Received for publication, January 24, 2003, and in revised form, April 29, 2003

1. 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

2. 名詞解釋 & Introduction Alternative Splicing：

3. SR protein : a part of Spliceosome complex To combine 3’-end exon and mediate the interaction between U1, U2 and U2AF

4. The relation of glucocorticoid (GC) and inflammation. activate inhibit arachidonic acid inflammation

5. Abstract Same 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 Which alternative splicing factor causes ? Authors focus on SR protein. more few

6. Method--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.

7. RESULTS chemiluminescence Fig.1 Polymorphonuclear netrophils Peripheral blood Mononuclear cell

8. 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.

9. 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

10. Method—3 • Freshly isolated neutrophils treat with IL-8 for 2 hoursor medium only. • Western Blot Analysis (primary and secondary antibody for SR protein) • Use flated scanner and NIH Image to measure chemiluminescence's intensity on each band.

11. Fig.3 Expression of SRp30 in neutrophils: Media only << After IL-8 stimulation

12. Method—4 1. PLB-985 cell exposure retinoic acid for 5 days. 2. Stained with Diff Qucik.

13. Fig.4 Neutrophilie differentiation of PLB-985 cells

14. Method—5 • PLB-985 cell exposure retinoic acid for 5 days. • Western Blot Analysis (primary and secondary antibody for GCR) • Use flated scanner and NIH Image to measure chemiluminescence's intensity on each band.

15. Fig.5 GCRαand GCRβ expression of PLB-985 cell after exposure in retinoic acid : day 0 < day 5.

16. Method—6 • PLB-985 cell exposure retinoic acid for 5 days. • Western Blot Analysis (primary and secondary antibody for SR protein) • Use flated scanner and NIH Image to measure chemiluminescence's intensity on each band.

17. 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.

18. Method—7 • The transfection of fluorescein-labeled antisense oligonucleotides for SRp30a, SRp30b,SRp30c using electroporation. And a control sample. • 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 GCRβ) • 4. Use flated scanner and NIH Image to measure chemiluminescence's intensity on each band.

19. Fig.7 ? Inhibit the expression of SRp30 also inhibit the expression of GCRβ

20. Method—8 • The transfection of fluorescein-tagged antisense oligonucleotides for SRp30c using electroporation. And a control sample. • 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.

21. Fig.8 Density units of GCRα Inhibit the expression of SRp30 will stimulation the expression of GCRα

22. 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.

23. The End