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Peter Liljeström - One of the best experts on this subject based on the ideXlab platform.
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Expression of proteins using Semliki Forest virus vectors.
Current protocols in molecular biology, 2001Co-Authors: Peter Liljeström, Henrik GaroffAbstract:Semliki Forest virus (SFV) vectors have been developed to provide a convenient system to express protein-encoding sequences in virtually any animal cell. This unit presents two strategies for protein expression using SFV vectors. In both cases the protein-coding sequence of interest is cloned into a plasmid vector, which is subsequently used to produce Recombinant RNA in vitro. This RNA, which is of positive polarity, is transfected into cells and there is amplified by virtue of its self-encoded RNA replicase. The same replicase also produces a shorter RNA species that encodes the protein of interest. In the first protocol, cells are transfected (either by electroporation or liposome-mediated transfection) and directly analyzed for expression of the heterologous protein. Accompanying support protocols provide methods for checking expression and transfection through galactosidase assays of transfected cells and cell lysates. The other strategy employs in vivo packaging of the RNA into SFV particles; Recombinant RNA is cotransfected with a special helper RNA that codes for the structural proteins needed for virus assembly. SFV particles carrying only Recombinant RNA are formed and are used to infect cells for analysis of protein expression. Accompanying support protocols describe methods for titrating and purifying Recombinant virus stocks. Although the protocols presented here are designed for use with BHK (baby hamster kidney) cells, the virus has a very broad host range and can be used with many different cell types.
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cloning of human il 12 p40 and p35 dna into the semliki forest virus vector expression of il 12 in human tumor cells
Gene Therapy, 1997Co-Authors: J Zhang, Peter Liljeström, Peter Berglund, C Asselinpaturel, Francoise Bex, Jacky Bernard, Jihed Chehimi, Fabienne Willems, Anne Caignard, Arsene BurnyAbstract:IL-12 can enhance the development of effective immune responses against tumors as well as against certain infectious agents. It is therefore a potential candidate for therapeutic use in cancer therapy and in the design of vaccines against several infectious diseases. Several studies have demonstrated that IL-12 could efficiently induce tumor regression in animal models. To investigate the antitumor effect of direct gene transfer of human IL-12 into tumors, human IL-12 p35 and p40 cDNAs were cloned into the Semliki Forest virus (SFV) vector pSFV1. In order to express the two subunits from the same vector, the p35 and the p40 cDNAs were cloned into pSFV1, each under the control of a subgenomic SFV promoter. Recombinant RNA produced by in vitro transcription of SFV-IL-12 construct, was packaged into SFV viral particles with the use of a non-packageable helper RNA. We show that human tumor cell lines infected in vitro in vivo with Recombinant SFV-IL-12 viral particles secrete high levels of biologically active heterodimeric p35/p40 IL-12, as demonstrated using ELISA and biological assays.
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Current Protocols in Molecular Biology - Expression of Proteins Using Semliki Forest Virus Vectors
Current Protocols in Molecular Biology, 1995Co-Authors: Peter Liljeström, Henrik GaroffAbstract:Semliki Forest virus (SFV) vectors have been developed to provide a convenient system to express protein-encoding sequences in virtually any animal cell. This unit presents two strategies for protein expression using SFV vectors. In both cases the protein-coding sequence of interest is cloned into a plasmid vector, which is subsequently used to produce Recombinant RNA in vitro. This RNA, which is of positive polarity, is transfected into cells and there is amplified by virtue of its self-encoded RNA replicase. The same replicase also produces a shorter RNA species that encodes the protein of interest. In the first protocol, cells are transfected (either by electroporation or liposome-mediated transfection) and directly analyzed for expression of the heterologous protein. Accompanying support protocols provide methods for checking expression and transfection through galactosidase assays of transfected cells and cell lysates. The other strategy employs in vivo packaging of the RNA into SFV particles; Recombinant RNA is cotransfected with a special helper RNA that codes for the structural proteins needed for virus assembly. SFV particles carrying only Recombinant RNA are formed and are used to infect cells for analysis of protein expression. Accompanying support protocols describe methods for titrating and purifying Recombinant virus stocks. Although the protocols presented here are designed for use with BHK (baby hamster kidney) cells, the virus has a very broad host range and can be used with many different cell types.
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self replicating semliki forest virus RNA as Recombinant vaccine
Vaccine, 1994Co-Authors: Xianzheng Zhou, Peter Berglund, Gary Rhodes, S E Parker, Mikael Jondal, Peter LiljeströmAbstract:Recombinant RNA based on the Semliki Forest virus (SFV) replicon was used to express the nucleoprotein of influenza virus in mice. Two strategies were employed to deliver the RNA. In the first, Recombinant RNA was packaged into infectious suicide SFV particles which were used directly for immunization. The second approach involved injection of in vitro-synthesized RNA directly into the quadriceps muscle. Both approaches resulted in the generation of humoral responses with high antibody titres. Immunization with suicide particles showed that a strong, class I-restricted cytotoxic T-cell response can be obtained using only 100 infectious units. We conclude that the self-replicative Recombinant SFV RNA may be quite useful as a nucleic acid vaccine.
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semliki forest virus expression system production of conditionally infectious Recombinant particles
Nature Biotechnology, 1993Co-Authors: Peter Berglund, Henrik Garoff, Mathilda Sjoberg, G J Atkins, B J Sheahan, Peter LiljeströmAbstract:In the recently developed Semliki Forest virus (SFV) DNA expression system, Recombinant RNA encoding the viral replicase, and helper RNA molecules encoding the structural proteins needed for virus assembly are cotransfected into cells. Since the helper RNA lacks the sequence needed for its packaging into nucleocapsids, only Recombinant RNAs should be packaged. We have found, however, that small amounts of replication-proficient SFV particles can still be produced. Here we describe the construction of a helper variant with a mutation in the gene encoding the viral spike protein such that its product cannot undergo normal proteolytic processing to activate viral entry functions. Hence, the Recombinant stock is noninfectious, but may be activated by cleavage with chymotrypsin. When Recombinant virus produced with the new helper was examined in a variety of assays, including sensitive animal tests, we were unable to detect any replication-competent SFV particles. We therefore conclude that this conditional expression system meets extremely stringent biosafety requirements.
Henrik Garoff - One of the best experts on this subject based on the ideXlab platform.
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eLS - Semliki Forest Virus Expression System
eLS, 2014Co-Authors: Maria Ekström, Henrik Garoff, Helena AnderssonAbstract:Publisher Summary The Semliki forest virus (SFV) is a positive-stranded RNA virus belonging to the alphaviruses. The SFV expression system is based on a cDNA copy of the viral genome. The cDNA has been cloned into an SP6-based transcription vector in such a way that exact copies of the alphavirus RNA genome can be transcribed in vitro. The Recombinant plasmid then serves as a template for in vitro synthesis of Recombinant RNA. When introduced into cells, the Recombinant RNA self-replicates, as it codes for its own replicase, leading to high synthesis of the heterologous protein while competing out the host protein synthesis. In the SFVC vector, the viral capsid gene is retained and the hierologist gene is cloned directly downstream of the capsid gene by polymerase chain reaction (PCR). One needs to avoid repeated freezing and thawing of the virus stock. This will reduce virus infectivity. For use in electroporation, the cells should not be passaged more than about 30 times.
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Expression of proteins using Semliki Forest virus vectors.
Current protocols in molecular biology, 2001Co-Authors: Peter Liljeström, Henrik GaroffAbstract:Semliki Forest virus (SFV) vectors have been developed to provide a convenient system to express protein-encoding sequences in virtually any animal cell. This unit presents two strategies for protein expression using SFV vectors. In both cases the protein-coding sequence of interest is cloned into a plasmid vector, which is subsequently used to produce Recombinant RNA in vitro. This RNA, which is of positive polarity, is transfected into cells and there is amplified by virtue of its self-encoded RNA replicase. The same replicase also produces a shorter RNA species that encodes the protein of interest. In the first protocol, cells are transfected (either by electroporation or liposome-mediated transfection) and directly analyzed for expression of the heterologous protein. Accompanying support protocols provide methods for checking expression and transfection through galactosidase assays of transfected cells and cell lysates. The other strategy employs in vivo packaging of the RNA into SFV particles; Recombinant RNA is cotransfected with a special helper RNA that codes for the structural proteins needed for virus assembly. SFV particles carrying only Recombinant RNA are formed and are used to infect cells for analysis of protein expression. Accompanying support protocols describe methods for titrating and purifying Recombinant virus stocks. Although the protocols presented here are designed for use with BHK (baby hamster kidney) cells, the virus has a very broad host range and can be used with many different cell types.
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Current Protocols in Molecular Biology - Expression of Proteins Using Semliki Forest Virus Vectors
Current Protocols in Molecular Biology, 1995Co-Authors: Peter Liljeström, Henrik GaroffAbstract:Semliki Forest virus (SFV) vectors have been developed to provide a convenient system to express protein-encoding sequences in virtually any animal cell. This unit presents two strategies for protein expression using SFV vectors. In both cases the protein-coding sequence of interest is cloned into a plasmid vector, which is subsequently used to produce Recombinant RNA in vitro. This RNA, which is of positive polarity, is transfected into cells and there is amplified by virtue of its self-encoded RNA replicase. The same replicase also produces a shorter RNA species that encodes the protein of interest. In the first protocol, cells are transfected (either by electroporation or liposome-mediated transfection) and directly analyzed for expression of the heterologous protein. Accompanying support protocols provide methods for checking expression and transfection through galactosidase assays of transfected cells and cell lysates. The other strategy employs in vivo packaging of the RNA into SFV particles; Recombinant RNA is cotransfected with a special helper RNA that codes for the structural proteins needed for virus assembly. SFV particles carrying only Recombinant RNA are formed and are used to infect cells for analysis of protein expression. Accompanying support protocols describe methods for titrating and purifying Recombinant virus stocks. Although the protocols presented here are designed for use with BHK (baby hamster kidney) cells, the virus has a very broad host range and can be used with many different cell types.
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semliki forest virus expression system production of conditionally infectious Recombinant particles
Nature Biotechnology, 1993Co-Authors: Peter Berglund, Henrik Garoff, Mathilda Sjoberg, G J Atkins, B J Sheahan, Peter LiljeströmAbstract:In the recently developed Semliki Forest virus (SFV) DNA expression system, Recombinant RNA encoding the viral replicase, and helper RNA molecules encoding the structural proteins needed for virus assembly are cotransfected into cells. Since the helper RNA lacks the sequence needed for its packaging into nucleocapsids, only Recombinant RNAs should be packaged. We have found, however, that small amounts of replication-proficient SFV particles can still be produced. Here we describe the construction of a helper variant with a mutation in the gene encoding the viral spike protein such that its product cannot undergo normal proteolytic processing to activate viral entry functions. Hence, the Recombinant stock is noninfectious, but may be activated by cleavage with chymotrypsin. When Recombinant virus produced with the new helper was examined in a variety of assays, including sensitive animal tests, we were unable to detect any replication-competent SFV particles. We therefore conclude that this conditional expression system meets extremely stringent biosafety requirements.
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a new generation of animal cell expression vectors based on the semliki forest virus replicon
Nature Biotechnology, 1991Co-Authors: Peter Liljeström, Henrik GaroffAbstract:We have developed a novel DNA expression system, based on the Semliki Forest virus (SFV) replicon, which combines a wide choice of animal cell hosts, high efficiency and ease of use. DNA of interest is cloned into SFV plasmid vectors that serve as templates for in vitro synthesis of Recombinant RNA. The RNA is transfected with virtually 100% efficiency into animal tissue culture cells by means of electroporation. Within the cell, the Recombinant RNA drives its own replication and capping and leads to massive production of the heterologous protein while competing out the host protein synthesis. The expression system also includes an in vivo packaging procedure whereby Recombinant RNA is packaged into infectious virus particles using cotransfection with packaging–deficient helper RNA molecules. The resulting high titer Recombinant virus stock can be used to infect a wide range of animal cells with subsequent high expression of the heterologous gene product, but without expression of any structural proteins of the helper. The infected cells produce protein for up to 75 hours post infection after which the heterologous product can constitute as much as 25% of the total cell protein. The general utility of the system is demonstrated through the expression of human transferrin receptor, mouse dihydrofolate reductase, chick lysozyme and Escherichia coli β–galactosidase.
Angel Onate - One of the best experts on this subject based on the ideXlab platform.
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vaccination with Recombinant semliki forest virus particles expressing translation initiation factor 3 of brucella abortus induces protective immunity in balb c mice
Immunobiology, 2009Co-Authors: Alex Cabrera, Sandra Céspedes, Edilia Andrews, Darwin Sáez, Angel OnateAbstract:Recombinant replicons of Semliki Forest virus (SFV) can be used to induce high-level, transient expression of heterologous proteins in vivo. We constructed infectious but replication-deficient SFV particles carrying Recombinant RNA encoding the Brucella abortus translation initiation factor 3 (IF3). The Recombinant SFV particles (SFV-IF3 particles) were then evaluated for their ability to induce immune responses and to protect BALB/c mice against a challenge with B. abortus 2308 following vaccination. Animals inoculated with SFV-IF3 developed IF3-specific IgM antibodies at day 14 post-immunization. In vitro stimulation of splenocytes from vaccinated mice with either Recombinant IF3 (rIF3) or crude Brucella protein extracts resulted in a T-cell proliferative response and induction of interferon gamma secretion, but not interleukin-4. In addition, mice immunized with SFV-IF3 exhibited a significant level of resistance against challenge with the virulent B. abortus strain 2308 (P<0.01). These findings indicate that an SFV-based vector carrying RNA encoding Brucella IF3 has potential for use as a vaccine to induce protection against B. abortus infections.
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Evaluation of Brucella abortus DNA and RNA vaccines expressing Cu–Zn superoxide dismutase (SOD) gene in cattle
Veterinary Microbiology, 2008Co-Authors: Darwin Sáez, Edilia Andrews, Alex Cabrera, Ingrid I. Guzmán, Angel OnateAbstract:Abstract This study was conducted to evaluate the immunogenicity of a DNA or RNA vaccines encoding Brucella abortus Cu–Zn superoxide dismutase (SOD) in cattle. Intramuscular injection of plasmid DNA carrying Brucella SOD gene (pcDNA-SOD) into animals elicited both humoral and cellular immune responses. Animals injected with pcDNA-SOD developed SOD IgG antibody with predominance of immunoglobulin G1 (IgG1) isotype over IgG2. In addition, the DNA vaccine elicited a specific T-cell-proliferative response. Furthermore, intraperitoneal injection of cattle with Recombinant Semliki Forest virus particles carrying Recombinant RNA encoding SOD (SFV-SOD) did not lead to the induction of SOD IgG 1 or 2 antibody, but induced specific T-cell activation. Both vaccines were able to induce a non-significant secretion of gamma interferon and did not induce the secretion of IL-4 or tumor necrosis factor (TNF)-α. These results suggest that SOD gene in a genetic vaccine formulation (DNA or RNA) might be of potential us as a vaccine to induce cell-mediated immunity in cattle. To our knowledge, this is the first study to evaluate a genetic vaccine against Brucella in cattle.
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an RNA vaccine based on Recombinant semliki forest virus particles expressing the cu zn superoxide dismutase protein of brucella abortus induces protective immunity in balb c mice
Infection and Immunity, 2005Co-Authors: Angel Onate, Gustavo Moragacid, Hugo Folch, Sandra Céspedes, Gabriel Donoso, Edilia AndrewsAbstract:We constructed infectious but replication-deficient Semliki Forest virus (SFV) particles carrying Recombinant RNA encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). The Recombinant SFV particles (SFV-SOD particles) were then evaluated for their ability to induce a T-cell immune response and to protect BALB/c mice against a challenge with B. abortus 2308. Intraperitoneal injection of mice with Recombinant SFV-SOD particles did not lead to the induction of SOD-specific antibodies, at least until week 6 after immunization (the end of the experiment). In vitro stimulation of splenocytes from the vaccinated mice with either Recombinant Cu,Zn SOD (rSOD) or crude Brucella protein resulted in a T-cell proliferative response and the induction of gamma interferon secretion but not interleukin-4. In addition, the splenocytes exhibited significant levels of cytotoxic T-lymphocyte activity against Brucella-infected cells. The SFV-SOD particles, but not the control virus particles, induced a significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308. These findings indicated that an SFV-based vector carrying the SOD gene has potential for use as a vaccine to induce resistance against B. abortus infections.
Edilia Andrews - One of the best experts on this subject based on the ideXlab platform.
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vaccination with Recombinant semliki forest virus particles expressing translation initiation factor 3 of brucella abortus induces protective immunity in balb c mice
Immunobiology, 2009Co-Authors: Alex Cabrera, Sandra Céspedes, Edilia Andrews, Darwin Sáez, Angel OnateAbstract:Recombinant replicons of Semliki Forest virus (SFV) can be used to induce high-level, transient expression of heterologous proteins in vivo. We constructed infectious but replication-deficient SFV particles carrying Recombinant RNA encoding the Brucella abortus translation initiation factor 3 (IF3). The Recombinant SFV particles (SFV-IF3 particles) were then evaluated for their ability to induce immune responses and to protect BALB/c mice against a challenge with B. abortus 2308 following vaccination. Animals inoculated with SFV-IF3 developed IF3-specific IgM antibodies at day 14 post-immunization. In vitro stimulation of splenocytes from vaccinated mice with either Recombinant IF3 (rIF3) or crude Brucella protein extracts resulted in a T-cell proliferative response and induction of interferon gamma secretion, but not interleukin-4. In addition, mice immunized with SFV-IF3 exhibited a significant level of resistance against challenge with the virulent B. abortus strain 2308 (P<0.01). These findings indicate that an SFV-based vector carrying RNA encoding Brucella IF3 has potential for use as a vaccine to induce protection against B. abortus infections.
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Evaluation of Brucella abortus DNA and RNA vaccines expressing Cu–Zn superoxide dismutase (SOD) gene in cattle
Veterinary Microbiology, 2008Co-Authors: Darwin Sáez, Edilia Andrews, Alex Cabrera, Ingrid I. Guzmán, Angel OnateAbstract:Abstract This study was conducted to evaluate the immunogenicity of a DNA or RNA vaccines encoding Brucella abortus Cu–Zn superoxide dismutase (SOD) in cattle. Intramuscular injection of plasmid DNA carrying Brucella SOD gene (pcDNA-SOD) into animals elicited both humoral and cellular immune responses. Animals injected with pcDNA-SOD developed SOD IgG antibody with predominance of immunoglobulin G1 (IgG1) isotype over IgG2. In addition, the DNA vaccine elicited a specific T-cell-proliferative response. Furthermore, intraperitoneal injection of cattle with Recombinant Semliki Forest virus particles carrying Recombinant RNA encoding SOD (SFV-SOD) did not lead to the induction of SOD IgG 1 or 2 antibody, but induced specific T-cell activation. Both vaccines were able to induce a non-significant secretion of gamma interferon and did not induce the secretion of IL-4 or tumor necrosis factor (TNF)-α. These results suggest that SOD gene in a genetic vaccine formulation (DNA or RNA) might be of potential us as a vaccine to induce cell-mediated immunity in cattle. To our knowledge, this is the first study to evaluate a genetic vaccine against Brucella in cattle.
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an RNA vaccine based on Recombinant semliki forest virus particles expressing the cu zn superoxide dismutase protein of brucella abortus induces protective immunity in balb c mice
Infection and Immunity, 2005Co-Authors: Angel Onate, Gustavo Moragacid, Hugo Folch, Sandra Céspedes, Gabriel Donoso, Edilia AndrewsAbstract:We constructed infectious but replication-deficient Semliki Forest virus (SFV) particles carrying Recombinant RNA encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). The Recombinant SFV particles (SFV-SOD particles) were then evaluated for their ability to induce a T-cell immune response and to protect BALB/c mice against a challenge with B. abortus 2308. Intraperitoneal injection of mice with Recombinant SFV-SOD particles did not lead to the induction of SOD-specific antibodies, at least until week 6 after immunization (the end of the experiment). In vitro stimulation of splenocytes from the vaccinated mice with either Recombinant Cu,Zn SOD (rSOD) or crude Brucella protein resulted in a T-cell proliferative response and the induction of gamma interferon secretion but not interleukin-4. In addition, the splenocytes exhibited significant levels of cytotoxic T-lymphocyte activity against Brucella-infected cells. The SFV-SOD particles, but not the control virus particles, induced a significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308. These findings indicated that an SFV-based vector carrying the SOD gene has potential for use as a vaccine to induce resistance against B. abortus infections.
Larry Simpson - One of the best experts on this subject based on the ideXlab platform.
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Reconstitution of full-round uridine-deletion RNA editing with three Recombinant proteins.
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Xuedong Kang, Guanghan Gao, Kestrel Rogers, Arnold M. Falick, Sharleen Zhou, Larry SimpsonAbstract:Uridine (U)-insertion/deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNAse III-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosoma brucei and Leishmania major were expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model U-deletion site and not a U-insertion site. Deletion or mutation of the RNAse III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of Recombinant MP90 and Recombinant RNA editing exonuclease I from L. major, and Recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNA-editing nuclease 1.
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Functional complementation of Trypanosoma brucei RNA in vitro editing with Recombinant RNA ligase.
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Guanghan Gao, Xuedong Kang, Kestrel Rogers, Agda M. Simpson, Martina Neboháčová, Larry SimpsonAbstract:The ≈20S RNA ligase-containing complex (L-complex) in trypanosomatid mitochondria interacts by means of RNA linkers with at least two other multiprotein complexes to mediate the editing of mitochondrial cryptogene transcripts. The L-complex contains ≈16 proteins, including the two RNA-editing ligases (RELs), REL1 and REL2. Leishmania tarentolae REL1 and REL2 and Trypanosoma brucei REL1 were expressed as enzymatically active tandem affinity purification-tagged proteins in a Baculovirus system. When these proteins were added to mitochondrial lysates from T. brucei procyclic cells that were depleted of the cognate endogenous ligase by RNA interference down-regulation of expression, the added proteins were integrated into the L-complex, and, in the case of REL1, there was a complementation of in vitro-precleaved U-insertion and U-deletion editing activities of the 20S L-complex. Integration of the Recombinant proteins did not occur or occurred at a very low level with noncognate ligase-depleted L-complex or with wild-type L-complex. A C-terminal region of the T. brucei Recombinant REL1 downstream of the catalytic domain was identified as being involved in integration into the L-complex. The ability to perform functional complementation in vitro provides a powerful tool for molecular dissection of the editing reaction.
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Reconstitution of uridine-deletion precleaved RNA editing with two Recombinant enzymes.
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Xuedong Kang, Guanghan Gao, Kestrel Rogers, Arnold M. Falick, Sharleen Zhou, Larry SimpsonAbstract:Uridine insertion/deletion RNA editing in trypanosomatid mitochondria is a posttranscriptional RNA modification phenomenon required for translation of mitochondrial mRNAs. This process involves guide RNA-mediated cleavage at a specific site, insertion or deletion of Us from the 3′ end of the 5′ mRNA fragment, and ligation of the two mRNA fragments. The Leishmania major RNA ligase-containing complex protein 2 expressed in insect cells has a 3′–5′ exoribonuclease activity and was therefore renamed RNA editing exonuclease 1 (REX1). Recombinant REX1 specifically trims 3′ overhanging Us and stops at a duplex region. Evidence is presented that REX1 is responsible for deletion of the 3′ overhanging Us from the bridged mRNA 5′ cleavage fragment and that RNA editing ligase 1 is responsible for the ligation of the two mRNA cleavage fragments in U-deletion editing. The evidence involves both in vivo down-regulation of REX1 expression in Trypanosoma brucei by RNA interference and the reconstitution of precleaved U-deletion in vitro editing with only two Recombinant enzymes: Recombinant REX1 and Recombinant RNA editing ligase 1.