BCMB2x01/MEDS2003

Lecture 25:
Post-transcriptional
processing
Dr Giselle Yeo
giselle.yeo@sydney.edu.au
Processing of rRNA (18S, 5.8S, 28S)
• Transcribed by RNA Pol I as a single 45S precursor
1. Nucleotides are modified
2. Pre-rRNA is assembled with ribosomal proteins
3. Pre-RNA is cleaved into 18S, 28S and 5.8S rRNA
• 5S rRNA is transcribed separately by RNA Pol III
Processing of tRNA
• Transcribed by RNA Pol III
1. Nucleotides from 5’ and 3’ ends are cleaved
2. Nucleotides CCA are added
3. Some nucleotides are modified
4. Intron is removed and products are ligated
Processing of mRNA
• In bacteria, transcription and translation occur in
the same space
• In eukaryotes, pre-mRNA transcripts are extensively
processed in the nucleus and transported into the
cytoplasm before translation
 mRNA processing events

1. Capping at 5’
end
2. Addition of 3’
poly(A) tail
3. Splicing
(removal of
introns)
Capping of the 5’ end
• Occurs when transcript is
~25 nt long
• Capping adds a methylated
guanine to the transcript
via a 5’-5’ linkage
• Done by 3 enzymatic
reactions
• Capping protects the 5’
end of mRNA from
phosphatases and
nucleases and enhances
mRNA translation
 Polyadenylation of the 3’ end
• Occurs after transcription has ended
• Pre-mRNA is cleaved and around 250 adenylate
residues are added using ATP as the substrate
• Poly(A) tail is
NOT encoded
by DNA
 Polyadenylation of the 3’ end
• Poly(A) tail increases mRNA stability and enhances
translation (but does not affect transport out of
nucleus)
• Deadenylation is associated with mRNA decay

Stable
α-globin
mRNA

Cleavage
enzymes

Waggoner and Liebhaber (2003) Experimental Biology and Medicine 228:387-395

Polyadenylation of the 3’ end
• All eukaryotic mRNAs are polyadenylated, except
histone mRNAs
• Histone mRNAs have a stem-loop structure
followed by a purine-rich sequence (A/G) to direct
cleavage

http://emboj.embopress.org/content/31/
19/3790.figures-only
Exploiting the poly(A) tail
• mRNA can be
purified using
Oligo-dTs that bind
to the poly(A) tail
gosocrative.com -> Room 1221
Which of the following statements about the 3’
poly(A) tail is TRUE?
A. The poly(A) tail is transcribed from a T-rich
region in DNA
B. Histone mRNA is polyadenylated by a series of
enzymes
C. Polyadenylation is a method of stabilising DNA
D. Polyadenylation increases the chance that
mRNA is translated
E. I don’t know
Splicing
• >90% of human genes
have exons (coding
regions) and introns
(non-coding regions)
• Introns can be 50-
10,000 nt long
• Splicing removes the
introns and links the
exons to form the
mature mRNA
Splice sites
• Consensus sequences at the ends of introns
identify splice sites
• Branch site – located 20-50 nt upstream of 3’ splice
site
• Mutations in splice sites or branch site lead to
aberrant splicing
10 U/C

Same in all introns

 Splicing machinery
• Spliceosome – large
splicing complex
(~4.8 MDa)
consisting of small
nuclear RNAs
(snRNAs), >300
proteins and pre-
mRNA
Incorrect splicing and disease
• Mutations affecting splicing cause at least 15% of
all genetic diseases
• Mutations can occur in the pre-mRNA (e.g.
thalassemia) or in the splicing factors
Alternative splicing
• One gene but many mRNAs = many proteins
• >70% of human protein-coding genes are
alternatively spliced

2n different mRNAs from one

pre-mRNA with n alternative
splice sites
Alternative splicing – example
• Alternative splicing gives rise to membrane-bound
and secreted variants of the same antibody
Transcription and mRNA processing
• These processes are coupled
• RNA Pol II recruits capping enzymes, splicing
machinery components, and polyadenylation
enzymes
mRNA transport
• After processing, mature mRNA is transported from
the nucleus to the cytosol through the nuclear pore
complex
 mRNA transport
• After processing, mature mRNA is transported from
the nucleus to the cytosol through the nuclear pore
complex
Nuclear
pore
complex

mRNA and
protein being
transported
out of
nucleus
What you’ve learned
• Processing events for rRNA, tRNA and mRNA
• Roles of of 5’ capping and 3’ polyadenylation
• Features and importance of splicing
• Alternative splicing as a means to generate diversity
• mRNA transport after processing