SNRPD2 Is a Novel Substrate for the Ubiquitin Ligase Activity of the Salmonella Type III Secretion Effector SlrP

Abstract

SlrP is a protein with E3 ubiquitin ligase activity that is translocated by Salmonella enterica serovar Typhimurium into eukaryotic host cells through a type III secretion system. A yeast two-hybrid screen was performed to find new human partners for this protein. Among the interacting proteins identified by this screen was SNRPD2, a core component of the spliceosome. In vitro ubiquitination assays demonstrated that SNRPD2 is a substrate for the catalytic activity of SlrP, but not for other members of the NEL family of E3 ubiquitin ligases, SspH1 and SspH2. The lysine residues modified by this activity were identified by mass spectrometry. The identification of a new ubiquitination target for SlrP is a relevant contribution to the understanding of the role of this Salmonella effector.

Keywords: RNA splicing; SNRPD2; SlrP; type III secretion; ubiquitination.

Figure 1

Analysis of interactions of SlrP with human proteins in the yeast two-hybrid system. Derivatives of plasmid pGAD1318 expressing the indicated proteins (or C-terminal fragments of these proteins) were introduced in yeast strain L40 together with pLEX10 or pLEX10-SlrP. The interaction between SlrP and human proteins is shown by the detection of blue color in the presence of X-Gal after a β-galactosidase filter assay. Empty vectors were used as negative controls.

Figure 2

Confirmation of the interaction of SNRPD2 with SlrP. (A) 6His-SlrP was incubated with GST or GST-SNRPD2 bound to glutathione-agarose beads. Copurification of SlrP with SNRPD2 was detected by immunoblot with anti-His antibodies. (B) 6His-SlrP or 6His-SseK1 bound to Ni-NTA agarose beads were incubated with a cell lysate obtained from HeLa cells expressing 3HA-SNRPD2. Copurification of SNRPD2 with SlrP was detected by immunoblot with anti-HA antibodies. Ponceau S red staining was used as loading control. (C) 6His-SlrP or 6His-SseK1 bound to Ni-NTA agarose beads were incubated with a cell lysate obtained from HeLa cells. Copurification of SNRPD2 with SlrP was detected by immunoblot with anti-SNRPD2 antibodies. Ponceau S red staining was used as loading control. Sizes of molecular weight markers are shown in kDa. Results are representative of two independent experiments.

Figure 3

In vitro ubiquitination of SNRPD2 catalyzed by SlrP. (A) Ubiquitination reactions carried out with HA-ubiquitin in the presence (+) or absence (−) of E1, E2, 6His-SlrP, and GST-SNRPD2, were submitted to immunoblot analysis with anti-HA monoclonal antibodies. (B) Ubiquitination reactions were carried out with GST or GST-SNRPD2 bound to glutathione-agarose beads, washed and subjected to immunoblot analysis with anti-HA monoclonal antibodies. Results shown are representative of three independent experiments.

Figure 4

Specificity of the interaction of SNRPD2. (A) Derivatives of pLEX10 and pGAD1318 were introduced in yeast strain L40 by transformation. Transformants were selected in media lacking tryptophan and leucine. The interactions between the indicated effectors and SNRPD2 are analyzed by growth in the absence of histidine. (B) 6His-SlrP, 6His-SspH1 or 6His-SspH2 bound to Ni-NTA agarose beads were incubated with a cell lysate obtained from HeLa cells expressing 3HA-SNRPD2. Copurification of SNRPD2 with Salmonella effectors was detected by immunoblot with anti-HA antibodies. Stain-free total protein staining was used as loading control. Sizes of molecular weight markers are shown in kDa. Results are representative of two independent experiments.

Figure 5

Specificity of the ubiquitination of SNRPD2. (A) The activity of 6His-tagged SlrP, SspH1 and SspH2 was tested with HA-ubiquitin in the presence or in the absence of E1, and E2. SlrP: 6His-SlrP, SspH1: 6His-SspH1, SspH2: 6His-SspH2, -: no E3 ligase effector added. (B) Ubiquitination of GST-SNRPD2 bound to glutathione-agarose beads was tested in the presence of HA-ubiquitin, E1, E2, and a Salmonella effector fused to 6His: wild-type SlrP, SlrP ∆N (lacking 139 N-terminal residues), SlrP C546A mutant, wild-type SspH1 or wild-type SspH2. GST was used as negative control. Beads were washed before the immunoblot analysis. Ponceau S red staining is shown in the lower panel. M, molecular weight markers (size of bands in kDa: 250, 150, 100, 75, 50, 37, 25, 20).

Figure 6

Identification of ubiquitinated residues in SNRPD2. (A) SDS-PAGE analysis of GST-SNRPD2 and GST-SNRPD2 subjected to the in vitro ubiquitination assay (GST-SNRPD2-Ub). The gel was stained with Coomassie blue and bands indicated by red arrows were cut for analyses by mass spectrometry. (B) Ubiquitination reactions catalyzed by SlrP on GST-SNRPD2 carried out in the presence of HA-ubiquitin were analyzed by Western blot with anti-HA antibodies. Different versions of GST-SNRPD2 used are indicated: wild-type (wt), point mutants K85A, K92A and K85A/K92A and deletion mutant Δ84-92. M, molecular weight markers (size of bands in kDa: 250, 150, 100, 75, 50, 37, 25, 20). (C) Quantification of ubiquitinated bands from three independent experiments. Means and standard deviations are represented relative to the wild-type values that were set to 1. * p < 0.05, for comparison with the wild type using ANOVA with Dunnett’s post-hoc test.

Figure 7

Stability of SNRPD2 in the presence of SlrP. HEK293T cells were transfected with 1, 5 or 10 μg of plasmids pCS2 or pIZ1720 (pCS2-SlrP-3xFLAG), as indicated. The level of SNRPD2 was detected by Western blot with anti-SNRPD2 antibodies. Expression of SlrP-3xFLAG was detected with anti-FLAG antibodies. Anti-β-actin antibodies were used as loading control. Sizes of molecular weight markers are shown in kDa. Results are representative of three independent experiments.