Syncrip facilitates porcine parvovirus viral dna replication through the alternative splicing of ns1 mrna to promote ns2 mrna formation
Electrophoretic mobility shift assay
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- Điều hướng trang này:
- Purification of GST‑tagged SYNCRIP protein
- Results PPV NS1 mRNA can specifically interact with SYNCRIP
| Electrophoretic mobility shift assay
NS1 mRNA sequence were synthesized, using a T7 in vitro transcription synthesized with biotinylation at 5′ end, following the manufacturer’s instruction. Glu- tathione (GST) and GST-SYNCRIP proteins were puri- fied using the GST protein purification kit (P2262; Beyotime, China), and further incubated with biotinyla- tion NS1 mRNA. Gel shift assays were performed using the Chemiluminescent EMSA Kit (GS009, Beyotime, China). Purification of GST‑tagged SYNCRIP protein Bacterially optimized SYNCRIP ORF (1–1689 bp) was cloned in pGEX-4 T-1 vector between Sal I and Not I restriction sites to express GST-SYNCRIP protein. The positive plasmids were transformed into bacteria strain Rosetta (DE3)plys. To induce fusion protein expres- sion, isopropul β-d-1-thiogalactopyranoside (IPTG) was added to bacteria culture medium at a final concentration of 1.0 mM for 6 h at 28 ℃. GST-SYNCRIP protein was purified as previously described [ 34 ]. Statistics These data are shown as means ± SEM (SD) values from the three independent experiments. Statistical analyses were performed with GraphPad Prism 5 software. Immu- nofluorescence values were calculated using Image-Pro Plus 6.0. A value of p < 0.05 was considered as significant. Results PPV NS1 mRNA can specifically interact with SYNCRIP Since NS2 mRNA is completely contained in NS1 mRNA, to determine how PPV NS1 mRNA regulate NS2 expres- sion through alternative splicing, we sought to identify intracellular NS1 mRNA binding factors using an unbi- ased approach. Full-length NS1 mRNA was transcribed with biotinylated nucleotides in vitro. Used partial LacZ mRNA without protein-coding potential served as a neg- ative control. Biotinylated NS1 mRNA or LacZ mRNA were incubated with total protein extracted from PK-15 cells and pulled down with streptavidin (Figure 2 A). The associated proteins were analyzed by SDS-PAGE and Coomassie blue staining. Two different strips specifically presented in the NS1 mRNA pull-down not LacZ mRNA samples were excised and analyzed by mass spectrometry (Figure 2 B), which identified sixty-four potential binding proteins (Additional file 1 ), six of them were identified to be involved in mRNA processing using gene ontology (Figure 2 C). To determine potential proteins that specifically interact with NS1 mRNA, first, we used online soft- ware catRAPID to predict the interaction between NS1 mRNA and the six host proteins. The results show that only SYNCRIP could interact with NS1 mRNA (Data not shown). In agreement with that, RNA-pulldown assay shows that only SYNCRIP could interact with NS1 mRNA in HEK293 cells, but not DHX29, PRPF40A, SFPQ, NOP56, DDX21 (Figure 2 D). In addition, bioti- nylated NS1 mRNA was also confirmed to interact with the endogenous SYNCRIP protein in PK-15 cells (Fig- ure 2 E). To substantiate this interaction, anti-SYNCRIP antibody was used to immunoprecipitate endogenous SYNCRIP and its binding RNA from the nuclear extracts of PPV-infected PK-15 cells, and RNA interacting with SYNCRIP were collected and analyzed. We detected the enrichment of NS1 mRNA, but not control GAPDH RNA, in the immunoprecipitates of anti-SYNCRIP com- pared with the IgG control (Figure 2 F). Furthermore, fluorescence in situ hybridization assay showed that SYNCRIP and NS1 mRNA co-localized in PPV-infected PK-15 cells (Figure 2 G). Meanwhile, purified GST-tagged SYNCRIP directly binded with NS1 mRNA in vitro (Fig- ure 2 H). Electrophoretic mobility shift assay shows that shift speed of biotinylated NS1 mRNA was slowed down with the increase of GST-SYNCRIP concentration (Fig- ure 2 I lines 3–6), suggesting that SYNCRIP can specifi- cally interact with NS1 mRNA. tải về 5.65 Mb. Chia sẻ với bạn bè của bạn:
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