A New Splice Variant of the Mouse SIRT3 Gene Encodes the Mitochondrial Precursor Protein
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Mendeley | Further Information

{"title"=>"A New Splice Variant of the Mouse SIRT3 Gene Encodes the Mitochondrial Precursor Protein", "type"=>"journal", "authors"=>[{"first_name"=>"Helen M.", "last_name"=>"Cooper"}, {"first_name"=>"Jing-Yi", "last_name"=>"Huang"}, {"first_name"=>"Eric", "last_name"=>"Verdin"}, {"first_name"=>"Johannes N.", "last_name"=>"Spelbrink"}], "year"=>2009, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"1932-6203", "doi"=>"10.1371/journal.pone.0004986", "isbn"=>"1932-6203 (Electronic)", "pmid"=>"19333382"}, "id"=>"bb649bee-a6ba-3a46-8bae-ca0bc3f41755", "abstract"=>"BACKGROUND: Mammals have seven NAD-dependent protein deacetylases. These proteins, called sirtuins, are homologous to yeast Sir2, and are emerging as important regulators of lifespan and intermediary metabolism. Three mammalian sirtuins, SIRT3-5 are mitochondrial. Sirtuins are highly conserved between species, yet mouse SIRT3 was reported to be markedly shorter than its human counterpart and to lack the N-terminal mitochondrial targeting signal present in the human protein. RESULTS: We have isolated a novel mouse SIRT3 splice variant. This cDNA contains two translation initiation codons upstream of the originally reported start site. We show, using immunofluorescence and protein expression analysis that these longer variants are expressed and efficiently targeted to mitochondria, and that the processed forms of these longer variants are identical in size to the endogenous mouse SIRT3. We also show that the previously described form of SIRT3 is not mitochondrial. CONCLUSIONS: Our observations point to a high level of conservation of SIRT3 as a mitochondrial protein in mice and human and indicate that several previous studies, which addressed mouse Sirt3 function, need to be re-evaluated.", "link"=>"http://www.mendeley.com/research/new-splice-variant-mouse-sirt3-gene-encodes-mitochondrial-precursor-protein", "reader_count"=>35, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>7, "Researcher"=>8, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>13, "Student > Postgraduate"=>1, "Student > Master"=>1, "Student > Bachelor"=>3}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>7, "Researcher"=>8, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>13, "Student > Postgraduate"=>1, "Student > Master"=>1, "Student > Bachelor"=>3}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>4, "Agricultural and Biological Sciences"=>25, "Medicine and Dentistry"=>3, "Neuroscience"=>2, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Neuroscience"=>{"Neuroscience"=>2}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>25}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>4}}, "reader_count_by_country"=>{"United States"=>4, "Finland"=>1, "France"=>1, "Portugal"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/446517"], "description"=>"<div><h3>Background</h3><p>Mammals have seven NAD-dependent protein deacetylases. These proteins, called sirtuins, are homologous to yeast Sir2, and are emerging as important regulators of lifespan and intermediary metabolism. Three mammalian sirtuins, SIRT3-5 are mitochondrial. Sirtuins are highly conserved between species, yet mouse SIRT3 was reported to be markedly shorter than its human counterpart and to lack the N-terminal mitochondrial targeting signal present in the human protein.</p><h3>Results</h3><p>We have isolated a novel mouse SIRT3 splice variant. This cDNA contains two translation initiation codons upstream of the originally reported start site. We show, using immunofluorescence and protein expression analysis that these longer variants are expressed and efficiently targeted to mitochondria, and that the processed forms of these longer variants are identical in size to the endogenous mouse SIRT3. We also show that the previously described form of SIRT3 is not mitochondrial.</p><h3>Conclusions</h3><p>Our observations point to a high level of conservation of SIRT3 as a mitochondrial protein in mice and human and indicate that several previous studies, which addressed mouse Sirt3 function, need to be re-evaluated.</p></div>", "links"=>[], "tags"=>["splice", "variant", "sirt3", "encodes", "mitochondrial", "precursor"], "article_id"=>148024, "categories"=>["Cell Biology", "Molecular Biology", "Genetics"], "users"=>["Helen M. Cooper", "Jing-Yi Huang", "Eric Verdin", "Johannes N. Spelbrink"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0004986", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/A_New_Splice_Variant_of_the_Mouse_SIRT3_Gene_Encodes_the_Mitochondrial_Precursor_Protein/148024", "title"=>"A New Splice Variant of the Mouse SIRT3 Gene Encodes the Mitochondrial Precursor Protein", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2009-03-31 02:13:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/902937"], "description"=>"<p>Met1 (M1) and Met15 (M2) are in exon 1B (E1B) (nomenclature according to Yang et al., <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004986#pone.0004986-Yang1\" target=\"_blank\">[12]</a>), Met78 (M3, equivalent to M143 in hSIRT3) in exon 2 (E2). M1 and M2 were cloned from cDNA derived from mouse NIH3T3 cells using exon 1B specific forward primers (1F and 2F in picture) and a reverse primer that included the sequence for the FLAG epitope-tag, binding at exon 7. Similarly, the second splice variant that can only result in an mSIRT3 protein when translation initiates at the third ATG (for M3) was obtained using the 1F and reverse oligo. Predicted proteins M1, M2 and M3 are indicated with a wave-line.</p>", "links"=>[], "tags"=>["coding", "msirt3", "splice"], "article_id"=>573390, "categories"=>["Cell Biology", "Molecular Biology", "Genetics"], "users"=>["Helen M. Cooper", "Jing-Yi Huang", "Eric Verdin", "Johannes N. Spelbrink"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0004986.g001", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schematic_representation_of_the_coding_sequence_of_mSIRT3_splice_variants_/573390", "title"=>"Schematic representation of the coding sequence of mSIRT3 splice variants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-03-31 00:56:30"}
  • {"files"=>["https://ndownloader.figshare.com/files/903233"], "description"=>"<p>FLAG-tagged mSIRT3 variants M1, M2 and M3 were expressed in 3T3 cells (A) and primary mouse hepatocytes from both wild type and ko-mice (B) while both FLAG-tagged M1–3 and untagged M1 and M2 were expressed in HEK293T cells (C). Crude mitochondrial lysates (3T3 cells, panel A) or total cell lysates (mouse hepatocytes and HEK293T cells, panels B and C) were analysed by western blot with an anti-FLAG antibody and an antibody against mSIRT3 as indicated in the various panels. In an attempt to boost expression, all three variants were also analyzed in 3T3 cells only, using constructs into which we introduced a consensus Kozak sequence, 5′CCACC (using PCR cloning), immediately upstream the respective ATG for M1–3 (Left part of panel A). A mouse liver extract was run alongside the HEK293T derived samples (panel C) to denote the size of endogenous mSIRT3 (indicated by →‘endogenous’). Similarly, non-transfected wild-type (wt) and mSirt3 knock-out (ko) mouse hepatocytes were run along transfected samples to clearly detect the endogenous protein (panel B, →‘endogenous’). In all cases both M1 and M2 constructs yielded an un-processed precursor (indicated by →M1 and M2 for the FLAG tagged protein, and →M1* and M2* for the untagged variants) and a processed mature protein (indicated by→proM1–2, and →proM1*–2*), the latter corresponding in size to the endogenous mSIRT3 (panel C). Translation of M2 but not M1 or M3, was greatly improved with the consensus Kozak sequence (panel A). M2 was processed more efficiently than M1 (all three panels). M2 processing may even account for all of the processed over-expressed protein in M1 lanes, since the M1 construct was also transcribed to yield a band the size of M2 in all cell types (all panels). In each panel we have emphasized detected proteins using small dots right from the appropriate bands. Over-expressed M3 is shorter than endogenous mSIRT3 and was undetected or only weakly detected in 3T3 total cell lysates (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004986#pone.0004986.s001\" target=\"_blank\">Figure S1</a> and not shown) or 3T3 mitochondrial lysates (panel A and not shown), but could clearly be detected in mouse hepatocytes and HEK293 cells (panels B and C, indicated by →M3). Membranes for total and mitochondrial 3T3 lysates were also analysed for a control cytoplasmic and mitochondrial protein in particular to illustrate the quality of the mitochondrial fractions (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004986#pone.0004986.s001\" target=\"_blank\">Figure S1</a>).</p>", "links"=>[], "tags"=>["variants", "m1", "m2"], "article_id"=>573690, "categories"=>["Cell Biology", "Molecular Biology", "Genetics"], "users"=>["Helen M. Cooper", "Jing-Yi Huang", "Eric Verdin", "Johannes N. Spelbrink"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0004986.g003", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Over_expressed_variants_M1_and_M2_appear_in_two_forms_/573690", "title"=>"Over-expressed variants M1 and M2 appear in two forms.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-03-31 01:01:30"}
  • {"files"=>["https://ndownloader.figshare.com/files/903076"], "description"=>"<p>Three FLAG-tagged mSIRT3 variants M1, starting at Met1 (A), M2, starting at Met15 (B) and M3, starting at Met78 (C), were over-expressed in mouse 3T3 cells. Two days after transfection, proteins were detected by immunofluorescence with an anti-FLAG antibody. The results show that M1 and M2 give a typically mitochondrial localization pattern, upper panel (confirmed by Mitotraker® Red staining, lower panel). M3, on the other hand is more uniformly distributed in the cytoplasm and does not show mitochondrial localization.</p>", "links"=>[], "tags"=>["sirt3", "variants", "m1", "m2", "mitochondrial", "m3"], "article_id"=>573530, "categories"=>["Cell Biology", "Molecular Biology", "Genetics"], "users"=>["Helen M. Cooper", "Jing-Yi Huang", "Eric Verdin", "Johannes N. Spelbrink"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0004986.g002", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Mouse_SIRT3_variants_M1_and_M2_show_mitochondrial_localization_whereas_M3_does_not_/573530", "title"=>"Mouse SIRT3 variants M1 and M2 show mitochondrial localization, whereas M3 does not.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-03-31 00:58:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/903341"], "description"=>"<p>Full length (fl) MycHis (Myc) and FLAG tagged versions of hSIRT3, or a truncated MycHis tagged version starting at Met143 (M3 in mouse) were expressed in human HEK293 EBNA cells and crude mitochondrial fractions were prepared for Western blot analysis using an hSIRT3 antibody. The results show that the truncated version is only weakly detected in the mitochondrial fraction in agreement with its mostly cytoplasmic localization <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004986#pone.0004986-Cooper1\" target=\"_blank\">[11]</a>. Over-expression of the full length proteins shows the clear presence of both the precursor and mature forms of hSIRT3.</p>", "links"=>[], "tags"=>["sirt3", "corresponds"], "article_id"=>573801, "categories"=>["Cell Biology", "Molecular Biology", "Genetics"], "users"=>["Helen M. Cooper", "Jing-Yi Huang", "Eric Verdin", "Johannes N. Spelbrink"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0004986.g004", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Human_SIRT3_corresponds_in_its_expression_pattern_to_mSIRT3_/573801", "title"=>"Human SIRT3 corresponds in its expression pattern to mSIRT3.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-03-31 01:03:21"}

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  • {"unique-ip"=>"12", "full-text"=>"8", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2014", "month"=>"9"}
  • {"unique-ip"=>"16", "full-text"=>"18", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"1", "cited-by"=>"1", "year"=>"2014", "month"=>"10"}
  • {"unique-ip"=>"4", "full-text"=>"4", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"1"}
  • {"unique-ip"=>"7", "full-text"=>"4", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"11"}
  • {"unique-ip"=>"5", "full-text"=>"6", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"12"}
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  • {"unique-ip"=>"5", "full-text"=>"4", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"1", "year"=>"2015", "month"=>"11"}
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  • {"unique-ip"=>"5", "full-text"=>"4", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"3"}
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  • {"unique-ip"=>"5", "full-text"=>"4", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"8", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"6"}
  • {"unique-ip"=>"1", "full-text"=>"1", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"7"}
  • {"unique-ip"=>"5", "full-text"=>"4", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"9"}
  • {"unique-ip"=>"6", "full-text"=>"7", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"10"}
  • {"unique-ip"=>"4", "full-text"=>"4", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"12"}
  • {"unique-ip"=>"8", "full-text"=>"8", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"1"}
  • {"unique-ip"=>"2", "full-text"=>"2", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2017", "month"=>"2"}
  • {"unique-ip"=>"4", "full-text"=>"4", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"3"}
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  • {"unique-ip"=>"7", "full-text"=>"6", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"5"}
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  • {"unique-ip"=>"3", "full-text"=>"2", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2017", "month"=>"9"}
  • {"unique-ip"=>"6", "full-text"=>"6", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"10"}
  • {"unique-ip"=>"4", "full-text"=>"3", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"11"}
  • {"unique-ip"=>"2", "full-text"=>"2", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"12"}
  • {"unique-ip"=>"4", "full-text"=>"4", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2018", "month"=>"1"}
  • {"unique-ip"=>"1", "full-text"=>"3", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"1", "year"=>"2018", "month"=>"2"}
  • {"unique-ip"=>"7", "full-text"=>"6", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"3"}
  • {"unique-ip"=>"5", "full-text"=>"4", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"1"}
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  • {"unique-ip"=>"3", "full-text"=>"3", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"4"}
  • {"unique-ip"=>"5", "full-text"=>"5", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"6"}
  • {"unique-ip"=>"4", "full-text"=>"4", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"7"}
  • {"unique-ip"=>"4", "full-text"=>"4", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"8"}
  • {"unique-ip"=>"6", "full-text"=>"7", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"10"}
  • {"unique-ip"=>"6", "full-text"=>"4", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2018", "month"=>"11"}
  • {"unique-ip"=>"4", "full-text"=>"5", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2018", "month"=>"9"}
  • {"unique-ip"=>"10", "full-text"=>"9", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"12"}
  • {"unique-ip"=>"10", "full-text"=>"12", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"2"}
  • {"unique-ip"=>"3", "full-text"=>"3", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"3"}
  • {"unique-ip"=>"3", "full-text"=>"3", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}
  • {"unique-ip"=>"8", "full-text"=>"9", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"5"}
  • {"unique-ip"=>"2", "full-text"=>"2", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"8"}
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Relative Metric

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