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. A complex (ATP dependent)
E complex is converted to the A complex (ATP dependent) when U2 snRNP binds to the branch site.
The U2 snRNA includes sequences complementary to the branch site.
Several proteins of the U2 snRNP are bound to the substrate RNA just upstream of the branch site.

4. A complex
Several proteins of the U2 snRNP are bound to the substrate RNA just upstream of the branch site.
The addition of U2 snRNP to the E complex generates the A presplicing complex.
The binding of U2 snRNP requires ATP hydrolysis and commits a pre-mRNA to the splicing pathway.

5. The commitment (E) complex forms by the successive addition of U1 snRNP to the 5' splice site, U2AF to the pyrimidine tract/3' splice site, and the bridging protein SF1/BBP.

6. Intron/exon definition
More than one way to form the E complex
Intron definition: The basic feature of this route for splicing is that the two splice sites are recognized without requiring any sequences outside of the intron.
Exon definition: The important feature of this route for splicing is that sequences downstream of the intron itself are required.

7. U1 snRNP initiates splicing
The E complex is converted to the A complex when U2 snRNP binds at the branch site.
Weak 3' splice sites may require a splicing enhancer located in the exon downstream to bind SR proteins directly.

8. U1 snRNP initiates splicing
The direct way of forming an E complex
U1 snRNP to bind at the 5' splice site and U2AF to bind at a pyrimidine tract between the branch site and the 3' splice site.
Another possibility is for the complex to form between U2AF at the pyrimidine tract and U1 snRNP at a downstream 5' splice site.

9. Intron definition
Ul snRNP at the 5' splice site is necessary for U2AF to bind at the pyrimidine tract downstream of the branch site, making it possible that the 5' and 3' ends of the intron are brought together in this complex.
The E complex is converted to the A complex when U2 snRNP binds at the branch site.

10. U1-independent pathway
SR proteins may enable U2AF/U2 snRNP to bind in vitro in the absence of U1, raising the possibility that there could be a U1-independent pathway for splicing.

11. Intron definition

12. Exon definition
5' splice site is recognized by U1 snRNA in the usual way
This U1 snRNA is connected by SR proteins to the U2AF at the pyrimidine tract
When U2 snRNP joins to generate the A complex, there is a rearrangement in the components

13. Intron/exon definition

14. Splicing Stages
Splicing Reaction

15. Splicing Stages
Binding of U5 and U4/U6 snRNPs converts the A complex to the B1 spliceosome, which contains all the components necessary for splicing.
The spliceosome passes through a series of further complexes as splicing proceeds.
Release of U1 snRNP allows U6 snRNA to interact with the 5‘ splice site and converts the B1 spliceosome to the B2 spliceosome.

16. Catalytic Center
When U4 dissociates from U6 snRNP, U6 snRNA can pair with U2 snRNA to form the catalytic active site.
snRNA components of the splicing apparatus interact both among themselves and with the substrate RNA by means of base pairing interactions
these interactions allow for changes in structure that may bring reacting groups into apposition and may even create catalytic centers.

18. snRNAs and the substrate RNA
Pairing reactions occurs b/w snRNAs and the substrate RNA in the course of splicing.
U1 pair with 5’ splice site
U2 pair with the branch site
U6 pair with 5’ splice site
U5 is close to both exons

20. Spliceosome Types (U2, U12)
The type of Spliceosomes is also influenced by sequences in the intron.
AU-AC introns spliced by U2-type spliceosomes
GU-AG introns spliced by U12-type spliceosomes

21. U12 spliceosome
A strong consensus seq at the left & conserved branch point end is require for U12 depended intron
UCCUUPuAPy, which pairs with U12.
For this reason, the term U12-dependent intron is used rather than AU-AC intron.
Two different types of intron may co-exist in the same genome or even in the same gene

22. 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

23. 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.

24. 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

25. 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

27. 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

28. 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

29. 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.

31. 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)

32. 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

34. 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

36. Thanks