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. 2009 Apr 15;23(8):963–974. doi: 10.1101/gad.1775609

Figure 6.

Figure 6.

Ribosomal fractions containing nonfunctional rRNAs are ubiquitinated in a Mms1–Rtt101-dependent manner. (A) Immunoblotting of ribosomal fractions purified from the wild-type strain expressing various tagged rRNAs, pMyc-Ubi, and RPL28-Flag. Ubiquitinated proteins were probed by anti-Myc polyclonal antibody. For lane 5, an empty vector was used instead of pMyc-Ubi. For lane 6, the wild-type strain with untagged RPL28 was used. (B) In addition to the wild-type strain (lanes 7,8), strain mms1Δ (lanes 9,10) and strain rtt101Δ (lanes 11,12) were used. Both deletion strains carry the Flag-tagged L28 gene. (C) The efficiency of immunoprecipitation with anti-Myc antibody for the wild-type strain expressing wild-type tagged rRNAs or A2451U. Efficiency was calculated based on the amounts of tagged and untagged rRNAs measured by qRT–PCR. The tagged rRNAs from strain A2451U were nonfunctional. All other rRNAs (tagged and untagged rRNAs from strain WT1, and untagged rRNAs from strain A2451U) are functional rRNAs. Myc-Ubi(−) columns indicate the background of anti-Myc. (D) The effect of overexpression of Myc-Ubi on NRD. The wild-type strain with pWT1 or pA2451U containing pMyc-Ubi, pUbi, or an empty vector was grown, and Myc-tagged or untagged ubiquitin was overexpressed. The tagged rRNAs at indicated time points were detected as in Figure 2C. The intensity of the bands was quantified and is summarized in E.