I. RNA processing
A. 5’ and 3’ end modifications
1. Capping at the 5’ end of mRNA occurs shortly after transcription begins.
Enzymes are recruited to phosphorylated C-terminal domain of RNA polymerase.
(1) Loss of P irom 5’ phosphate group.
(2) Addition of Gp from GTP (excess is just 2 P) i
(3) Addition of methyl group to 5’ end of Gp.
(4) (Only on some caps) Methylation of most 5’ ribose.
b) Result: CH -3GpppNpNp - exon
c) Capping protects against degradation by ribonucleases, and the 5’ cap
binds to translation initiation factors.
2. Addition of polyA tail to 3’ end of mRNA
(1) RNA polymerase is transcribing DNA and codes AAUAAA.
(2) AAUAAA is the cleavage signal, which recruits a specific
endonuclease to cleave the extra DNA to the 3’ of signal.
(3) PolyA polymerase adds the tail using energy from a split ATP.
ATP is split into Ap and PP, ihe former is used in the polyA tail.
b) The polyA tail is untemplated, meaning that it is not transcribed directly
from template DNA. It is assembled post-cleavage.
c) Result: AAUAAA (cleavage signal) - AAAAA(A) (polyA tanl) - OH 3’
B. Intron splicing
1. Primary transcript
a) Introns are generally large sequences and make up most of the DNA.
Introns are the RNA sequences that will be removed from the completed
RNA before translation, and are therefore not included in coding for
proteins. b) Exons are small sequences. They are the sequences that remain after
RNA modification and will ultimately code for proteins.
2. Splicing consensus sequences and chemistry
a) Important locations
(1) The 5’ splice site occurs at the 5’ position of the GU of the intron.
(2) Branch site A is where the 5’ splice site of the spliced intron will
ultimately attach to form a “lariat”. The “A” is the Adenine base
that is approximately 15-45 bases to the 5’ of the 3’ splice site.
(3) The 3’ splice site occurs at the 3’ position of the AG of the intron.
(4) 5’ exon - GU intron intron intron A 15-45 bases (still intron) AG -
b) Transesterification: reactions that occur in splicing; phosphodiester bond
at each splice site is broken and then remade to form lariat and attached
c) Lariat is formed when branch point A attaches to the G from the 5’ splice
site. Bond is between G’s 5’ phosphate and A’s 2’ OH.
3. Splicing mechanism
a) snRNP: small nuclear ribonucleoprotein made up of snRNA and 10 or
more proteins. snRNPs involved in the splicing mechanism are U1, U2,
U4, U5, and U6.
(1) U1 base pairs with 5’ splice site.
(2) U2 base pairs with branch site.
(3) U5 binds to both 5’ and 3’ splice sites and brings exon 1 and
exon 2 into close proximity.
(4) U4 releases U6, which then binds to U2. U4 dissociates.
(5) U1 is released and U6 binds to the 5’ splice site. (6) Exons are clipped and ligated together. 5’ splice site is ligated to
(7) Lariat is released.
4. Alternative splicing
a) Alternative splicing results in different sequences being considered
exons and introns based on what protein they will be coding for.
b) Alternative 5’ or 3’ splice sites can result in changing size and sequences
for introns and exons.