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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PPV.NS

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.

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