Sergey E. Dmitriev
Research interests
-
InterestsTranslational Control Of Gene Expression, Translation Initiation Factors, eIF2D, IRES...), 5' CITE, eIF4B, eIF2, HCV
Publications
-
7.48Impact points
Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation.
Nucleic acids research. 02/2012;
Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are spe... [more] Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are specific RNA structures that facilitate the 5' end-independent internal initiation of translation both under normal conditions and when the cap-dependent host protein synthesis is shut-down in infected cells. A longstanding issue is which host factors play a major role in this internal initiation. Here, we show that the functionally most important domain V of the poliovirus IRES uses tRNA(Gly) anticodon stem-loop mimicry to recruit glycyl-tRNA synthetase (GARS) to the apical part of domain V, adjacent to the binding site of the key initiation factor eIF4G. The binding of GARS promotes the accommodation of the initiation region of the IRES in the mRNA binding site of the ribosome, thereby greatly enhancing the activity of the IRES at the step of the 48S initiation complex formation. Moonlighting functions of GARS that may be additionally needed for other events of the virus-host cell interaction are discussed.
-
3.87Impact points
Archaeal translation initiation factor aIF2 can substitute for eukaryotic eIF2 in ribosomal scanning during mammalian 48S complex formation.
Journal of molecular biology. 08/2011; 413(1):106-14.
Heterotrimeric translation initiation factor (IF) a/eIF2 (archaeal/eukaryotic IF 2) is present in both Eukarya and Archaea. Despite strong structural similarity between a/eIF2 orthologs from the two domains of life, their functional relationship is obscure. Here, we show that aIF2 from Sulfolobus so... [more] Heterotrimeric translation initiation factor (IF) a/eIF2 (archaeal/eukaryotic IF 2) is present in both Eukarya and Archaea. Despite strong structural similarity between a/eIF2 orthologs from the two domains of life, their functional relationship is obscure. Here, we show that aIF2 from Sulfolobus solfataricus can substitute for its mammalian counterpart in the reconstitution of eukaryotic 48S initiation complexes from purified components. aIF2 is able to correctly place the initiator Met-tRNA(i) into the P-site of the 40S ribosomal subunit and accompany the entire set of eukaryotic translation IFs in the process of cap-dependent scanning and AUG codon selection. However, it seems to be unable to participate in the following step of ribosomal subunit joining. In accordance with this, aIF2 inhibits rather than stimulates protein synthesis in mammalian cell-free system. The ability of recombinant aIF2 protein to direct ribosomal scanning suggests that some archaeal mRNAs may utilize this mechanism during translation initiation.
-
7.48Impact points
Unidirectional constant rate motion of the ribosomal scanning particle during eukaryotic translation initiation.
Nucleic acids research. 03/2011; 39(13):5555-67.
According to the model of translation initiation in eukaryotes, the 40S ribosomal subunit binds to capped 5'-end of mRNA and subsequently migrates along 5'-UTR in searching for initiation codon. However, it remains unclear whether the migration is the result of a random one-dimensional diffu... [more] According to the model of translation initiation in eukaryotes, the 40S ribosomal subunit binds to capped 5'-end of mRNA and subsequently migrates along 5'-UTR in searching for initiation codon. However, it remains unclear whether the migration is the result of a random one-dimensional diffusion, or it is an energy-driven unidirectional movement. To address this issue, the method of continuous monitoring of protein synthesis in situ was used for high precision measurements of the times required for translation of mRNA with 5'-UTRs of different lengths and structures in mammalian and plant cell-free systems. For the first time, the relationship between the scanning time and the 5'-UTR length was determined and their linear correlation was experimentally demonstrated. The conclusion is made that the ribosome migration is an unidirectional motion with the rate being virtually independent of a particular mRNA sequence and secondary structure.
-
4.41Impact points
Design of targeted B cell killing agents.
PloS one. 01/2011; 6(6):e20991.
B cells play an important role in the pathogenesis of both systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce autoantibodies, but also are capable to efficiently present specific autoantigens to T cells. Furthermore, B cells can secrete proinflammatory cytokines a... [more] B cells play an important role in the pathogenesis of both systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce autoantibodies, but also are capable to efficiently present specific autoantigens to T cells. Furthermore, B cells can secrete proinflammatory cytokines and amplify the vicious process of self-destruction. B cell-directed therapy is a potentially important approach for treatment of various autoimmune diseases. The depletion of B cells by anti-CD20/19 monoclonal antibody Retuximab® used in autoimmune diseases therapy leads to systemic side effects and should be significantly improved. In this study we designed a repertoire of genetically engineered B cell killers that specifically affected one kind of cells carrying a respective B cell receptor. We constructed immunotoxins (ITs), fused with c-myc epitope as a model targeting sequence, based on barnase, Pseudomonas toxin, Shiga-like toxin E.coli and Fc domain of human antibody IgGγ1. C-MYC hybridoma cell line producing anti-c-myc IgG was chosen as a model for targeted cell depletion. C-myc sequence fused with toxins provided addressed delivery of the toxic agent to the target cells. We demonstrated functional activity of designed ITs in vitro and showed recognition of the fusion molecules by antibodies produced by targeted hybridoma. To study specificity of the proposed B cells killing molecules, we tested a set of created ITs ex vivo, using C-MYC and irrelevant hybridoma cell lines. Pseudomonas-containing IT showed one of the highest cytotoxic effects on the model cells, however, possessed promiscuous specificity. Shiga-like toxin construct demonstrated mild both cytotoxicity and specificity. Barnase and Fc-containing ITs revealed excellent balance between their legibility and toxic properties. Moreover, barnase and Fc molecules fused with c-myc epitope were able to selectively deplete c-myc-specific B cells and decrease production of anti-c-myc antibodies in culture of native splenocytes, suggesting their highest therapeutic potential as targeted B cell killing agents.
-
1.88Impact points
Cap- and IRES-independent scanning mechanism of translation initiation as an alternative to the concept of cellular IRESs.
Molecules and cells. 10/2010; 30(4):285-93.
During the last decade the concept of cellular IRES-elements has become predominant to explain the continued expression of specific proteins in eukaryotic cells under conditions when the cap-dependent translation initiation is inhibited. However, many cellular IRESs regarded as cornerstones of the c... [more] During the last decade the concept of cellular IRES-elements has become predominant to explain the continued expression of specific proteins in eukaryotic cells under conditions when the cap-dependent translation initiation is inhibited. However, many cellular IRESs regarded as cornerstones of the concept, have been compromised by several recent works using a number of modern techniques. This review analyzes the sources of artifacts associated with identification of IRESs and describes a set of control experiments, which should be performed before concluding that a 5' UTR of eukaryotic mRNA does contain an IRES. Hallmarks of true IRES-elements as exemplified by well-documented IRESs of viral origin are presented. Analysis of existing reports allows us to conclude that there is a constant confusion of the cap-independent with the IRES-directed translation initiation. In fact, these two modes of translation initiation are not synonymous. We discuss here not numerous reports pointing to the existence of a cap- and IRES-independent scanning mechanism of translation initiation based on utilization of special RNA structures called cap-independent translational enhancers (CITE). We describe this mechanism and suggest it as an alternative to the concept of cellular IRESs.
-
5.33Impact points
GTP-independent tRNA delivery to the ribosomal P-site by a novel eukaryotic translation factor.
The Journal of biological chemistry. 08/2010; 285(35):26779-87.
During translation, aminoacyl-tRNAs are delivered to the ribosome by specialized GTPases called translation factors. Here, we report the tRNA binding to the P-site of 40 S ribosomes by a novel GTP-independent factor eIF2D isolated from mammalian cells. The binding of tRNA(i)(Met) occurs after the AU... [more] During translation, aminoacyl-tRNAs are delivered to the ribosome by specialized GTPases called translation factors. Here, we report the tRNA binding to the P-site of 40 S ribosomes by a novel GTP-independent factor eIF2D isolated from mammalian cells. The binding of tRNA(i)(Met) occurs after the AUG codon finds its position in the P-site of 40 S ribosomes, the situation that takes place during initiation complex formation on the hepatitis C virus internal ribosome entry site or on some other specific RNAs (leaderless mRNA and A-rich mRNAs with relaxed scanning dependence). Its activity in tRNA binding with 40 S subunits does not require the presence of the aminoacyl moiety. Moreover, the factor possesses the unique ability to deliver non-Met (elongator) tRNAs into the P-site of the 40 S subunit. The corresponding gene is found in all eukaryotes and includes an SUI1 domain present also in translation initiation factor eIF1. The versatility of translation initiation strategies in eukaryotes is discussed.
-
7.48Impact points
Differential contribution of the m7G-cap to the 5' end-dependent translation initiation of mammalian mRNAs.
Nucleic acids research. 09/2009;
Many mammalian mRNAs possess long 5' UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised. To test this hypothesis, we have c... [more] Many mammalian mRNAs possess long 5' UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised. To test this hypothesis, we have compared the translation initiation efficiencies of some cellular 5' UTRs reported to have IRES-activity with those lacking IRES-elements in RNA-transfected cells and cell-free systems. Unlike viral IRESes, the tested 5' UTRs with so-called 'cellular IRESes' demonstrate only background activities when placed in the intercistronic position of dicistronic RNAs. In contrast, they are very active in the monocistronic context and the cap is indispensable for their activities. Surprisingly, in cultured cells or cytoplasmic extracts both the level of stimulation with the cap and the overall translation activity do not correlate with the cumulative energy of the secondary structure of the tested 5' UTRs. The cap positive effect is still observed under profound inhibition of translation with eIF4E-BP1 but its magnitude varies for individual 5' UTRs irrespective of the cumulative energy of their secondary structures. Thus, it is not mandatory to invoke the IRES hypothesis, at least for some mRNAs, to explain their preferential translation when eIF4E is partially inactivated.
-
Efficient cap-dependent translation of mammalian mRNAs with long and highly structured 5'-untranslated regions in vitro and in vivo
Mol Biol (Mosk). 01/2009; 43(1):108-113.
According to generally accepted scanning model proposed by M. Kozak, the secondary structure of 5'-untranslated regions (5'-UTR) of eukaryotic mRNAs can only cause an inhibitory effect on the translation initiation since it would counteract migration of the 40S ribosomal subunit along the mR... [more] According to generally accepted scanning model proposed by M. Kozak, the secondary structure of 5'-untranslated regions (5'-UTR) of eukaryotic mRNAs can only cause an inhibitory effect on the translation initiation since it would counteract migration of the 40S ribosomal subunit along the mRNA polynucleotide chain. Thus, the existence of efficiently translatable mRNAs with long and highly structured 5'-UTRs is not compatible with the cap-dependent scanning mechanism. It is expected that such mRNAs should use alternative ways of translation initiation to be efficiently translated, first of all the mechanism of the internal ribosome entry mediated by special RNA structures called IRESes (for Internal Ribosome Entry Sites), which have been proposed to reside within their 5'-UTRs. In this paper, it is shown that this point of view is not correct and most probably based on experiments of mRNA translation in rabbit reticulocyte lysate. This cell free system does not reflect correctly the ratio of translation efficiencies of various mRNAs which is observed in the living cell. Using five different mRNAs of similar design which possess either relatively short leaders of cellular mRNAs (beta-globin and beta-actin mRNAs) or long and highly structured 5'-UTRs (c-myc, LINE-1, Apaf-1 mRNAs), we show that the translation activities of all tested 5'-UTRs are comparable, both in transfected cells and in a whole cytoplasmic extract of cultivated cells. This activity is strongly dependent on the presence of the cap at their 5'-ends.
-
12.27Impact points
Eukaryotic translation initiation machinery can operate in a bacterial-like mode without eIF2.
Nature structural & molecular biology. 08/2008;
Unlike bacteria, a specialized eukaryotic initiation factor (eIF)-2, in the form of the ternary complex eIF2-GTP-Met-tRNA(i) (Met), is used to deliver the initiator tRNA to the ribosome in all eukaryotic cells. Here we show that the hepatitis C virus (HCV) internal ribosome entry site (IRES) can dir... [more] Unlike bacteria, a specialized eukaryotic initiation factor (eIF)-2, in the form of the ternary complex eIF2-GTP-Met-tRNA(i) (Met), is used to deliver the initiator tRNA to the ribosome in all eukaryotic cells. Here we show that the hepatitis C virus (HCV) internal ribosome entry site (IRES) can direct translation without eIF2 and its GTPase-activating protein eIF5. In addition to the general eIF2- and eIF5-dependent pathway of 80S complex assembly, the HCV IRES makes use of a bacterial-like pathway requiring as initiation factors only eIF5B (an analog of bacterial IF2) and eIF3. The switch from the conventional eukaryotic mode of translation initiation to the eIF2-independent mechanism occurs when eIF2 is inactivated by phosphorylation under stress conditions.
-
5.20Impact points
The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome.
RNA (New York, N.Y.). 03/2008; 14(2):233-9.
RelE/RelB is a well-characterized toxin-anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. RelE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is simi... [more] RelE/RelB is a well-characterized toxin-anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. RelE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes also in eukaryotes. RelE cleavage in the A-site codon of a stalled eukaryote ribosome is precise and easily monitored, making "RelE printing" a useful complement to toeprinting to determine the exact mRNA location on the eukaryote ribosome and to probe the occupancy of its A site.
-
5.20Impact points
Differential factor requirement to assemble translation initiation complexes at the alternative start codons of foot-and-mouth disease virus RNA.
RNA (New York, N.Y.). 09/2007; 13(8):1366-74.
The foot-and-mouth disease virus (FMDV) RNA contains two in-frame AUG codons separated by 84 nt that direct translation initiation of the viral polyprotein. The mechanism of initiation at the IRES-proximal AUG codon (AUG1) has been previously analyzed, whereas no data on factor requirements for AUG2... [more] The foot-and-mouth disease virus (FMDV) RNA contains two in-frame AUG codons separated by 84 nt that direct translation initiation of the viral polyprotein. The mechanism of initiation at the IRES-proximal AUG codon (AUG1) has been previously analyzed, whereas no data on factor requirements for AUG2 have been reported. Here, using the method of 48S translation initiation complex reconstitution, we show that eIF1 is indispensable in forming the 48S initiation complex at AUG2. In contrast, it reduces the assembly of this complex at AUG1. Stabilization of a stem-loop between the initiation triplets induces a small decrease in the toeprint intensity at AUG2, accompanied by an increase in the AUG1/AUG2 ratio as well as a moderate reduction of protein synthesis initiated at AUG2 in transfected cells. PTB and ITAF45 exerted an additive positive effect on the 48S complex at AUG2, although a substantial reconstitution on both AUGs occurs on omission of either of these proteins. Relative to the beta-globin mRNA, the 48S complex formation at AUG1 and AUG2 is slow and occurs with the same kinetics as revealed by the "kinetic" toeprint assay. Mutation of AUG1 to AUA does not abrogate protein synthesis in transfected cells, and has no effect on the rate of the 48S complex formation at AUG2. We conclude that the AUG2 initiation region is selected independently of 48S complex formation at the upstream AUG1. The kinetic toeprint assay also shows that cap-dependent assembly of the 48S complex in vitro occurs faster than the FMDV IRES-mediated complex assembly.
-
6.06Impact points
Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated.
Molecular and cellular biology. 08/2007; 27(13):4685-97.
Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5' untranslated region (5'UTR) directs synthes... [more] Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5' untranslated region (5'UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5'UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5'UTR-Fluc) or bicistronic (Rluc-L1 5'UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5'UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5'UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5'UTR. Nevertheless, this cap-dependent initiation activity of the L1 5'UTR was unexpectedly high and resembles that of the beta-actin 5'UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5'UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5'UTRs and call into question the conception that every long GC-rich 5'UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.
-
6.06Impact points
A leaderless mRNA can bind to mammalian 80S ribosomes and direct polypeptide synthesis in the absence of translation initiation factors.
Molecular and cellular biology. 05/2006; 26(8):3164-9.
Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes th... [more] Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis. We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred. The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such an mRNA was supplied with a 5' leader, irrespective of whether it was in a totally single-stranded conformation or was prone to base pairing. The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial 70S ribosomes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents a relic from early steps in the evolution of the translation apparatus.
-
6.06Impact points
A cross-kingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry.
Molecular and cellular biology. 10/2005; 25(17):7879-88.
Rhopalosiphum padi virus (RhPV) is an insect virus of the Dicistroviridae family. Recently, the 579-nucleotide-long 5' untranslated region (UTR) of RhPV has been shown to contain an internal ribosome entry site (IRES) that functions efficiently in mammalian, plant, and insect in vitro translatio... [more] Rhopalosiphum padi virus (RhPV) is an insect virus of the Dicistroviridae family. Recently, the 579-nucleotide-long 5' untranslated region (UTR) of RhPV has been shown to contain an internal ribosome entry site (IRES) that functions efficiently in mammalian, plant, and insect in vitro translation systems. Here, the mechanism of action of the RhPV IRES has been characterized by reconstitution of mammalian 48S initiation complexes on the IRES from purified components combined with the toeprint assay. There is an absolute requirement for the initiation factors eIF2 and eIF3 and the scanning factor eIF1 to form 48S complexes on the IRES. In addition, eIF1A, eIF4F (or the C-terminal fragment of eIF4G), and eIF4A strongly stimulated the assembly of this complex, whereas eIF4B had no effect. Although the eIF4-dependent pathway is dominant in the RhPV IRES-directed cell-free translation, omission of either eIF4G or eIF4A or both still allowed the assembly of 48S complexes from purified components with approximately 23% of maximum efficiency. Deletions of up to 100 nucleotides throughout the 5'-UTR sequence produced at most a marginal effect on the IRES activity, suggesting the absence of specific binding sites for initiation factors. Only deletion of the U-rich unstructured 380-nucleotide region proximal to the initiation codon resulted in a complete loss of the IRES activity. We suggest that the single-stranded nature of the RhPV IRES accounts for its strong but less selective potential to bind key mRNA recruiting components of the translation initiation apparatus from diverse origins.
-
6.06Impact points
Assembly of 48S translation initiation complexes from purified components with mRNAs that have some base pairing within their 5' untranslated regions.
Molecular and cellular biology. 12/2003; 23(24):8925-33.
The reconstitution of translation initiation complexes from purified components is a reliable approach to determine the complete set of essential canonical initiation factors and auxiliary proteins required for the 40S ribosomal subunit to locate the initiation codon on individual mRNAs. Until now, ... [more] The reconstitution of translation initiation complexes from purified components is a reliable approach to determine the complete set of essential canonical initiation factors and auxiliary proteins required for the 40S ribosomal subunit to locate the initiation codon on individual mRNAs. Until now, it has been successful mostly for formation of 48S translation initiation complexes with viral IRES elements. Among cap-dependent mRNAs, only globin mRNAs and transcripts with artificial 5' leaders were amenable to this assembly. Here, with modified conditions for the reconstitution, 48S complexes have been successfully assembled with the 5' UTR of beta-actin mRNA (84 nucleotides) and the tripartite leader of adenovirus RNAs (232 nucleotides), though the latter has been able to use only the scanning rather then the shunting model of translation initiation with canonical initiation factors. We show that initiation factor 4B is essential for mRNAs that have even a rather moderate base pairing within their 5' UTRs (with the cumulative stability of the secondary structure within the entire 5' UTR < -13 kcal/mol) and not essential for beta-globin mRNA. A recombinant eIF4B poorly substitutes for the native factor. The 5' UTRs with base-paired G residues reveal a very sharp dependence on the eIF4B concentration to form the 48S complex. The data suggest that even small variations in concentration or activity of eIF4B in mammalian cells may differentially affect the translation of different classes of cap-dependent cellular mRNAs.
-
5.33Impact points
Distinctive properties of the 5'-untranslated region of human hsp70 mRNA.
The Journal of biological chemistry. 06/2003; 278(25):22350-6.
A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5'-untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dic... [more] A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5'-untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5'-UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES (100-fold over the empty vector) is similar to that of the classical picornaviral IRESs. Additional controls indicate that this high expression of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The effect of small deletions within the 5'-UTR of Hsp70 mRNA on the IRES activity varies in dependence on their position within the 5'-UTR sequence. With the exception of deletion of nt 33-50, it is small for the 5'-terminal half of the 5'-UTR and rather strong for the 3'-terminal section. However, neither of these small deletions abolishes the IRES activity completely. Excision of larger sections (>50 nt) by truncation of the 5'-UTR from the 5'-end or by internal deleting results in a dramatic impairment of the IRES function. Taken together, these data suggest that the IRES activity of the 5'-UTR of Hsp70 mRNA requires integrity of almost the entire sequence of the 5'-UTR. The data are discussed in terms of a model that allows a three-dimensional rather than linear mode of selection of the initiation region surrounding the start codon of Hsp70 mRNA.
-
3.54Impact points
Conversion of 48S translation preinitiation complexes into 80S initiation complexes as revealed by toeprinting.
FEBS letters. 02/2003; 533(1-3):99-104.
A method of analysis of translation initiation complexes by toeprinting has recently acquired a wide application to investigate molecular mechanisms of translation initiation in eukaryotes. So far, this very fruitful approach was used when researchers did not aim to discriminate between patterns of ... [more] A method of analysis of translation initiation complexes by toeprinting has recently acquired a wide application to investigate molecular mechanisms of translation initiation in eukaryotes. So far, this very fruitful approach was used when researchers did not aim to discriminate between patterns of toeprints for 48S and 80S translation initiation complexes. Here, using cap-dependent and internal ribosomal entry site (IRES)-dependent mRNAs, we show that the toeprint patterns for 48S and 80S complexes are distinct whether the complexes are assembled in rabbit reticulocyte lysate or from fully purified individual components. This observation allowed us to demonstrate for the first time a delay in the conversion of the 48S complex into the 80S complex for beta-globin and encephalomyocarditis virus (EMCV) RNAs, and to assess the potential of some 80S antibiotics to block polypeptide elongation. Besides, additional selection of the authentic initiation codon among three consecutive AUGs that follow the EMCV IRES was revealed at steps subsequent to the location of the initiation codon by the 40S ribosomal subunit.
-
[Analysis of Suffix--short Drosophila retroelement--in the heterochromatin region]
Doklady Akademii nauk / [Rossiĭskaia akademii nauk]. 06/1998; 360(1):124-7.
-
Eukaryotic translation initiation machinery can operate in a prokaryotic-like mode without eIF2
Nature Precedings.
Unlike prokaryotes, a specialized eukaryotic initiation factor 2 (eIF2), in the form of the ternary complex eIF2*GTP*Met-tRNAiMet is utilized to deliver the initiator tRNA to the ribosome within all eukaryotic cells1. Phosphorylation of eIF2 is known to be central to the global regulation of protein... [more] Unlike prokaryotes, a specialized eukaryotic initiation factor 2 (eIF2), in the form of the ternary complex eIF2*GTP*Met-tRNAiMet is utilized to deliver the initiator tRNA to the ribosome within all eukaryotic cells1. Phosphorylation of eIF2 is known to be central to the global regulation of protein synthesis under stress conditions and infection2. Another distinctive feature of eukaryotic translation is scanning of mRNA 5'-leaders, whose origin in evolution may be relevant to the appearance of eIF2 in eukaryotes. Translation initiation on the hepatitis C virus (HCV) internal ribosome entry site (IRES) occurs without scanning3,4. Whether these unique features of the HCV IRES account for the formation of the final 80S initiation complex is unknown. Here we show that the HCV IRES-directed translation can occur without either eIF2 or its GTPase activating protein eIF5. In addition to the general eIF2- and eIF5-dependent pathway of 80S complex assembly, the HCV IRES makes use of a prokaryotic-like pathway which involves eIF5B, the analogue of bacterial IF25,6, instead of eIF2. This switch from a eukaryotic-like mode of AUG selection to a "bacterial" one occurs when eIF2 is inactivated by phosphorylation, a way with which host cells counteract infection. The relative resistance of HCV IRES-directed translation to eIF2 phosphorylation may represent one more line of defense used by this virus against host antiviral responses and can contribute to the well known resistance of HCV to interferon based therapy.
-
[Adequate system for investigation of translation initiation of the human retrotransposon L1 mRNA in vitro]
Molekuliarnaia biologiia. 40(1):25-30.
Retrotransposon L1 codes for a unique dicistronic mRNA which serves both a transposition intermediate and a template for the synthesis of two proteins of this mobile element. According to preliminary data, the translation initiation of both cistrons of L1 occurs by non-canonical mechanisms. When tra... [more] Retrotransposon L1 codes for a unique dicistronic mRNA which serves both a transposition intermediate and a template for the synthesis of two proteins of this mobile element. According to preliminary data, the translation initiation of both cistrons of L1 occurs by non-canonical mechanisms. When translating the L1 mRNA in rabbit reticulocyte lysate (RRL), a standard system routinely used by many researchers to study mechanisms of translation initiation in eukaryotes, we observed along with expected products a number of polypeptides resulted from aberrant initiation at internal AUG codons. Such products are absent on translation of L1 mRNA in vivo. Addition to the system of a cytoplasmic extract from HeLa cells resulted in disappearance of these abberant products whereas the efficiency of translation of the first cistron remained unchanged. The level of translation of the second cistron became significantly lower. This also made the picture closer to that observed in vivo. These and other experiments allowed us to clearly demonstrate that the new combined cell-free system is much more adequate to study mechanisms of translation initiation than a regular RRL.
Following (8)
-
Alexander E Ivliev
Moscow State University -
Evgeniy Galimov
Moscow State University -
Ksenia Fominykh
Moscow State University -
Daria Ivanova
Moscow State University -
Alexandra Ryazanova
Moscow State University
Topics (6)
