Serine-Arginine-rich Protein p30 Directs
Alternative Splicing of Glucocorticoid Receptor
Pre-mRNA to Glucocorticoid Receptor β in
Neutrophils*
Received for publication, January 24, 2003, and in
revised form, April 29, 2003
Published, JBC Papers in Press, May 8, 2003, DOI
10.1074/jbc.M300824200
Qing Xu‡, Donald Y. M. Leung‡§, and Kevin O. Kisich¶
班級：生科四甲
學號：91390170
姓名：張祐睿
名詞解釋 & Introduction
Alternative Splicing：
SR protein : a part of Spliceosome complex
To combine 3’-end
exon and mediate the
interaction between U1,
U2 and U2AF
The relation of glucocorticoid (GC) and inflammation.
inhibit
arachidonic acid
inflammation
activate
Abstract
Same pre-mRNA
Alternative splicing
more
Which alternative splicing
factor causes ? Authors focus
on SR protein.
few
β-isoform mRNA
>
α-isoform mRNA
↓
translation
↓
Glucocorticoid receptorβ
>
Glucocorticoid receptorα
(GRβ or GCRβ)
(GRα or GCRα)
↓
GCRβ can not bind Glucocorticoid, and it is a inhibitor of GCRα
↓
Glucocorticoid (GC) insenitivity
↓
Can not treat for Inflammatory diseases
Method--1
1. 從六個健康人體中各取出40ml的靜脈血液
2. Use Percoll density gradient 來分離、純化出PBMC (peripheral blood
mononuclear cells)和Neutrophils。
3. Western Blot Analysis with PBMC and Neutrophils:
Celllysis buffer(10mM Tris-HCl,1 mM EDTA,mixture of protease inhibitors) 30min
centrifuged at 12000 rpm for 10min at 4℃收集上清液 protein resolved by
electrophoresis and transfer to polyvinylidene difluoride membranes in Tris-glycine
buffer Membrane were block with blocking buffer(5% milk,10% 50mM sodium
phosphate, 3% 5M NaCl, and 0.05% Tween 20)  incubated with primary antibody to
SR proteins for 2 hours wash incubated with secondary antibody for 1 hour  wash
and developed with ECL Western blotting detection reagents.
4.Use flated scanner and NIH Image to measure chemiluminescence's (化學冷光)
intensity oneach band.
RESULTS
chemiluminescence
Polymorphonuclear
netrophils
Fig.1
Peripheral blood
Mononuclear cell
Method—2
1.RNA Isolation and cDNA Preparation : Use RNA-Bee to isolation total RNA from
netrophils, and use SuperScript II reverse transcriptase to reverse transcript RNA to
cDNA.
2.Plasmid Construction : Plamids used to create standard curves for real-time PCR.
SRp30a, SRp30b, SRp30c cDNA region spanning nucleotides from Genbank.
Use Macvector to design SRp30a, SRp30b, SRp30c forward primer and reverse primer to
amplifed. The PCR products cloned into the PGEM-T vector.
3.Real-time PCR : Use different Taqman probe to quantify mRNA levels of SRp30a, SRp30b,
SRp30c. And each sequence was quantified relative to a standard curve of its cognate
cloned cDNA sequence.
以 two-tailed paired t test，p值<0.05具統計學上的明顯差異
Fig.2
SRp30c >> SRp30a > SRp30b
Method—3
1. Freshly isolated neutrophils treat with IL-8 for 2 hours or medium only.
2. Western Blot Analysis (primary and secondary antibody for SR protein)
3. Use flated scanner and NIH Image to measure chemiluminescence's intensity on
each band.
Fig.3
Expression of SRp30 in neutrophils: Media only << After IL-8
stimulation
Method—4
1. PLB-985 cell exposure retinoic acid for 5 days.
2. Stained with Diff Qucik.
Fig.4
Neutrophilie
differentiation of PLB985 cells
Method—5
1. PLB-985 cell exposure retinoic acid for 5 days.
2. Western Blot Analysis (primary and secondary antibody for GCR)
3. Use flated scanner and NIH Image to measure chemiluminescence's intensity on
each band.
Fig.5
GCRαand GCRβ expression of PLB-985 cell after exposure in retinoic
acid : day 0 < day 5.
Method—6
1. PLB-985 cell exposure retinoic acid for 5 days.
2. Western Blot Analysis (primary and secondary antibody for SR protein)
3. Use flated scanner and NIH Image to measure chemiluminescence's intensity on
each band.
Fig.6
day 0
day 5
Day of retinoic acid exposure
SRp30 expression of PLB-985 cell after exposure in retinoic acid : day 0 <
day5.
Method—7
1. The transfection of fluorescein-labeled antisense oligonucleotides for SRp30a,
SRp30b,SRp30c using electroporation. And a control sample.
2. Flow Cytometry: Use FACSCaliber to select PLB-985 cells which are positive
for fluorescein. Then place into cultures for differentiation with retinoic acid.
3. Western Blot Analysis (primary and secondary antibody for GCR β)
4. Use flated scanner and NIH Image to measure chemiluminescence's intensity on
each band.
Fig.7
?
Inhibit the expression of SRp30 also inhibit the expression of GCRβ
Method—8
1. The transfection of fluorescein-tagged antisense oligonucleotides for SRp30c
using electroporation. And a control sample.
2. Flow Cytometry: Use FACSCaliber to select PLB-985 cells which are positive
for fluorescein. Then place into cultures for differentiation with retinoic acid.
3. Western Blot Analysis (primary and secondary antibody for GCRα)
4. Use flated scanner and NIH Image to measure chemiluminescence's intensity on
each band.
Fig.8
Density units of GCRα
Inhibit the expression of SRp30 will stimulation the expression of GCRα
DISCUSSION
1. SRp30c stimulate the expression of GCRβ,inhibit
the expression of GCRα.
2. Conclude that SRp30c is required for alternative
splicing to generate GCRβ mRNA.
GCR β is dependent on the presence of SRp30c,
this SR protein may make an attractive target for
therapeutic intervention.
The End