“Gene splicing”

1. “Gene splicing” Anumkamal(BB-307-016) Umm-e-Habiba(BB307-035)

2. Gene splicing • “Gene splicing is the removal of introns from the primary trascript of a discontinuous gene during the process of Transcription.” • Gene splicing is a post-transcriptional modification in which a single gene can code for multiple proteins.

3. Gene splicing is an important source of protein diversity. • One gene can lead to different mature mRNA molecules that generate multiple functional proteins. • Thus, gene splicing enables a single gene to increase its coding capacity.

4. Introns: The areas of the gene that are spliced out are representing noncoding regions that are intervening sequences also known as introns. • Exons: The DNA that remains in the processed RNA is referred to as the coding regions and each coding regions of the gene are known as exons.

6. There are two phenomenons by which gene splicing occur one is natural i.e.  • Post transcriptional modification. • Artificially or by chemical agent used generally in recombinant technology.

7. History of gene splicing: Berg was the first scientist to splice together segments of DNA from different organismsand his work was published in a landmark paper in the Proceedings of the National Academy of Science in 1972.

8. Gene Splicing Mechanism • There are several types of common gene splicing events. • Exon Skipping: • In which exons are included or excluded from the final gene transcript leading to extended or shortened mRNA variants. • The exons are responsible for producing proteins that are utilized in various cell types for a number of functions.

9. 2.Intron Retention: • In which an intron is retained in the final transcript. • In humans 2-5 % of the genes have been reported to retain introns. • It leads to a demornity in the protein structure and functionality.

10. 3.Alternative 3' splice site and 5' splice site: • Alternative gene splicing includes joining of different 5' and 3' splice site. • Two or more alternative 5' splice site compete for joining to two or more alternate 3' splice site.  • “Introns are first cut at their 5' end and then at their 3' end.

12. Spliceosomes • “The molecules or molecular complexes that actually splice RNA in the cellular nucleus are called spliceosomes”. • The spliceosome is an enzymatic complex.

13. Structure of spliceosomes • Spliceosomes are made of small sequences of RNAs bound by additional small proteins. • The small RNAs which make up the spliceosome are small nuclear RNAs (snRNA's). • The snRNAs combine with proteins to comprise, small nuclear ribonuclearprotein particles. • The absence of individual snRNP components can inhibit splicing.

15. Alternative splicing • “A single gene can be processed to create numerous gene products, or proteins and this process is referred to as alternative splicing”. • In eukaryotes information can be stored much more economically. • Several proteins can be encoded by a single gene, thus allowing a more varied proteome from a genome of limited size.

16. Alternative splicing was first observed in the late 1970s. • In humans, over 80 % of genes are alternatively spliced. • Alternate splicing is used to create the five antibody-types from the same gene. • Alternate splicing controls sex determination in  Drosophila melanogaster flies. • The gene Traencodes a protein that is expressed only in females.

18. Alternative splicing and disease • over 60% of human disease causing mutations affect splicing rather than directly affecting coding sequences. • Cancerous cells show higher levels of intron retention than normal cells, but lower levels of exon skipping.

19. Splicing out introns • Most introns begin with the nucleotide sequence G-T and end with the sequence A-G. • Are described as conforming to the GT-AG rule. • Within the intron is another highly conserved sequence this region (called the branch site) is the area that connects to the 5' end of the intron as it is cut and then curls around to form a lariat shape. • which is removed from the maturing RNA.

20. mRNA splicing mechanism • Splicing of mRNA is performed by spliceosome, containing snRNPs designated • U1 • U2 • U4 • U5 • U6 • (U3 is not involved in mRNA splicing).

23. Self-splicing • “Self-splicing occurs for rare introns that form a ribozyme, performing the functions of the spliceosome by RNA alone”. • There are three kinds of self-splicing introns. •  Group I •  Group II  • Group III

25. Other splicing events

26. tRNA splicing • tRNA (also tRNA-like) splicing is another rare form of splicing that usually occurs in tRNA. • The splicing reaction involves a different biochemistry • Ribonucleases cleave the RNA and ligases join the exons together.

27. Protein splicing • Proteins can also undergo splicing. • The biomolecular mechanisms is different, but principle is the same. • Inteins instead of introns, are removed. • The remaining parts, called exteins instead of exons. • Protein splicing has been observed in all sorts of organisms, including bacteria, archaea, plants, yeast and human.

28. Common errors • Mutation of a splice site resulting in loss of function of that site. • Mutation of a splice site reducing specificity. • Displacement of a splice site, leading to inclusion or exclusion of more RNA than expected, resulting in longer or shorter exons.

29. Applications of gene splicing • Using gene-splicing technology, vaccines have been produced. • Another application of gene spicing technology is related to the gene involved in Vitamin B production. • Human insulin-producing genes have been spliced into plasmids.

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