Modulating Pre-mRNA Splicing to Restore Genetic Mutants to Wild-Type

Sachin KhisteSchool of Pharmacy,

University of Louisiana at Monroe.

30, 000 genesIn HumanGenome

One geneone DNAsequence

So, synthesis30, 000

Proteins, right ?

But why humanshave more than

100,000 proteins ?

Monoclonal antibody but why two bands ??

Protein Synthesis

Pre-mRNA splicing

Alternative Pre-mRNA splicing

Spliceosome Machinery

A spliceosome machinery is a large and complex molecular machine which removes introns and links exons to form mature mRNA

SR - Proteins

1. Family of protein involved in pre mRNA splicing.

2. Long repeats of Serine (S) and Arginine (R) amino acid residue.

3. Consist of two domains RNA recognition motif ( RRM) and RS binding domains.

Structure of different SR-Proteins

Mechanism and phosphorylation of SR-Proteins

Pre-mRNA Splicing and Cancer: Exon skipping in BRCA1:

Function: Repairs double-strand breaks in DNA.

Mutation : Hereditary risk of breast cancer females.

Study 1

1. Amiloride “normalizes” the isoform RNA splicing of BcL-X, HIPK3 and RON/MISTR1 in Huh-7 cells

2. Proteomic detection of mostly hypo-phosphorylated splicing factor SF2/ASF in the cytoplasm and nucleus of amiloride-treated cells

3. Amiloride down-regulates phosphorylation of AKT kinase and PP1 phosphatase

4. Decreased cell mobility/invasion activities and depleted cytoskeletal structures following AS changes of involved genes after amiloride

exposure

5. Perturbation of cell cycle progression and mitotic cytokinesis by amiloride

6. Cell death with severe DNA degradation subsequent to amiloride-induced altered splicing of functional molecules in apoptosis and DNA

repair pathways

7. Similar modulating effects of amiloride on oncogenic RNA splicing in other human solid tumor and leukemic cell lines

Conclusion of study 1

1. Amiloride promotes pro-apoptotic isoform expression of BcL-x, HIPK3 and flRON in Huh-7 hepatic cancer cells by modulating pre-mRNA splicing.

2. Amiloride modulates phosphorylation status of SR proteins by protein kinase and phosphatase and further regulates pre-mRNA splicing.

Study 2

Exon skipping in BRCA1

Function: Repairs double-strand breaks in DNA.

Mutation : Hereditary risk of breast cancer females.

1. Design of molecules that promote exon inclusion

2. Splicing correction by synthetic effectors

Wt

Mt

3. The RS minidomain is activated in phosphorylated state

Spinal muscle atrophy

4. Promotion of SMN2 exon 7 inclusion

Conclusion of Study 2

1. Alternative Pre-mRNA splicing results in deletion mutation of BRCA1 and SMN2.

2. Enhancement of SF1 and spliceosomes binding to specific exon-18 of BRCA1 and exon-7 of SMN2 by ESSECE, a oligo-peptide compound can effectively restore the expression of wild-type BRCA1 and SMN2.

3. ESSENCE (exon-specific splicing enhancement by small chimeric effectors) recognizes specific elements in pre-mRNA and enhances the phosphorylation of SR-proteins for wild-type BRCA1 and SMN2 pre-mRNA splicing.

Conclusion

1. Alternative RNA splicing is a cause of anti-apoptotic isoform proteins and deletion mutants presenting in cancer cells and genetic diseases.

2. Modulating pre-mRNA splicing by Amiloride in SR protein phosphorylation that can restore pro-apoptotic proteins in gene expression level is a new therapeutic approach to target cancer cells.

3. ESSENCE is a specific approach to restore wild-type protein in deletion mutant cells.

Acknowledgement

Major Adviser:Dr. Yong Yu Liu

Lab Colleagues:SalmanBurhan

and

ULM College of Pharmacy

Thank you

Questions and Comments