RESUMO
Eukaryotic release factor eRF1, encoded by the ETF1 gene, recognizes stop codons and induces peptide release during translation termination. ETF1 produces several different transcripts as a result of alternative splicing, from which two eRF1 isoforms can be formed. Isoform 1 codes well-studied canonical eRF1, and isoform 2 is 33 amino acid residues shorter than isoform 1 and completely unstudied. Using a reconstituted mammalian in vitro translation system, we showed that the isoform 2 of human eRF1 is also involved in translation. We showed that eRF1iso2 can interact with the ribosomal subunits and pre-termination complex. However, its codon recognition and peptide release activities have decreased. Additionally, eRF1 isoform 2 exhibits unipotency to UGA. We found that eRF1 isoform 2 interacts with eRF3a but stimulated its GTPase activity significantly worse than the main isoform eRF1. Additionally, we studied the eRF1 isoform 2 effect on stop codon readthrough and translation in a cell-free translation system. We observed that eRF1 isoform 2 suppressed stop codon readthrough of the uORFs and decreased the efficiency of translation of long coding sequences. Based on these data, we assumed that human eRF1 isoform 2 can be involved in the regulation of translation termination. Moreover, our data support previously stated hypotheses that the GTS loop is important for the multipotency of eRF1 to all stop codons. Whereas helix α1 of the N-domain eRF1 is proposed to be involved in conformational rearrangements of eRF1 in the A-site of the ribosome that occur after GTP hydrolysis by eRF3, which ensure hydrolysis of peptidyl-tRNA at the P site of the ribosome.
Assuntos
Códon de Terminação , Fatores de Terminação de Peptídeos , Isoformas de Proteínas , Humanos , Fatores de Terminação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Códon de Terminação/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ribossomos/metabolismo , Biossíntese de Proteínas , Terminação Traducional da Cadeia Peptídica , Ligação ProteicaRESUMO
The poly(A) tail plays an important role in maintaining mRNA stability and influences translation efficiency via binding with PABP. However, the impact of poly(A) tail length on mRNA translation remains incompletely understood. This study explores the effects of poly(A) tail length on human translation. We determined the translation rates in cell lysates using mRNAs with different poly(A) tails. Cap-dependent translation was stimulated by the poly(A) tail, however, it was largely independent of poly(A) tail length, with an exception observed in the case of the 75 nt poly(A) tail. Conversely, cap-independent translation displayed a positive correlation with poly(A) tail length. Examination of translation stages uncovered the dependence of initiation and termination on the presence of the poly(A) tail, but the efficiency of initiation remained unaffected by poly(A) tail extension. Further study unveiled that increased binding of eRFs to the ribosome with the poly(A) tail extension induced more efficient hydrolysis of peptidyl-tRNA. Building upon these findings, we propose a crucial role for the 75 nt poly(A) tail in orchestrating the formation of a double closed-loop mRNA structure within human cells which couples the initiation and termination phases of translation.
Assuntos
Poli A , Biossíntese de Proteínas , RNA Mensageiro , Ribossomos , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Poli A/metabolismo , Ribossomos/metabolismo , Ribossomos/genética , Iniciação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , Células HeLa , Capuzes de RNA/metabolismo , Estabilidade de RNARESUMO
The genetic code is a set of instructions that determine how the information in our genetic material is translated into amino acids. In general, it is universal for all organisms, from viruses and bacteria to humans. However, in the last few decades, exceptions to this rule have been identified both in pro- and eukaryotes. In this review, we discuss the 16 described alternative eukaryotic nuclear genetic codes and observe theories of their appearance in evolution. We consider possible molecular mechanisms that allow codon reassignment. Most reassignments in nuclear genetic codes are observed for stop codons. Moreover, in several organisms, stop codons can simultaneously encode amino acids and serve as termination signals. In this case, the meaning of the codon is determined by the additional factors besides the triplets. A comprehensive review of various non-standard coding events in the nuclear genomes provides a new insight into the translation mechanism in eukaryotes.
Assuntos
Código Genético , Biossíntese de Proteínas , RNA Mensageiro , Código Genético/genética , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Biossíntese de Proteínas/genética , Animais , Códon de Terminação/genética , Núcleo Celular/genética , Evolução Molecular , Códon/genética , Eucariotos/genéticaRESUMO
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is a revolutionary tool for precise genome editing across various cell types. Ribonucleoproteins (RNPs), encompassing the Cas9 protein and guide RNA (gRNA), have emerged as a promising technique due to their increased specificity and reduced off-target effects. This method eliminates the need for plasmid DNA introduction, thereby preventing potential integration of foreign DNA into the target cell genome. Given the requirement for large quantities of highly purified protein in various Cas9 studies, we present an efficient and simple method for the preparation of recombinant Streptococcus pyogenes Cas9 (SpCas9) protein. This method leverages the Small Ubiquitin Like Modifier(SUMO) tag system, which includes metal-affinity chromatography followed by anion-exchange chromatography purification. Furthermore, we compare two methods of CRISPR-Cas9 system delivery into cells: transfection with plasmid DNA encoding the CRISPR-Cas9 system and RNP transfection with the Cas9-gRNA complex. We estimate the efficiency of genomic editing and protein lifespan post-transfection. Intriguingly, we found that RNP treatment of cells, even in the absence of a transfection system, is a relatively efficient method for RNP delivery into cell culture. This discovery is particularly promising as it can significantly reduce cytotoxicity, which is crucial for certain cell cultures such as induced pluripotent stem cells (iPSCs).
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , DNARESUMO
Nonsense variants underlie many genetic diseases. The phenotypic impact of nonsense variants is determined by Nonsense-mediated mRNA decay (NMD), which degrades transcripts with premature termination codons (PTCs). NMD activity varies across transcripts and cellular contexts via poorly understood mechanisms. Here, by leveraging human genetic datasets, we uncover that the amino acid preceding the PTC dramatically affects NMD activity in human cells. We find that glycine codons in particular support high levels of NMD and are enriched before PTCs but depleted before normal termination codons (NTCs). Gly-PTC enrichment is most pronounced in human genes that tolerate loss-of-function variants. This suggests a strong biological impact for Gly-PTC in ensuring robust elimination of potentially toxic truncated proteins from non-essential genes. Biochemical assays revealed that the peptide release rate during translation termination is highly dependent on the identity of the amino acid preceding the stop codon. This release rate is the most critical feature determining NMD activity across our massively parallel reporter assays. Together, we conclude that NMD activity is significantly modulated by the "window of opportunity" offered by translation termination kinetics. Integrating the window of opportunity model with the existing framework of NMD would enable more accurate nonsense variant interpretation in the clinic.
RESUMO
The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3' contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3' stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3' nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3' nucleotides. Moreover, the efficiency of translation termination in weak 3' contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3' nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs.
Assuntos
Nucleotídeos , Biossíntese de Proteínas , Animais , Códon de Terminação/genética , Códon de Terminação/metabolismo , Eucariotos/metabolismo , Humanos , Mamíferos/metabolismo , Nucleotídeos/genética , Nucleotídeos/metabolismo , Elongação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica/genética , Fatores de Terminação de Peptídeos/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.
Assuntos
Biossíntese de Proteínas , SARS-CoV-2 , Proteínas não Estruturais Virais/fisiologia , Animais , Sistema Livre de Células , Códon de Terminação/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Mutação , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , Peptídeos/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , RNA Mensageiro/metabolismo , Coelhos , Ribossomos/metabolismoRESUMO
Programmed cell death 4 protein (PDCD4) regulates many vital cell processes, although is classified as a tumor suppressor because it inhibits neoplastic transformation and tumor growth. For example, PCDC4 has been implicated in the regulation of transcription and mRNA translation. PDCD4 is known to inhibit translation initiation by binding to eukaryotic initiation factor 4A and elongation of oncogenic c- and A-myb mRNAs. Additionally, PDCD4 has been shown to interact with poly(A)-binding protein (PABP), which affects translation termination, although the significance of this interaction is not fully understood. Considering the interaction between PABP and PDCD4, we hypothesized that PDCD4 may also be involved in translation termination. Using in vitro translation systems, we revealed that PDCD4 directly activates translation termination. PDCD4 stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the PABP, which also stimulates peptide release, PDCD4 activity in translation termination increases. PDCD4 regulates translation termination by facilitating the binding of release factors to the ribosome, increasing the GTPase activity of eRF3, and dissociating eRF3 from the posttermination complex. Using a toe-printing assay, we determined the first stage at which PDCD4 functions-binding of release factors to the A-site of the ribosome. However, preventing binding of eRF3 with PABP, PDCD4 suppresses subsequent rounds of translation termination. Based on these data, we assumed that human PDCD4 controls protein synthesis during translation termination. The described mechanism of the activity of PDCD4 in translation termination provides a new insight into its functioning during suppression of protein biosynthesis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Terminação Traducional da Cadeia Peptídica , Proteínas de Ligação a RNA/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sistema Livre de Células/metabolismo , Humanos , Fatores de Terminação de Peptídeos/metabolismo , Proteínas de Ligação a Poli(A)/metabolismoRESUMO
eIF3j is one of the eukaryotic translation factors originally reported as the labile subunit of the eukaryotic translation initiation factor eIF3. The yeast homolog of this protein, Hcr1, has been implicated in stringent AUG recognition as well as in controlling translation termination and stop codon readthrough. Using a reconstituted mammalian in vitro translation system, we showed that the human protein eIF3j is also important for translation termination. We showed that eIF3j stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the initiation factor eIF3, which also stimulates peptide release, eIF3j activity in translation termination increases. We found that eIF3j interacts with the pre-termination ribosomal complex, and eRF3 destabilises this interaction. In the solution, these proteins bind to each other and to other participants of translation termination, eRF1 and PABP, in the presence of GTP. Using a toe-printing assay, we determined the stage at which eIF3j functions - binding of release factors to the A-site of the ribosome before GTP hydrolysis. Based on these data, we assumed that human eIF3j is involved in the regulation of translation termination by loading release factors into the ribosome.
Assuntos
Fator de Iniciação 3 em Eucariotos/genética , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/genética , Proteína I de Ligação a Poli(A)/genética , Ribossomos/genética , Animais , Sistema Livre de Células , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fator de Iniciação 3 em Eucariotos/química , Fator de Iniciação 3 em Eucariotos/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Modelos Moleculares , Fatores de Terminação de Peptídeos/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reticulócitos/química , Reticulócitos/metabolismo , Ribossomos/metabolismo , Transdução de SinaisRESUMO
Pairs of unnatural nucleotides are used to expand the genetic code and create artificial DNA or RNA templates. In general, an approach is used to engineer orthogonal systems capable of reading codons comprising artificial nucleotides; however, DNA and RNA polymerases capable of recognizing unnatural nucleotides are required for amplification and transcription of templates. Under favorable conditions, in the presence of modified nucleotide triphosphates, DNA polymerases are able to synthesize unnatural DNA with high efficiency; however, the currently available RNA polymerases reveal high specificity to the natural nucleotides and may not easily recognize the unnatural nucleotides. Due to the absence of simple and rapid methods for testing the activity of mutant RNA polymerases, the development of RNA polymerase recognizing unnatural nucleotides is limited. To fill this gap, we developed a method for rapid analysis of mutant RNA polymerase activity on templates containing unnatural nucleotides. Herein, we optimized a coupled cell-free translation system and tested the ability of three unnatural nucleotides to be transcribed by different T7 RNA polymerase mutants, by demonstrating high sensitivity and simplicity of the developed method. This approach can be applied to various unnatural nucleotides and can be simultaneously scaled up to determine the activity of numerous polymerases on different templates. Due to the simplicity and small amounts of material required, the developed cell-free system provides a highly scalable and versatile tool to study RNA polymerase activity.
Assuntos
Bacteriófago T7/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Mutação , Nucleotídeos/análise , Moldes Genéticos , Proteínas Virais/metabolismo , Sistema Livre de Células , RNA Polimerases Dirigidas por DNA/genética , Transcrição Gênica , Proteínas Virais/genéticaRESUMO
Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3' UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a "leaky" stop codon context, which likely defines the basis of nonsense suppression.
Assuntos
Bioensaio/métodos , Códon sem Sentido , Taxa de Mutação , Terminação Traducional da Cadeia Peptídica/genética , Sistema Livre de Células/fisiologia , Códon de Terminação/genética , Análise Mutacional de DNA , Genes Reporter , Humanos , Técnicas In Vitro , Luciferases/genética , Luciferases/metabolismo , Terminação Traducional da Cadeia Peptídica/fisiologia , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas/genéticaRESUMO
Classical toeprinting is generally used to determine the position of ribosomes on mRNA; however, it has several disadvantages. We describe a fluorescent toeprinting assay that enables easier identification of ribosomal complexes bound to mRNA in vitro. The procedure involves the use of stable and safe fluorescently labeled oligonucleotides for reverse transcription reactions as primers, followed by the analysis of cDNA products using an automatic sequencer. This procedure allows the multiplexing and simultaneous analysis of a large number of samples. Over the past ten years, fluorescent toeprinting was applied to determine the activities of eukaryotic release factors and additional proteins involved in translation termination, to study the dynamics of translation initiation and elongation complexes, and to quantitatively evaluate the observed ribosomal complexes. Because of the simplicity and small amounts of material required, fluorescent toeprinting provides a highly scalable and versatile tool to study ribosomal complexes.
Assuntos
Bioensaio/métodos , Técnicas Genéticas , Ribossomos/metabolismo , Fluorescência , Células HeLa , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Biossíntese de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transcrição Reversa , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/isolamento & purificação , Proteínas Ribossômicas/metabolismoRESUMO
Polyadenylate-binding protein (PABP) stimulates translation termination via interaction of its C-terminal domain with eukaryotic polypeptide chain release factor, eRF3. Additionally, two other proteins, poly(A)-binding protein-interacting proteins 1 and 2 (PAIP1 and PAIP2), bind the same domain of PABP and regulate its translation-related activity. To study the biochemistry of eRF3 and PAIP1/2 competition for PABP binding, we quantified the effects of PAIPs on translation termination in the presence or absence of PABP. Our results demonstrated that both PAIP1 and PAIP2 prevented translation termination at the premature termination codon, by controlling PABP activity. Moreover, PAIP1 and PAIP2 inhibited the activity of free PABP on translation termination in vitro However, after binding the poly(A) tail, PABP became insensitive to suppression by PAIPs and efficiently activated translation termination in the presence of eRF3a. Additionally, we revealed that PAIP1 binds eRF3 in solution, which stabilizes the post-termination complex. These results indicated that PAIP1 and PAIP2 participate in translation termination and are important regulators of readthrough at the premature termination codon.
Assuntos
Terminação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Humanos , Fatores de Iniciação de Peptídeos/química , Fatores de Terminação de Peptídeos/química , Poli A/química , Poli A/metabolismo , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Proteínas Repressoras/químicaRESUMO
Eukaryotic translation initiation is tightly regulated, requiring a set of conserved initiation factors (eIFs). Translation of a capped mRNA depends on the trimeric eIF4F complex and eIF4B to load the mRNA onto the 43S pre-initiation complex comprising 40S and initiation factors 1, 1A, 2, 3 and 5 as well as initiator-tRNA. Binding of the mRNA is followed by mRNA scanning in the 48S pre-initiation complex, until a start codon is recognised. Here, we use a reconstituted system to prepare human 48S complexes assembled on capped mRNA in the presence of eIF4B and eIF4F. The highly purified h-48S complexes are used for cross-linking/mass spectrometry, revealing the protein interaction network in this complex. We report the electron cryo-microscopy structure of the h-48S complex at 6.3 Å resolution. While the majority of eIF4B and eIF4F appear to be flexible with respect to the ribosome, additional density is detected at the entrance of the 40S mRNA channel which we attribute to the RNA-recognition motif of eIF4B. The eight core subunits of eIF3 are bound at the 40S solvent-exposed side, as well as the subunits eIF3d, eIF3b and eIF3i. elF2 and initiator-tRNA bound to the start codon are present at the 40S intersubunit side. This cryo-EM structure represents a molecular snap-shot revealing the h-48S complex following start codon recognition.
Assuntos
Fatores de Iniciação em Eucariotos/química , Modelos Moleculares , Subunidades Ribossômicas Menores de Eucariotos/química , Microscopia Crioeletrônica , Fator de Iniciação 3 em Eucariotos/química , Humanos , Espectrometria de Massas , Iniciação Traducional da Cadeia Peptídica , Capuzes de RNA/química , RNA Mensageiro/química , RNA Ribossômico 18S/química , Proteínas Ribossômicas/químicaRESUMO
During protein synthesis, a ribosome moves along the mRNA template and, using aminoacyl-tRNAs, decodes the template nucleotide triplets to assemble a protein amino acid sequence. This movement is accompanied by shifting of mRNA-tRNA complexes within the ribosome in a process called translocation. In living cells, this process proceeds in a unidirectional manner, bringing the ribosome to the 3' end of mRNA, and is catalyzed by the GTPase translation elongation factor 2 (EF-G in prokaryotes and eEF2 in eukaryotes). Interestingly, the possibility of spontaneous backward translocation has been shown in vitro for bacterial ribosomes, suggesting a potential reversibility of this reaction. However, this possibility has not yet been tested for eukaryotic ribosomes. Here, using a reconstituted mammalian translation system, we show that the eukaryotic elongation factor eEF2 catalyzes ribosomal reverse translocation at one mRNA triplet. We found that this process requires a cognate tRNA in the ribosomal E-site and cannot occur spontaneously without eEF2. The efficiency of this reaction depended on the concentrations of eEF2 and cognate tRNAs and increased in the presence of nonhydrolyzable GTP analogues. Of note, ADP-ribosylation of eEF2 domain IV blocked reverse translocation, suggesting a crucial role of interactions of this domain with the ribosome for the catalysis of the reaction. In summary, our findings indicate that eEF2 is able to induce ribosomal translocation in forward and backward directions, highlighting the universal mechanism of tRNA-mRNA movements within the ribosome.
Assuntos
Elongação Traducional da Cadeia Peptídica/fisiologia , Fator 2 de Elongação de Peptídeos/metabolismo , Ribossomos/metabolismo , Animais , Escherichia coli/metabolismo , Eucariotos/metabolismo , Células Eucarióticas/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Modelos Moleculares , Fator 2 de Elongação de Peptídeos/fisiologia , Fator G para Elongação de Peptídeos/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Coelhos , Proteínas RecombinantesRESUMO
The human DEAD-box RNA-helicase DDX19 functions in mRNA export through the nuclear pore complex. The yeast homolog of this protein, Dbp5, has been reported to participate in translation termination. Using a reconstituted mammalian in vitro translation system, we show that the human protein DDX19 is also important for translation termination. It is associated with the fraction of translating ribosomes. We show that DDX19 interacts with pre-termination complexes (preTCs) in a nucleotide-dependent manner. Furthermore, DDX19 increases the efficiency of termination complex (TC) formation and the peptide release in the presence of eukaryotic release factors. Using the eRF1(AGQ) mutant protein or a non-hydrolysable analog of GTP to inhibit subsequent peptidyl-tRNA hydrolysis, we reveal that the activation of translation termination by DDX19 occurs during the stop codon recognition. This activation is a result of DDX19 binding to preTC and a concomitant stabilization of terminating ribosomes. Moreover, we show that DDX19 stabilizes ribosome complexes with translation elongation factors eEF1 and eEF2. Taken together, our findings reveal that the human RNA helicase DDX19 actively participates in protein biosynthesis.
Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Elongação Traducional da Cadeia Peptídica/fisiologia , Terminação Traducional da Cadeia Peptídica/fisiologia , Ribossomos/metabolismo , Códon de Terminação , RNA Helicases DEAD-box/genética , Células HEK293 , Humanos , Mutação , Proteínas de Transporte Nucleocitoplasmático/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Polirribossomos/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The genetic code determines how amino acids are encoded within mRNA. It is universal among the vast majority of organisms, although several exceptions are known. Variant genetic codes are found in ciliates, mitochondria, and numerous other organisms. All revealed genetic codes (standard and variant) have at least one codon encoding a translation stop signal. However, recently two new genetic codes with a reassignment of all three stop codons were revealed in studies examining the protozoa transcriptomes. Here, we discuss this finding and the recent studies of variant genetic codes in eukaryotes. We consider the possible molecular mechanisms allowing the use of certain codons as sense and stop signals simultaneously. The results obtained by studying these amazing organisms represent a new and exciting insight into the mechanism of stop codon decoding in eukaryotes. Also see the video abstract here.
Assuntos
Códon de Terminação , Terminação Traducional da Cadeia Peptídica , Regiões 3' não Traduzidas , Animais , Eucariotos/genética , Evolução Molecular , Humanos , Fatores de Terminação de Peptídeos/fisiologiaRESUMO
For protein synthesis that occurs locally in dendrites, the translational control mechanisms are much more important for neuronal functioning than the transcription levels. Here, we show that uORFs (upstream open reading frames) in the 5' untranslated region (5'UTR) play a critical role in regulation of the translation of protein kinase Mζ (PKMζ). Elimination of these uORFs activates translation of the reporter protein in vitro and in primary cultures of rat hippocampal neurons. Using cell-free translation systems, we demonstrate that translational initiation complexes are formed only on uORFs. Further, we address the mechanism of translational repression of PKMζ translation, by uORFs. We observed an increase in translation of the reporter protein under the control of PKMζ leader in neuronal culture during non-specific activation by picrotoxin. We also show that such a mechanism is similar to the mechanism seen in cell stress, as application of sodium arsenite to neuron cultures induced translation of mRNA carrying PKMζ 5'UTR similarly to picrotoxin activation. Therefore, we suppose that phosphorylation of eIF2a, like in cell stress, is a main regulator of PKMζ translation. Altogether, our findings considerably extend our understanding of the role of uORF in regulation of PKMζ translation in activated neurons, important at early stages of LTP.
RESUMO
Poly(A)-binding protein (PABP) is a major component of the messenger RNA-protein complex. PABP is able to bind the poly(A) tail of mRNA, as well as translation initiation factor 4G and eukaryotic release factor 3a (eRF3a). PABP has been found to stimulate translation initiation and to inhibit nonsense-mediated mRNA decay. Using a reconstituted mammalian in vitro translation system, we show that PABP directly stimulates translation termination. PABP increases the efficiency of translation termination by recruitment of eRF3a and eRF1 to the ribosome. PABP's function in translation termination depends on its C-terminal domain and its interaction with the N-terminus of eRF3a. Interestingly, we discover that full-length eRF3a exerts a different mode of function compared to its truncated form eRF3c, which lacks the N-terminal domain. Pre-association of eRF3a, but not of eRF3c, with pre-termination complexes (preTCs) significantly increases the efficiency of peptidyl-tRNA hydrolysis by eRF1. This implicates new, additional interactions of full-length eRF3a with the ribosomal preTC. Based on our findings, we suggest that PABP enhances the productive binding of the eRF1-eRF3 complex to the ribosome, via interactions with the N-terminal domain of eRF3a which itself has an active role in translation termination.
Assuntos
Códon de Terminação/metabolismo , Terminação Traducional da Cadeia Peptídica/genética , Fatores de Terminação de Peptídeos/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Humanos , Hidrólise , Modelos Biológicos , Ligação Proteica , Aminoacil-RNA de TransferênciaRESUMO
Stabilization of the ribosomal complexes plays an important role in translational control. Mechanisms of ribosome stabilization have been studied in detail for initiation and elongation of eukaryotic translation, but almost nothing is known about stabilization of eukaryotic termination ribosomal complexes. Here, we present one of the mechanisms of fine-tuning of the translation termination process in eukaryotes. We show that certain deacylated tRNAs, remaining in the E site of the ribosome at the end of the elongation cycle, increase the stability of the termination and posttermination complexes. Moreover, only the part of eRF1 recognizing the stop codon is stabilized in the A site of the ribosome, and the stabilization is not dependent on the hydrolysis of peptidyl-tRNA. The determinants, defining this property of the tRNA, reside in the acceptor stem. It was demonstrated by site-directed mutagenesis of tRNA(Val) and construction of a mini-helix structure identical to the acceptor stem of tRNA. The mechanism of this stabilization is different from the fixation of the unrotated state of the ribosome by CCA end of tRNA or by cycloheximide in the E site. Our data allow to reveal the possible functions of the isodecoder tRNAs in eukaryotes.