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Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A.

Andersen PL, Zhou H, Pastushok L, Moraes T, McKenna S, Ziola B, Ellison MJ, Dixit VM, Xiao W - J. Cell Biol. (2005)

Bottom Line: In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2.Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains.Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.

ABSTRACT
Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13-Mms2 is required for DNA damage repair but not nuclear factor kappaB (NF-kappaB) activation, whereas Ubc13-Uev1A is involved in NF-kappaB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

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Spontaneous Rad51 nuclear foci formation after RNAi treatment. (A) Western blot analysis of purified recombinant hMms2 (lane 1), hUbc13 (lane 2), and 3T3 total cell extracts (lanes 3–7) using 4E11 (anti-Ubc13) or 2H11 (anti-hMms2) as primary antibodies. Lanes 4–7 represent cultures transfected with various RNAi constructs as indicated. Note that 2H11 (and other anti-hMms2 mAbs) detected only one band from mammalian cell extracts, which corresponds to the migration of Mms2, but was found to contain both Mms2 and Uev1A by mass spectrometry (not depicted). (B–F) 4 d after transfection of 3T3 cells with the indicated RNAi constructs against specific target genes, cells were stained with either 4E11 (B and C) or 2H11 (D–F) plus Alexa488 to reveal Ubc13 and Uev immunoreactivity, respectively. The same cells were also stained with anti-Rad51 plus Alexa546 to reveal Rad51 nuclear foci and with DAPI to reveal nuclei of all cells. Bar, 10 μm.
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fig5: Spontaneous Rad51 nuclear foci formation after RNAi treatment. (A) Western blot analysis of purified recombinant hMms2 (lane 1), hUbc13 (lane 2), and 3T3 total cell extracts (lanes 3–7) using 4E11 (anti-Ubc13) or 2H11 (anti-hMms2) as primary antibodies. Lanes 4–7 represent cultures transfected with various RNAi constructs as indicated. Note that 2H11 (and other anti-hMms2 mAbs) detected only one band from mammalian cell extracts, which corresponds to the migration of Mms2, but was found to contain both Mms2 and Uev1A by mass spectrometry (not depicted). (B–F) 4 d after transfection of 3T3 cells with the indicated RNAi constructs against specific target genes, cells were stained with either 4E11 (B and C) or 2H11 (D–F) plus Alexa488 to reveal Ubc13 and Uev immunoreactivity, respectively. The same cells were also stained with anti-Rad51 plus Alexa546 to reveal Rad51 nuclear foci and with DAPI to reveal nuclei of all cells. Bar, 10 μm.

Mentions: To further address whether the Ubc13–Mms2 and Ubc13–Uev1A complexes play a role in protecting mammalian cells from genomic instability under normal growth conditions, we attempted to experimentally suppress target gene expression by an RNA interference (RNAi) technology (Yu et al., 2002) and examine its effects on endogenous DNA damage, as measured by spontaneous Rad51 and Mre11 nuclear foci formation. The effects of this method were examined by Western blot analysis using Ubc13 and Mms2/Uev1-specific mAbs. As shown in Fig. 5 A, transfection of mouse 3T3 cells with RNAi against Ubc13 (iUbc13) reduced target Ubc13 to an undetectable level (lane 7) with no effect on Mms2/Uev1A expression. This suppression is highly specific, as a single nucleotide mismatch (iUbc13m) completely abolished the target gene suppression (Fig. 5 A, lane 6). Transfection with iMms2 (Fig. 5 A, lane 4) or iUev1 (lane 5) resulted in partial reduction of 2H11 immunoreactivity; the remaining immunoreactivity is presumably due to cross-reaction of 2H11 to both Mms2 and Uev1. Similar results were also obtained by immunocytochemical analysis (Fig. 5, D–F), which together demonstrate that, like iUbc13, the suppression of target genes by iMms2 and iUev1 was also highly efficient and specific. Experimental ablation of either Ubc13 or Mms2 in 3T3 cells was accompanied by an increased number of cells exhibiting Rad51 nuclear foci from a basal level of ∼8 to 33 and 32%, respectively; the difference is consistent in three independent experiments and statistically significant (P < 0.001; Fig. 5, C and E; and Table I). In contrast, cells transfected with iUbc13m (Fig. 5, B and D) did not affect Rad51 foci formation, suggesting that the induced Rad51 nuclear foci were indeed due to the increased spontaneous DNA damage when Ubc13 or Mms2 activity was compromised. As expected, iUev1 transfection did not result in an increased number of cells containing Rad51 nuclear foci (Fig. 5 F and Table I), suggesting that Uev1 is not involved in DNA damage avoidance. It was noted that within the experimental period, cell viability, doubling time, and the percentage of Mre11-positive nuclei were not altered in any of the aforementioned RNAi transfectants (unpublished data). Thus, we conclude from the aforementioned observations that Ubc13 and Mms2 are required to prevent endogenous DNA damage and double strand break formation, whereas Uev1 is not involved in this process. To our knowledge, this is the first experimental evidence that a mammalian Ubc13–Uev complex protects cells from spontaneous DNA damage.


Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A.

Andersen PL, Zhou H, Pastushok L, Moraes T, McKenna S, Ziola B, Ellison MJ, Dixit VM, Xiao W - J. Cell Biol. (2005)

Spontaneous Rad51 nuclear foci formation after RNAi treatment. (A) Western blot analysis of purified recombinant hMms2 (lane 1), hUbc13 (lane 2), and 3T3 total cell extracts (lanes 3–7) using 4E11 (anti-Ubc13) or 2H11 (anti-hMms2) as primary antibodies. Lanes 4–7 represent cultures transfected with various RNAi constructs as indicated. Note that 2H11 (and other anti-hMms2 mAbs) detected only one band from mammalian cell extracts, which corresponds to the migration of Mms2, but was found to contain both Mms2 and Uev1A by mass spectrometry (not depicted). (B–F) 4 d after transfection of 3T3 cells with the indicated RNAi constructs against specific target genes, cells were stained with either 4E11 (B and C) or 2H11 (D–F) plus Alexa488 to reveal Ubc13 and Uev immunoreactivity, respectively. The same cells were also stained with anti-Rad51 plus Alexa546 to reveal Rad51 nuclear foci and with DAPI to reveal nuclei of all cells. Bar, 10 μm.
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Related In: Results  -  Collection

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fig5: Spontaneous Rad51 nuclear foci formation after RNAi treatment. (A) Western blot analysis of purified recombinant hMms2 (lane 1), hUbc13 (lane 2), and 3T3 total cell extracts (lanes 3–7) using 4E11 (anti-Ubc13) or 2H11 (anti-hMms2) as primary antibodies. Lanes 4–7 represent cultures transfected with various RNAi constructs as indicated. Note that 2H11 (and other anti-hMms2 mAbs) detected only one band from mammalian cell extracts, which corresponds to the migration of Mms2, but was found to contain both Mms2 and Uev1A by mass spectrometry (not depicted). (B–F) 4 d after transfection of 3T3 cells with the indicated RNAi constructs against specific target genes, cells were stained with either 4E11 (B and C) or 2H11 (D–F) plus Alexa488 to reveal Ubc13 and Uev immunoreactivity, respectively. The same cells were also stained with anti-Rad51 plus Alexa546 to reveal Rad51 nuclear foci and with DAPI to reveal nuclei of all cells. Bar, 10 μm.
Mentions: To further address whether the Ubc13–Mms2 and Ubc13–Uev1A complexes play a role in protecting mammalian cells from genomic instability under normal growth conditions, we attempted to experimentally suppress target gene expression by an RNA interference (RNAi) technology (Yu et al., 2002) and examine its effects on endogenous DNA damage, as measured by spontaneous Rad51 and Mre11 nuclear foci formation. The effects of this method were examined by Western blot analysis using Ubc13 and Mms2/Uev1-specific mAbs. As shown in Fig. 5 A, transfection of mouse 3T3 cells with RNAi against Ubc13 (iUbc13) reduced target Ubc13 to an undetectable level (lane 7) with no effect on Mms2/Uev1A expression. This suppression is highly specific, as a single nucleotide mismatch (iUbc13m) completely abolished the target gene suppression (Fig. 5 A, lane 6). Transfection with iMms2 (Fig. 5 A, lane 4) or iUev1 (lane 5) resulted in partial reduction of 2H11 immunoreactivity; the remaining immunoreactivity is presumably due to cross-reaction of 2H11 to both Mms2 and Uev1. Similar results were also obtained by immunocytochemical analysis (Fig. 5, D–F), which together demonstrate that, like iUbc13, the suppression of target genes by iMms2 and iUev1 was also highly efficient and specific. Experimental ablation of either Ubc13 or Mms2 in 3T3 cells was accompanied by an increased number of cells exhibiting Rad51 nuclear foci from a basal level of ∼8 to 33 and 32%, respectively; the difference is consistent in three independent experiments and statistically significant (P < 0.001; Fig. 5, C and E; and Table I). In contrast, cells transfected with iUbc13m (Fig. 5, B and D) did not affect Rad51 foci formation, suggesting that the induced Rad51 nuclear foci were indeed due to the increased spontaneous DNA damage when Ubc13 or Mms2 activity was compromised. As expected, iUev1 transfection did not result in an increased number of cells containing Rad51 nuclear foci (Fig. 5 F and Table I), suggesting that Uev1 is not involved in DNA damage avoidance. It was noted that within the experimental period, cell viability, doubling time, and the percentage of Mre11-positive nuclei were not altered in any of the aforementioned RNAi transfectants (unpublished data). Thus, we conclude from the aforementioned observations that Ubc13 and Mms2 are required to prevent endogenous DNA damage and double strand break formation, whereas Uev1 is not involved in this process. To our knowledge, this is the first experimental evidence that a mammalian Ubc13–Uev complex protects cells from spontaneous DNA damage.

Bottom Line: In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2.Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains.Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.

ABSTRACT
Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13-Mms2 is required for DNA damage repair but not nuclear factor kappaB (NF-kappaB) activation, whereas Ubc13-Uev1A is involved in NF-kappaB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

Show MeSH
Related in: MedlinePlus