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. 2021 Mar 21;49(7):3796–3813. doi: 10.1093/nar/gkab155

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — MKRN3 ubiquitinates PABP family members that contain PABC domain and CPP-associated mutations of MKRN3 comprised this ubiquitination. (A) MKRN3 ubiquitinated PABPC1, PABPC3 and PABPC4, but not PABPC5 in vitro. An in vitro ubiquitination assay was carried out using the recombinant Flag tagged PABPC1, PABPC3, PABPC4 or PABPC5, His6-tagged UBA1 (E1) and UBCH5A (E2), and Gst-tagged MKRN3 (E3) or the E3 ligase dead mutant MKRN3 (C340G), together with the indicated components. Usp2cc, the catalytic core of human deubiquitinase Usp2; PABPCX represents PABPC1, PABPC3, PABPC4 or PABPC5. (B) Flag-tagged PABPC1 was efficiently ubiquitinated by 3XMyc-tagged MKRN3. MKRN3⁻^/⁻ HEK293T cells were co-transfected with indicated plasmids, and cell lysates were immunoprecipitated with anti-HA affinity gels, treated with Usp2cc or not, followed by immunoblotting with anti-Flag antibody to detect ubiquitinated PABPC1. (C) The ubiquitination of PABPC1 was reduced in Mkrn3^m+/p⁻ mouse hypothalamus at postnatal day 15, compared to those from the age-matched wild-type littermates (n = 3). Endogenous PABPC1 proteins of hypothalamic lysates from wild-type or Mkrn3^m+/p⁻ mice were immunoprecipitated using anti-PABPC1 antibody, followed by immunoblotting with anti-Ub or other indicated antibodies. (D) CPP-associated mutations of MKRN3 compromised its E3 ligase activity towards PABPC1 in MKRN3⁻^/⁻ HEK293T cells. Cells were transfected with HA-tagged Ub, Flag-tagged PABPC1, and 3xMyc-tagged wild-type MKRN3 or the indicated mutants (CPP-associated mutations, C340G, R365S, P417I and H420C). Cell lysates were immunoprecipitated with anti-HA affinity gels, followed by immunoblotting with anti-Flag antibody to detect ubiquitinated PABPC1 and other antibodies indicated.