CSB349H1 Lecture Notes - Lecture 9: Exon Skipping, Polypyrimidine Tract, Spliceosome
14 May 2018
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2’-5’ bond is only possible in RNA because we require the O; DNA is de-oxy
Lecture 9(a): RNA Splicing
Introns:
• When eukaryotic DNA is transcribed it generates a primary RNA
transcript which consists of alternating introns and exons
o To generate a mRNA it must be processed:
§ (1) 5’ Cap | (2) Intron Splicing | (3) 5’-Poly-A-tail
• Capping and tail function to stabilize and
protect mRNA when found in the cytoplasm
as it can be degraded rapidly without them
ð mRNA consists of intrinsic features that determine its stability; they have an ‘internal clock’
that determines its lifetime – genes vary in terms of their mRNA turnover rate
Primary Transcript:
• Primary transcript is interrupted by non-coding
sequences (introns); number and size of the introns vary dramatically between different genes
o Most common intron is the AGGU type – has a GU at the start of the intron, and
an AG at the end of the intron, with a conserved adenine (A) residue at the end
ð Humans have more intron sequences than exons: 6x more introns than exons
Distinguishing Introns From Exons:
• Borders of introns and exons are marked by specific nucleotides within the pre-mRNA
known as consensus sequences:
o 5’ splice site (donor) => GT/U
o 3’ splice site (acceptor) => AG
o Branch Point
ð Branch point is necessary for splicing and is located within the intron close to the 3’ splice site:
Ø Begins with an A, and is followed by a stretch known as a poly-pyrimidine tract
(rich with C’s and T’s)
Splicing Reaction:
• Splicing is achieved by two successive transesterification (TE) reactions:
o Phosphodiester linkages within the pre-mRNA are broken and new ones are formed
First Reaction:
• 2’ OH group of the conserved A (on the branch site) undergoes a nucleophilic
attack with the phosphoryl group of the conserved G in the 5’ splice site
o Generate an unusual 2’-5’ bond forming a lariat structure
Second Reaction:
• 3’ OH of the 5’ exon undergoes another nucleophilic attack with phosphoryl
group at the 3’ splice site
o Cause the liberation of the intron in the shape of a lariat
