Serine-Arginine-rich Protein p30 Directs Alternative Splicing of Glucocorticoid Receptor Pre-mRNA t

o Glucocorticoid Receptor β in Neutrophils*

Received for publication, January 24, 2003, and in revised form, April 29, 2003Published, 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 & IntroductionAlternative 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.

activate inhibit

arachidonic acid

inflammation

AbstractAbstractSame pre-mRNA

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

Alternative splicing

more

few

Which alternative splicing factor causes ? Authors focus on SR protein.

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: Celllysis buffer(10mM Tris-HCl,1 mM EDTA,mixture of protease inhibito

rs) 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 dev

eloped 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

Fig.1

Polymorphonuclear netrophils

Peripheral blood Mononuclear cell

chemiluminescence

Method—2

1.RNA Isolation and cDNA Preparation : Use RNA-Bee to isolation total RNA from netrophils, and use SuperScript II reverse transcriptase t

o 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 v

ector.

3.Real-time PCR : Use different Taqman probe to quantify mRNA levels of SRp30a, SRp30b, SRp30c. And each sequence was quantified relativ

e 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.

Neutrophilie differentiation of PLB-9

85 cells

Fig.4

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

SRp30 expression of PLB-985 cell after exposure in retinoic acid : day 0 < day5.

Day of retinoic acid exposure

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 diffe

rentiation with retinoic acid.

3. Western Blot Analysis (primary and secondary antibody for GCRβ)

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 diffe

rentiation 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