for long RNAs up-to-date. Here, we demonstrate that novel complementary-addressed SDSL approach allows efficient spin labeling and following structural EPR studies of long RNAs. We succeeded to spin-label Hepatitis C Virus RNA internal ribosome entry site consisting of ≈330 nucleotides and having a complicated spatial structure. Application of pulsed double electron-electron resonance provided spin-spin distance distribution, which agrees well with the results of molecular dynamics (MD) calculations. Thus, novel SDSL approach in conjunction with EPR and MD allows structural studies of long natural RNAs with nanometer resolution and can be applied to systems of biological and biomedical significance.
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Hepacivirus/genética , ARN Viral/metabolismo , Marcadores de Spin , Alquilación , Secuencia de Bases , Espectroscopía de Resonancia por Spin del Electrón , Sitios Internos de Entrada al Ribosoma , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/metabolismo , ARN Viral/química , ARN Viral/genética , TemperaturaRESUMEN
Exosomes, membranous vesicles secreted by various cells, are involved in intercellular communication and carry vast repertoires of RNAs and proteins. Processes mediating RNA sorting into exosomes are currently poorly understood. Using bioinformatics approaches, three structural motifs ACCAGCCU, CAGUGAGC and UAAUCCCA have been discovered as enriched in exosomal mRNAs and long noncoding RNAs. Here, utilizing short RNA hairpins, each containing one of the motifs, in a pull-down assay of cytosolic extract of human embryonic kidney 293 (HEK293) cells, we prove that multifunctional RNA-binding protein YB-1 specifically interacts with all three motifs, whereas methyltransferase NSUN2 recognizes only the motif CAGUGAGC. RNA hairpins other than those mentioned above pull out neither YB-1 nor NSUN2. Both these proteins are found in exosomes secreted by HEK293 cells. YB-1 for all that is detected as a form having a slightly higher electrophoretic mobility than that of YB-1 associated with the above RNA hairpins, assuming changes in posttranslational modifications of the protein during its transfer from cytoplasm into exosomes. Next generation sequencing of total exosomal RNA (eRNA) reveals a large representative set of RNA species, including mRNAs containing the above-mentioned motifs. The degree of enrichment in exosomes with this kind of mRNAs strongly depends on the locations of eRNA-specific motifs within the mRNA sequences. Altogether, our findings point to YB-1 and NSUN2 as possible mediators of the process of transfer of specific mRNAs into exosomes, allowing us to speculate on an involvement of these proteins in the mRNA sorting via the recognition of the above motifs.
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Citosol/metabolismo , Exosomas/metabolismo , Metiltransferasas/metabolismo , ARN Mensajero/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Secuencia de Aminoácidos , Citometría de Flujo , Células HEK293 , Humanos , Metiltransferasas/química , Microscopía Inmunoelectrónica , ARN Mensajero/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteína 1 de Unión a la Caja Y/químicaRESUMEN
Association of ribosomal proteins with rRNA during assembly of ribosomal subunits is an intricate process, which is strictly regulated in vivo. As for the assembly in vitro, it was reported so far only for prokaryotic subunits. Bacterial ribosomal proteins are capable of selective binding to 16S rRNA as well as to its separate morphological domains. In this work, we explored binding of total protein of human 40S ribosomal subunit to the RNA transcript corresponding to the major 3'-domain of 18S rRNA. We showed that the resulting ribonucleoprotein particles contained almost all of the expected ribosomal proteins, whose binding sites are located in this 18S rRNA domain in the 40S subunit, together with several nonspecific proteins. The binding in solution was accompanied with aggregation of the RNA-protein complexes. Ribosomal proteins bound to the RNA transcript protected from chemical modification mostly those 18S rRNA nucleotides that are known to be involved in binding with the proteins in the 40S subunit and thereby demonstrated their ability to selectively bind to the rRNA in vitro. The possible implication of unstructured extensions of eukaryotic ribosomal proteins in their nonspecific binding with rRNA and in subsequent aggregation of the resulting complexes is discussed.
Asunto(s)
ARN Ribosómico 18S/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/genética , Sitios de Unión , Humanos , Estructura Terciaria de Proteína , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/ultraestructura , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/ultraestructura , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/ultraestructura , Ribosomas/ultraestructuraRESUMEN
In this work, intimate contacts of riboses of mRNA stretch from nucleotides in positions +3 to +12 with respect to the first nucleotide of the P site codon were studied using cross-linking of short mRNA analogs with oxidized 3'-terminal riboses bound to human ribosomes in the complexes stabilized by codon-anticodon interactions and in the binary complexes. It was shown that in all types of complexes cross-links of the mRNA analogs to ribosomal protein (rp) uS3 occur and the yield of these cross-links does not depend on the presence of tRNA and on sequences of the mRNA analogs. Site of the mRNA analogs cross-linking in rp uS3 was mapped to the peptide in positions 55-64 that is located away from the mRNA binding site. Additionally, in complexes with P site-bound tRNA, riboses of mRNA nucleotides in positions +4 to +7 cross-linked to the C-terminal tail of rp uS19 displaying a contact specific to the decoding site of the mammalian ribosome, and tRNA bound at the A site completely blocked this cross-linking. Remarkably, rps uS3 and uS19 were also able to cross-link to the fragment of HCV IRES containing unstructured 3'-terminal part restricted by the AUGC tetraplet with oxidized 3'-terminal ribose. However, no cross-linking to rp uS3 was observed in the 48S preinitiation complex assembled in reticulocyte lysate with this HCV IRES derivative. The results obtained show an ability of rp uS3 to interact with single-stranded RNAs. Possible roles of rp uS3 region 55-64 in the functioning of ribosomes are discussed.
Asunto(s)
ARN Mensajero/metabolismo , Ribosamonofosfatos/metabolismo , Ribosomas/metabolismo , Anticodón/química , Secuencia de Bases , Sitios de Unión/efectos de los fármacos , Codón/química , Codón/metabolismo , Reactivos de Enlaces Cruzados/química , Hepacivirus/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Mensajero/química , ARN de Transferencia/química , ARN de Transferencia/metabolismo , ARN Viral/química , ARN Viral/metabolismo , Ribosamonofosfatos/química , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Ribosomas/química , Sitio de Iniciación de la TranscripciónRESUMEN
mRNAs are involved in complicated supramolecular complexes with human 40S and 80S ribosomes responsible for the protein synthesis. In this work, a derivative of nonaribonucleotide pUUCGUAAAA with nitroxide spin labels attached to the 5'-phosphate and to the C8 atom of the adenosine in sixth position (mRNA analog) was used for studying such complexes using double electron-electron resonance/pulsed electron-electron double resonance spectroscopy. The complexes were assembled with participation of tRNA(Phe), which targeted triplet UUC of the derivative to the ribosomal peptidyl site and predetermined location of the adjacent GUA triplet coding for Val at the aminoacyl (A) site. The interspin distances were measured between the two labels of mRNA analog attached to the first nucleotide of the peptidyl site bound codon and to the third nucleotide of the A site bound codon, in the absence/presence of second tRNA bound at the A site. The values of the obtained interspin distances agree with those calculated for available near-atomic structures of similar complexes of 40S and 80S ribosomes, showing that neither 60S subunit nor tRNA at the A site have a noticeable effect on arrangement of mRNA at the codon-anticodon interaction area. In addition, the shapes of distance distributions in four studied ribosomal complexes allowed conclusions on conformational flexibility of mRNA in these complexes. Overall, the results of this study are the first, to our knowledge, demonstration of double electron-electron resonance/pulsed electron-electron double resonance application for measurements of intramolecular distances in multicomponent supramolecular complexes involving intricate cellular machineries and for evaluating dynamic properties of ligands bound to these machineries.
Asunto(s)
Marcadores de Spin , Secuencia de Bases , Espectroscopía de Resonancia por Spin del Electrón , Femenino , Humanos , Óxidos de Nitrógeno/química , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/química , Ribosomas/metabolismoRESUMEN
Previous analyses of complexes of 40S ribosomal subunits with the hepatitis C virus (HCV) internal ribosome entry site (IRES) have revealed contacts made by the IRES with ribosomal proteins. Here, using chemical probing, we show that the HCV IRES also contacts the backbone and bases of the CCC triplet in the 18S ribosomal RNA (rRNA) expansion segment 7. These contacts presumably provide interplay between IRES domain II and the AUG codon close to ribosomal protein S5, which causes a rearrangement of 18S rRNA structure in the vicinity of the universally conserved nucleotide G1639. As a result, G1639 becomes exposed and the corresponding site of the 40S subunit implicated in transfer RNA discrimination can select . These data are the first demonstration at nucleotide resolution of direct IRES-rRNA interactions and how they induce conformational transition in the 40S subunit allowing the HCV IRES to function without AUG recognition initiation factors.
Asunto(s)
Regiones no Traducidas 5' , Hepacivirus/genética , Iniciación de la Cadena Peptídica Traduccional , ARN Ribosómico 18S/metabolismo , ARN Viral/química , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Secuencia de Bases , Codón , Humanos , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , ARN Ribosómico 18S/químicaRESUMEN
Site-directed spin labeling (SDSL) is widely applied for structural studies of biopolymers by electron paramagnetic resonance (EPR). However, SDSL of long RNA sequences still remains a challenging task. Here, we propose a novel SDSL approach potentially suitable for long natural RNAs, which is based on the attachment of a linker containing an aliphatic amino group to the target nucleotide residue followed by selective coupling of a spin label to this amino group. Such a linker can be attached to the desired RNA residue via a sequence-specific reaction with the derivatives of oligodeoxyribonucleotides. To verify this approach, we applied it to model RNA duplex with known structure and expected distance between corresponding residues. A new 2,5-bis(spirocyclohexane)-substituted spin label with advanced stability and relaxation properties has been used, and the distance distribution measured using Q-band (34 GHz) pulsed double electron-electron resonance corresponds well to the expected one. We have additionally validated the obtained results by studying a similar RNA duplex, where the linker with the aliphatic amino group was introduced via solid-phase synthesis. Although this novel SDSL approach does not provide an advantage in precision of molecular distance measurements, we believe that its applicability to long RNAs is a crucial benefit for future structural studies using pulse EPR.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , ARN/química , Marcadores de Spin , Alquilación , Secuencia de Bases , ADN Complementario/genética , Electrones , Datos de Secuencia Molecular , Conformación de Ácido NucleicoRESUMEN
Ribosomal protein eL42 (formerly known as L36A), a small protein of the large (60S) subunit of the eukaryotic ribosome, is a component of its exit (E) site. The residue K53 of this protein resides within the motif QSGYGGQTK mainly conserved in eukaryotes, and it is located in the immediate vicinity of the CCA-terminus of the ribosome-bound tRNA in the hybrid P/E state. To examine the role of this eL42 motif in translation, we obtained HEK293T cells producing the wild-type FLAG-tagged protein or its mutant forms with either single substitutions of conserved amino acid residues in the above motif, or simultaneous replacements in positions 45 and 51 or 45 and 53. Examination of the level of exogenous eL42 in fractions of polysome profiles from the target protein-producing cells by the Western blotting revealed that neither single substitution affects the assembly of 60S ribosomal subunits and 80S ribosomes or critically decreases the level of polysomes, but the latter was observed with the double replacements. Analysis of tRNAs bound to 80S ribosomes containing eL42 with double substitutions and examination their peptidyl transferase activity enabled estimation the stage of the elongation cycle, in which amino acid residues of the conserved eL42 motif are involved. We clearly show that cooperative interactions implicating the eL42 residues Q45, Q51, and K53 play a critical role in the ability of the human ribosome to perform properly elongation cycle at the step of deacylated tRNA dissociation from the E site in the human cell.