1. Dr. Peter John M.Phil, PhD Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences & Technology (NUST)

2. RNA Splicing & Processing

3. Alternative splicing
Alternative Splicing: When a single gene give rise to more than one type of mRNA
Use of different start points/ generation of alternative 3’ ends alter the pattern of splicing
In some cases the multiple products are made in the same cell but in other the splicing is normal

4. Alternative splicing
An alternative splicing pathway uses another set of snRNPs that comprise the U12 spliceosome.
The target introns are defined by longer consensus sequences at the splice junctions, but usually include the same GU-AG junctions.
Some introns have the splice junctions AU-AC, including some that are U1-dependent and some that are U12-dependent.

5. Alternative Splicing in E1A
Adeno virus E1A region by using alternative 5’ sites generates different size products
T antigen is larger in size
t antigen is smaller in size

6. Alternative Splicing in E1A
t antigen producing cell
From these cell extract protein ASF (Alternative Splicing factor)
This protein (ASF) (alternative splicing factor) have same role as splicing factor SF2, required for early steps in spliceosome assembly and for the first cleavage-ligation reaction

8. Alternative Splicing in Mouse
Exon 2 & 3 of the Mouse Tropnin T gene are mutantly exclusive
Exon-2 used in smooth muscle & Exon -3 used in other tissue
Smooth muscle contain protein that binds to repeated elements located on either side of the exon-3 & prevent use of the 3’ & 5’ sites that are needed to include it

9. Drosophila Sex Determination
Male include exon-3 of sxl gene which have termination ultimately no SXL protein
Female exclude exon-3 of sxl gene
SXL protein in female
SXL protein change the splicing of Tra gene
Tra produce Tra2 gene
Tra + Tra 2 produce dsx (double sex) gene
DSX protein suppress male genes & promote female development

10. Drosophila Sex Determination
Sex determination in D. melanogaster involves a pathway in which different splicing events occur in females.
Blocks at any stage of the pathway result in male development.

12. Splicing and mRNA export
Spliceosome also may provide the initial point of contact for the export apparatus.
A protein complex binds to the RNA via the splicing apparatus
The complex consists of >9 proteins and is called the EJC (exon junction complex)

13. Exon Junction Complex EJC
EJC is involved in several functions
Some of the proteins of the EJC are directly involved in association with splicing Factors
Others recruit additional proteins for particular functions

15. mRNA Transport EJC includes a group of proteins called the REF family
REF proteins in turn interact with a transport protein (called TAP and Mex)
TAP and Mex has direct responsibility for interaction with the nuclear pore

17. Thanks