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G G Karpova - One of the best experts on this subject based on the ideXlab platform.
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Molecular environment of the subdomain IIIe loop of the RNA IRES element of hepatitis C virus on the human 40S Ribosomal Subunit
Bioorganicheskaia khimiia, 2020Co-Authors: E S Laletina, A A Malygin, I N Shatskiĭ, D M Graĭfer, G G KarpovaAbstract:The molecular environment of the internal ribosome entry site (IRES) element of hepatitis C viral (HCV) RNA in the binary complex with the human 40S Ribosomal Subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA of the 40S Subunit. It was found that the photoreactive group of IRES nucleotide A296 was crosslinked to the 40S Subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group in nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S Subunit is located on the outer Subunit surface between the head and the body next to the "beak" near the entrance into the mRNA-binding channel. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.
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Binding of the IRES of hepatitis C virus RNA to the 40S Ribosomal Subunit: role of protein p40
Molekuliarnaia biologiia, 2020Co-Authors: A A Malygin, Z V Bochkaeva, E I Bondarenko, O A Kosinova, V B Loktev, I N Shatskiĭ, G G KarpovaAbstract:Ribosomal protein p40 is a structural component of the 40S Ribosomal Subunit, which is partially homologuos to prokaryotic Ribosomal protein S2 and has a long eukaryote-specific C-terminal region. In the present work, we have studied the binding of the Internal Ribosome Entry Site (IRES) of the hepatitis C virus (HCV) RNA to the 40S Ribosomal Subunit either deficient on protein p40, or saturated with the recombinant p40, or pre-bound to monoclonal antibodies (MAB) 4F6 against p40. It was shown that the apparent association constant of HCV IRES binding to 40S Subunits directly depends on p40 content in the Subunits. Binding of MAB 4F6 against p40 to 40S Subunits prevented the HCV IRES binding by the Subunits and blocked translation of the IRES-containing RNA in cell-free translation system. The data obtained point to the involvement of the Ribosomal protein p40 in the binding of the HCV IRES by ribosomes and therefore in initiation of translation of RNA of this virus.
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Molecular environment of the IIId subdomain of the IRES element of hepatitits C virus RNA on the human 40S Ribosomal Subunit
Bioorganicheskaia khimiia, 2020Co-Authors: E S Babaĭlova, A A Malygin, I N Shatskiĭ, D M Graĭfer, I Shtal, G G KarpovaAbstract:The molecular environment of the key subdomain IIId of the internal ribosome entry site (IRES) element of hepatitis C virus (HCV) RNA in the binary complex with the human 40S Ribosomal Subunit was studied. To this end, HCV IRES derivatives bearing perfluorophenylazido groups activatable by mild UV at nucleotide G263 or A275 in the subdomain IIId stem were used. They were prepared by the complementarily addressed modification of the corresponding RNA transcript with alkylating oligodeoxynucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA. It was found that the photoreactive groups of the IRES G263 and A275 nucleotides are crosslinked to Ribosomal proteins S3a, S14, and S16. For the IRES derivative with the photoreactive group in nucleotide G263, the degree of modification of proteins S14 and S16 was greater than that of S3a, whereas the derivative containing the same photoreactive group in nucleotide A275 was mainly crosslinked to proteins S3a and S14. An analysis of the data led to the conclusion that, in the binary complex of HCV IRES elements with the small Subunit of the 80S ribosome, its subdomain IIId stem is located on the outer Subunit surface between the head and the body next to the "beak" near the exit of mRNA from the ribosome.
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exploring the interactions of short rnas with the human 40S Ribosomal Subunit near the mrna entry site by epr spectroscopy
Nucleic Acids Research, 2019Co-Authors: Alexey A Malygin, Dmitri M Graifer, Olesya A Krumkacheva, Ivan O Timofeev, Anastasia S Ochkasova, Maria I Meschaninova, Alya G Venyaminova, Matvey V Fedin, Michael K Bowman, G G KarpovaAbstract: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.
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the es26 protein is involved in the formation of a nucleophosmin binding site on the human 40S Ribosomal Subunit
Biochimica et Biophysica Acta, 2018Co-Authors: Anton V Ivanov, A A Malygin, G G Karpova, Alexander V GopanenkoAbstract:Abstract 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.
A A Malygin - One of the best experts on this subject based on the ideXlab platform.
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Molecular environment of the subdomain IIIe loop of the RNA IRES element of hepatitis C virus on the human 40S Ribosomal Subunit
Bioorganicheskaia khimiia, 2020Co-Authors: E S Laletina, A A Malygin, I N Shatskiĭ, D M Graĭfer, G G KarpovaAbstract:The molecular environment of the internal ribosome entry site (IRES) element of hepatitis C viral (HCV) RNA in the binary complex with the human 40S Ribosomal Subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA of the 40S Subunit. It was found that the photoreactive group of IRES nucleotide A296 was crosslinked to the 40S Subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group in nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S Subunit is located on the outer Subunit surface between the head and the body next to the "beak" near the entrance into the mRNA-binding channel. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.
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Binding of the IRES of hepatitis C virus RNA to the 40S Ribosomal Subunit: role of protein p40
Molekuliarnaia biologiia, 2020Co-Authors: A A Malygin, Z V Bochkaeva, E I Bondarenko, O A Kosinova, V B Loktev, I N Shatskiĭ, G G KarpovaAbstract:Ribosomal protein p40 is a structural component of the 40S Ribosomal Subunit, which is partially homologuos to prokaryotic Ribosomal protein S2 and has a long eukaryote-specific C-terminal region. In the present work, we have studied the binding of the Internal Ribosome Entry Site (IRES) of the hepatitis C virus (HCV) RNA to the 40S Ribosomal Subunit either deficient on protein p40, or saturated with the recombinant p40, or pre-bound to monoclonal antibodies (MAB) 4F6 against p40. It was shown that the apparent association constant of HCV IRES binding to 40S Subunits directly depends on p40 content in the Subunits. Binding of MAB 4F6 against p40 to 40S Subunits prevented the HCV IRES binding by the Subunits and blocked translation of the IRES-containing RNA in cell-free translation system. The data obtained point to the involvement of the Ribosomal protein p40 in the binding of the HCV IRES by ribosomes and therefore in initiation of translation of RNA of this virus.
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Molecular environment of the IIId subdomain of the IRES element of hepatitits C virus RNA on the human 40S Ribosomal Subunit
Bioorganicheskaia khimiia, 2020Co-Authors: E S Babaĭlova, A A Malygin, I N Shatskiĭ, D M Graĭfer, I Shtal, G G KarpovaAbstract:The molecular environment of the key subdomain IIId of the internal ribosome entry site (IRES) element of hepatitis C virus (HCV) RNA in the binary complex with the human 40S Ribosomal Subunit was studied. To this end, HCV IRES derivatives bearing perfluorophenylazido groups activatable by mild UV at nucleotide G263 or A275 in the subdomain IIId stem were used. They were prepared by the complementarily addressed modification of the corresponding RNA transcript with alkylating oligodeoxynucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA. It was found that the photoreactive groups of the IRES G263 and A275 nucleotides are crosslinked to Ribosomal proteins S3a, S14, and S16. For the IRES derivative with the photoreactive group in nucleotide G263, the degree of modification of proteins S14 and S16 was greater than that of S3a, whereas the derivative containing the same photoreactive group in nucleotide A275 was mainly crosslinked to proteins S3a and S14. An analysis of the data led to the conclusion that, in the binary complex of HCV IRES elements with the small Subunit of the 80S ribosome, its subdomain IIId stem is located on the outer Subunit surface between the head and the body next to the "beak" near the exit of mRNA from the ribosome.
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the es26 protein is involved in the formation of a nucleophosmin binding site on the human 40S Ribosomal Subunit
Biochimica et Biophysica Acta, 2018Co-Authors: Anton V Ivanov, A A Malygin, G G Karpova, Alexander V GopanenkoAbstract:Abstract 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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proteins of the human 40S Ribosomal Subunit involved in hepatitis c ires binding as revealed from fluorescent labeling
Biochemistry, 2013Co-Authors: A A Malygin, Ivan N Shatsky, G G KarpovaAbstract:Initiation of translation of genomic RNA (gRNA) of hepatitis C virus (HCV) is provided by a highly structured fragment in its 5′-untranslated region, the so-called Internal Ribosome Entry Site (IRES). In this work, the exposed NH2-groups of proteins in the 40S Subunit of the human ribosome and in its binary complexes with RNA transcripts corresponding to the full-size HCV IRES or its fragments were probed using the N-hydroxysuccinimide derivative of the fluorescent dye Cy3. Comparison of efficiencies of modification of Ribosomal proteins in free Subunits and in their binary complexes with the RNA transcripts revealed Ribosomal proteins involved in the HCV IRES binding. It was found that binding of the 40S Subunits with the RNA transcript corresponding to full-size HCV IRES results in a decrease in modification levels of Ribosomal protein (rp) S27 and, to a lesser extent of rpS10; also, a noticeable decrease in the efficiency of labeling of proteins RACK1/S2/S3a was observed. When a fragment of HCV IRES containing the initial part of the open reading frame (ORF) of the viral gRNA was deleted, the level of rpS10 modification became the same as in free Subunits, whereas the levels of modification of rpS27 and the RACK1/S2/S3a group remained virtually unchanged compared to those observed in the complex of 40S Subunit with the full-size HCV IRES. Binding of 40S Subunits to a fragment of the HCV IRES lacking an ORF and domain II increased the modification level of the RACK1/S2/S3a proteins, while the efficiencies of labeling of rpS10 and rpS27 remained the same as upon the deletion of the ORF fragment. Comparison of these results with known structural and biochemical data on the organization of 40S Subunit and the location of the HCV IRES on it revealed structural elements of the IRES contacting exposed lysine residues of the above-mentioned Ribosomal proteins. Thus, it was found that the majority of exposed lysine residues of rpS27 are involved in the binding of the HCV IRES region formed by the junction of subdomains IIIa, IIIb, and IIIc with the central stalk of domain III, and that several lysine residues of rpS10 participate in the binding of the HCV IRES region corresponding to the initial part of the ORF of the viral gRNA. In addition, we concluded that lysine residues of rpS3a are involved in the binding of domains II and III of HCV IRES.
Ulrike Kutay - One of the best experts on this subject based on the ideXlab platform.
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usp16 counteracts mono ubiquitination of rps27a and promotes maturation of the 40S Ribosomal Subunit
eLife, 2020Co-Authors: Christian Montellese, Ivo Zemp, Stefanie Jonas, Jasmin Van Den Heuvel, Caroline Ashiono, Kerstin Dorner, Andre Melnik, Paola Picotti, Ludovic C Gillet, Ulrike KutayAbstract:Establishment of translational competence represents a decisive cytoplasmic step in the biogenesis of 40S Ribosomal Subunits. This involves final 18S rRNA processing and release of residual biogenesis factors, including the protein kinase RIOK1. To identify novel proteins promoting the final maturation of human 40S Subunits, we characterized pre-Ribosomal Subunits trapped on RIOK1 by mass spectrometry, and identified the deubiquitinase USP16 among the captured factors. We demonstrate that USP16 constitutes a component of late cytoplasmic pre-40S Subunits that promotes the removal of ubiquitin from an internal lysine of Ribosomal protein RPS27a/eS31. USP16 deletion leads to late 40S Subunit maturation defects, manifesting in incomplete processing of 18S rRNA and retarded recycling of late-acting ribosome biogenesis factors, revealing an unexpected contribution of USP16 to the ultimate step of 40S synthesis. Finally, ubiquitination of RPS27a appears to depend on active translation, pointing at a potential connection between 40S maturation and protein synthesis.
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human aatf che 1 forms a nucleolar protein complex with ngdn and nol10 required for 40S Ribosomal Subunit synthesis
Nucleic Acids Research, 2016Co-Authors: Lukas Bammert, Stefanie Jonas, Rosemarie Ungricht, Ulrike KutayAbstract:Mammalian AATF/Che-1 is essential for embryonic development, however, the underlying molecular mechanism is unclear. By immunoprecipitation of human AATF we discovered that AATF forms a salt-stable protein complex together with neuroguidin (NGDN) and NOL10, and demonstrate that the AATF-NGDN-NOL10 (ANN) complex functions in ribosome biogenesis. All three ANN complex members localize to nucleoli and display a mutual dependence with respect to protein stability. Mapping of protein-protein interaction domains revealed the importance of both the evolutionary conserved WD40 repeats in NOL10 and the UTP3/SAS10 domain in NGDN for complex formation. Functional analysis showed that the ANN complex supports nucleolar steps of 40S Ribosomal Subunit biosynthesis. All complex members were required for 18S rRNA maturation and their individual depletion affected the same nucleolar cleavage steps in the 5′ETS and ITS1 regions of the Ribosomal RNA precursor. Collectively, we identified the ANN complex as a novel functional module supporting the nucleolar maturation of 40S Ribosomal Subunits. Our data help to explain the described role of AATF in cell proliferation during mouse development as well as its requirement for malignant tumor growth.
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Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S Ribosomal Subunit synthesis
Nucleic Acids Research, 2016Co-Authors: Lukas Bammert, Stefanie Jonas, Rosemarie Ungricht, Ulrike KutayAbstract:Mammalian AATF/Che-1 is essential for embryonic development, however, the underlying molecular mechanism is unclear. By immunoprecipitation of human AATF we discovered that AATF forms a salt-stable protein complex together with neuroguidin (NGDN) and NOL10, and demonstrate that the AATF-NGDN-NOL10 (ANN) complex functions in ribosome biogenesis. All three ANN complex members localize to nucleoli and display a mutual dependence with respect to protein stability. Mapping of protein-protein interaction domains revealed the importance of both the evolutionary conserved WD40 repeats in NOL10 and the UTP3/SAS10 domain in NGDN for complex formation. Functional analysis showed that the ANN complex supports nucleolar steps of 40S Ribosomal Subunit biosynthesis. All complex members were required for 18S rRNA maturation and their individual depletion affected the same nucleolar cleavage steps in the 5′ETS and ITS1 regions of the Ribosomal RNA precursor. Collectively, we identified the ANN complex as a novel functional module supporting the nucleolar maturation of 40S Ribosomal Subunits. Our data help to explain the described role of AATF in cell proliferation during mouse development as well as its requirement for malignant tumor growth.
Jennifer A Doudna - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of the HCV IRES central domain reveals strategy for start-codon positioning
Structure, 2011Co-Authors: Katherine E Berry, Shruti Waghray, Stefanie A. Mortimer, Jennifer A DoudnaAbstract:Summary Translation of hepatitis C viral proteins requires an internal ribosome entry site (IRES) located in the 5′ untranslated region of the viral mRNA. The core domain of the hepatitis C virus (HCV) IRES contains a four-way helical junction that is integrated within a predicted pseudoknot. This domain is required for positioning the mRNA start codon correctly on the 40S Ribosomal Subunit during translation initiation. Here, we present the crystal structure of this RNA, revealing a complex double-pseudoknot fold that establishes the alignment of two helical elements on either side of the four-helix junction. The conformation of this core domain constrains the open reading frame's orientation for positioning on the 40S Ribosomal Subunit. This structure, representing the last major domain of HCV-like IRESs to be determined at near-atomic resolution, provides the basis for a comprehensive cryoelectron microscopy-guided model of the intact HCV IRES and its interaction with 40S Ribosomal Subunits.
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the hcv ires pseudoknot positions the initiation codon on the 40S Ribosomal Subunit
RNA, 2010Co-Authors: Katherine E Berry, Shruti Waghray, Jennifer A DoudnaAbstract:The hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) in its 5′ untranslated region, the structure of which is essential for viral protein translation. The IRES includes a predicted pseudoknot interaction near the AUG start codon, but the results of previous studies of its structure have been conflicting. Using mutational analysis coupled with activity and functional assays, we verified the importance of pseudoknot base pairings for IRES-mediated translation and, using 35 mutants, conducted a comprehensive study of the structural tolerance and functional contributions of the pseudoknot. Ribosomal toeprinting experiments show that the entirety of the pseudoknot element positions the initiation codon in the mRNA binding cleft of the 40S Ribosomal Subunit. Optimal spacing between the pseudoknot and the start site AUG resembles that between the Shine–Dalgarno sequence and the initiation codon in bacterial mRNAs. Finally, we validated the HCV IRES pseudoknot as a potential drug target using antisense 2′-OMe oligonucleotides.
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mass spectrometric analysis of the human 40S Ribosomal Subunit native and hcv ires bound complexes
Protein Science, 2009Co-Authors: Julie A Leary, Jennifer A Doudna, Yonghao Yu, Hong JiAbstract:Hepatitis C virus uses an internal ribosome entry site (IRES) in the viral RNA to directly recruit human 40S ribosome Subunits during cap-independent translation initiation. Although IRES-mediated translation initiation is not subject to many of the regulatory mechanisms that control cap-dependent translation initiation, it is unknown whether other noncanonical protein factors are involved in this process. Thus, a global protein composition analysis of native and IRES-bound 40S Ribosomal complexes has been conducted to facilitate an understanding of the IRES ribosome recruitment mechanism. A combined top-down and bottom-up mass spectrometry approach was used to identify both the proteins and their posttranslational modifications (PTMs) in the native 40S Subunit and the IRES recruited translation initiation complex. Thirty-one out of a possible 32 Ribosomal proteins were identified by combining top-down and bottom-up mass spectrometry techniques. Proteins were found to contain PTMs, including loss of methionine, acetylation, methylation, and disulfide bond formation. In addition to the 40S Ribosomal proteins, RACK1 was consistently identified in the 40S fraction, indicating that this protein is associated with the 40S Subunit. Similar methodology was then applied to the hepatitis C virus IRES-bound 40S complex. Two 40S Ribosomal proteins, RS25 and RS29, were found to contain different PTMs than those in the native 40S Subunit. In addition, RACK1, eukaryotic initiation factor 3 proteins and nucleolin were identified in the IRES-mediated translation initiation complex.
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eif3j is located in the decoding center of the human 40S Ribosomal Subunit
Molecular Cell, 2007Co-Authors: Christopher S Fraser, John W B Hershey, Katherine E Berry, Jennifer A DoudnaAbstract:Summary Protein synthesis in all cells begins with the ordered binding of the small Ribosomal Subunit to messenger RNA (mRNA) and transfer RNA (tRNA). In eukaryotes, translation initiation factor 3 (eIF3) is thought to play an essential role in this process by influencing mRNA and tRNA binding through indirect interactions on the backside of the 40S Subunit. Here we show by directed hydroxyl radical probing that the human eIF3 Subunit eIF3j binds to the aminoacyl (A) site and mRNA entry channel of the 40S Subunit, placing eIF3j directly in the Ribosomal decoding center. eIF3j also interacts with eIF1A and reduces 40S Subunit affinity for mRNA. A high affinity for mRNA is restored upon recruitment of initiator tRNA, even though eIF3j remains in the mRNA-binding cleft in the presence of tRNA. These results suggest that eIF3j functions in part by regulating access of the mRNA-binding cleft in response to initiation factor binding.
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Structural roles for human translation factor eIF3 in initiation of protein synthesis.
Science, 2005Co-Authors: Bunpote Siridechadilok, Christopher S Fraser, Jennifer A Doudna, Richard J. Hall, Eva NogalesAbstract:Protein synthesis in mammalian cells requires initiation factor eIF3, a ∼750-kilodalton complex that controls assembly of 40S Ribosomal Subunits on messenger RNAs (mRNAs) bearing either a 5′-cap or an internal ribosome entry site (IRES). Cryo–electron microscopy reconstructions show that eIF3, a five-lobed particle, interacts with the hepatitis C virus (HCV) IRES RNA and the 5′-cap binding complex eIF4F via the same domain. Detailed modeling of eIF3 and eIF4F onto the 40S Ribosomal Subunit reveals that eIF3 uses eIF4F or the HCV IRES in structurally similar ways to position the mRNA strand near the exit site of 40S, promoting initiation complex assembly.
Patrick Linder - One of the best experts on this subject based on the ideXlab platform.
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has1p a member of the dead box family is required for 40S Ribosomal Subunit biogenesis in saccharomyces cerevisiae
Molecular Microbiology, 2004Co-Authors: Bertrand Emery, Jesus De La Cruz, Sanda Rocak, Olivier Deloche, Patrick LinderAbstract:Summary The Has1 protein, a member of the DEAD-box family of ATP-dependent RNA helicases in Saccharomyces cerevisiae , has been found by different proteomic approaches to be associated with 90S and several pre-60S Ribosomal complexes. Here, we show that Has1p is an essential trans- acting factor involved in 40S Ribosomal Subunit biogenesis. Polysome analy- ses of strains genetically depleted of Has1p or carry- ing a temperature-sensitive has1-1 mutation show a clear deficit in 40S Ribosomal Subunits. Analyses of pre-rRNA processing by pulse-chase labelling, North- ern hybridization and primer extension indicate that these strains form less 18S rRNA because of inhibi- tion of processing of the 35S pre-rRNA at the early cleavage sites A 0 , A 1 and A 2 . Moreover, processing of the 27SA 3 and 27SB pre-rRNAs is delayed in these strains. Therefore, in addition to its role in the biogen- esis of 40S Ribosomal Subunits, Has1p is required for the optimal synthesis of 60S Ribosomal Subunits. Consistent with a role in ribosome biogenesis, Has1p is localized to the nucleolus. On sucrose gradients, Has1p is associated with a high-molecular-weight complex sedimenting at positions equivalent to 60S and pre-60S Ribosomal particles. A mutation in the ATP-binding motif of Has1p does not support growth of a has1 null strain, suggesting that the enzymatic activity of Has1p is required in ribosome biogenesis. Finally, sequence comparisons suggest that Has1p homologues exist in all eukaryotes, and we show that a has1 null strain can be fully complemented by the Candida albicans homologue.
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Has1p, a member of the DEAD‐box family, is required for 40S Ribosomal Subunit biogenesis in Saccharomyces cerevisiae†
Molecular Microbiology, 2004Co-Authors: Bertrand Emery, Jesus De La Cruz, Sanda Rocak, Olivier Deloche, Patrick LinderAbstract:Summary The Has1 protein, a member of the DEAD-box family of ATP-dependent RNA helicases in Saccharomyces cerevisiae , has been found by different proteomic approaches to be associated with 90S and several pre-60S Ribosomal complexes. Here, we show that Has1p is an essential trans- acting factor involved in 40S Ribosomal Subunit biogenesis. Polysome analy- ses of strains genetically depleted of Has1p or carry- ing a temperature-sensitive has1-1 mutation show a clear deficit in 40S Ribosomal Subunits. Analyses of pre-rRNA processing by pulse-chase labelling, North- ern hybridization and primer extension indicate that these strains form less 18S rRNA because of inhibi- tion of processing of the 35S pre-rRNA at the early cleavage sites A 0 , A 1 and A 2 . Moreover, processing of the 27SA 3 and 27SB pre-rRNAs is delayed in these strains. Therefore, in addition to its role in the biogen- esis of 40S Ribosomal Subunits, Has1p is required for the optimal synthesis of 60S Ribosomal Subunits. Consistent with a role in ribosome biogenesis, Has1p is localized to the nucleolus. On sucrose gradients, Has1p is associated with a high-molecular-weight complex sedimenting at positions equivalent to 60S and pre-60S Ribosomal particles. A mutation in the ATP-binding motif of Has1p does not support growth of a has1 null strain, suggesting that the enzymatic activity of Has1p is required in ribosome biogenesis. Finally, sequence comparisons suggest that Has1p homologues exist in all eukaryotes, and we show that a has1 null strain can be fully complemented by the Candida albicans homologue.
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fal1p is an essential dead box protein involved in 40S Ribosomal Subunit biogenesis in saccharomyces cerevisiae
Molecular and Cellular Biology, 1997Co-Authors: Dieter Kressler, Jesus De La Cruz, Manuel Rojo, Patrick LinderAbstract:A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A). Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family. Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization. An HA epitope-tagged Fal1p is localized predominantly in the nucleolus. Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S Ribosomal Subunits. Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin. Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2. Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S Ribosomal Subunits. Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation.
