Fig. 7: SUMOylation stabilizes DKC1 and promotes its interaction with snoRNP partners.

A DKC1 protein levels were detected by immunoblot assay. Cell lysates from Patu-8988t cell lines infected with lentivirus expressing vector, wild-type SENP3 or catalytically deficient SENP3 were subjected to SDS-PAGE and then visualized by immunoblot using indicated antibodies. B DKC1 transcriptional levels were assessed by RT-qPCR. C Assessment of ubiquitination among DKC1 under distinct SUMOylation degrees. HEK293T cells were transfected with indicated panel, in which SUMOylation degrees was modulated by UBC9 (up) and SENP3 (down), UB WT constructs were used to reveal total ubiquitination levels in DKC1 while an UB K48 variant was responsible for detecting K48-polyubiquitination status of this protein. Negative control did not receive DKC1-encoding vectors. Cell lysates were collected after four-hour treatment with MG132 and then subjected to pulling down with anti-Flag gels. Ubiquitination levels were visualized by immunoblot (anti-HA) following SDS-PAGE in denaturing condition. D Determination of ubiquitination status of wild-type and mutant DKC1 molecules. HEK293T cells received empty vectors or expressing constructs encoding either wild-type or SUMOylation-resistant DKC1. These cells were transfected with UB molecules as shown in (C) to reveal different ubiquitination types. Cell lysates were recovered after treatment with MG132 and then subjected to IP with anti-Flag gels. Proteins were resolved by SDS-PAGE and subsequently detected by immunoblot with anti-HA antibodies. E Sequence alignments of two conserved SUMO-interacting (SIM) sites in NHP2 among different species. The indicated amino acids in red (I/L/V) within the SIM were mutated to alanine in NHP2 mutants. F Interaction between DKC1 and NHP2 variants was assessed. HEK293T cells were transduced with expression vectors encoding wild-type NHP2 or mutated NHP2 in one or two SIM sites. These cells were co-transfected with MYC-labelled DKC1 construct and then subjected to co-IP test. After resolution in SDS-PAGE, DKC1 and NHP2 levels were revealed by immunoblot assay with anti-MYC and anti-Flag antibodies respectively. G Interaction between NHP2 and DKC1 variants was tested. Wild-type and SUMOylation-resistant (3KR) DKC1 were transfected into HEK293T cells, which were epigenous expressing MYC-tagged NHP2 by plasmid transduction. An empty vector was used as control. The co-IP test was carried out using lysates of these transfected cells, and the protein levels were detected by immunoblot with indicated antibodies. H Sequence alignment of SIM site in GAR1 among different species. The highlighted red amino acids were mutated to alanine. I Mutation of SIM in GAR1 did not disrupt the interaction between DKC1 and GAR1. Wild-type or mutated GAR1 was co-transfected into HEK293T cells with Myc-tagged DKC1. DKC1 was detected in the pooled complexes IPed by anti-Flag gels by SDS/PAGE resolution and immunoblot. J GAR1 could comparably interact with SUMOylation-resistant (3KR) DKC1. Interaction between GAR1 with wild-type or SUMOylation-resistant DKC1 was assessed by co-IP assay. HEK293T cells were transfected with Myc-tagged GAR1 and Flag-tagged DKC1 variants. After being pulled down with anti-Flag gels, the associated GAR1 was visualized by SDS/PAGE and immunoblot assay. HC, high chains of antibodies used for IP. LC, low chains of antibodies used for IP. Unpaired t test, ns, no significance.