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CHAPTER 13. RNA Splicing. 2 conceptions: What is intron ? And what is exon ?. Recall the structuer of DNA.
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CHAPTER 13 RNA Splicing
2 conceptions: • What is intron ? • And what is exon ? Recall the structuer of DNA
In the vast majority of cases in bacteria and their phage the codon for one amino acid is immediately adjacent to the codon for the next amino acidin the polypeptide chain. But in many eukaryotic gene consisting of blocks of coding sequences separated by blocks of non-coding sequences. The coding sequences are called “extron” The non-coding sequences are called “intron”
Question • Why do eukaryotic gene contain so much non-coding sequences ? Is that a waste? Try to find out the answer after finish the study of this chapter!
What is RNA splicing? • The primary transcript for a typical eukaryotic gene contained introns as well as exon calls pre- mRNA The process which removes the introns from the pre-mRNA is called RNA Splicing
Objectives • Understand the mechanism of splicing by splicesome. • Understand how the splicesome direct the splicing.
Topic 1 The chemistry of RNA splicing
Sequences within the RNA determing where splicing occurs • 5’ splice site (contain GU) • 3’ splice site (contain AG) • Branch point site (contain A)
How the intron is removed? • Twotransesterification reactions: 1.The 2’OH of the conserved A at the branch site attack the phosphoryl group of the conserved G in the 5’ splice site so the freed 5’ end is joined to the A. 2. The 5’exon attacks the phosphoryl group at the 3’splice site so the 5’ is joined to the 3’.
Notice that the newly liberated intron has the shape of a lariat
The structure of the three-way junction formed during the splicing reaction
What ensures that splicing only goes forward • First ,the forward reaction involves an increase in entropy . • Second ,the excised intron is rapidly degraded after its removal .
Exon from different RNA molecules can be fused by thans-splicing
Splicing is carried out by spliceosome • The spliceosome complex contain : 1.150 proteins 2.5 small nuclear RNAs(U1 U2 U4 U5 U6) complexed with small nuclear ribonuclear protein(snRNPs)
Three roles of snRNPs in splicing • Recognize the 5’splice site and the branch site • Bring those sites together as required • Catalyzed the RNA cleavages and joining reaction. To perform these functions, RNA-RNA,RNA-protein, and protein-protein interactions are all important
Assembly, rearrangements, and catalysis within the spliceosome • Assembly 1.U1 snRNP recognize the 5’ splice site 2.One subunit of U2AF binds to the Py and the other to the 3’splice site 3.The BBP bind to the branch site This arrangement of RNA and protein are called early (E) complex.
4.U2 snRNPs then binds to the branch site ,aided by U2AF and displacing BBP. This arrangement is called A comlex
5.The U4 and U6 snRNPs, alone with the U5 snRNPs, joined the complex. The three snRNPs are called the tri-snRNP particle. The A complex is converted into B complex
6.U1 leaves the complex, and U6 replaces it at the 5’ splice site .This the base-pairingbetween the U1 and the pre-mRNA be broken, allowing the U6 to anneal with the same region. This steps complete the assembly pathway .
Rearrangement 1.U4 is released from the complex ,allowing U6 to interact with U2 This arrangement is called C complex ,producing the active site, it also ensure the substrate RNA is the active site primarily formed of RNA
Catalysis 1.The formation of the active site juxtaposes the 5’ splice site of the pre-mRNA and the branch site. 2.The second reaction ,the U5 snRNPs helps to bring the two exons together. 3.Final step involves release of the mRNA product and the snRNPs.
Self-splicing introns reveal that RNA can catalyze RNA splicing • Self-splicing introns The intron itself folds into a specific conformation within the precursor RNA and catalyzes the chemistry of its own release. Strictly speaking, they are not enzymes for they mediate only one round of RNA processing.
The self-splicing introns are grouped into two classes on the basis of their structure and splicing mechanism • Group I • Group II In the case of group II introns, the chemistry of splicing, and the RNA intermediates produced ,are the same as for nuclear pre-mRNA.
The secondary structure folds into a tertiary stureture • The Guanine-binding pocket • Internal guide sequence.
Guanine-binding pocket can bind any G-containing ribonucleotide
The path way of group I introns splicing 1.It use a free G nucleotide instead of a branch point A residue. The same type of transesterific- ation reaction that leads to the lariat formation in the earlier example fuses the “G” to the 5’end of the intron 2.The freed 3’end of the exon attack the 5’splice site and fuse the two exon and release the intron
Group I introns release a linear intron rather than a lariat Linear ↓
Group II introns • The chemistry is essentially the same as in the spliceosome case, with a highly reactive Adenine within the intron initiating splicing, and leading to the formation of a lariat product.
Proposed folding of the RNA catalytic region for splicing of group II introns and pre-mRNA
How does the spliceosome find the splice site reliably • The two kinds of errors in splice-site recognition 1.The splice sites can be skipped 2.Other site, close in sequence but not legitimate splice sites, could be mistakenly recognized.
Why does the problem of appropriate splice site recognition remain formidable? • The average extron is only some 150 nucleotides long, whereas the average intron is approximately 3,000 nucleotides long. Thus ,the extron must be identified within a vast ocean of intronic sequences ,so it seems inevitable that many errors would occur.
