6.
RNA Processing
a). Steps in mRNA processing
i). Capping
ii). Cleavage and polyadenylation
iii). Splicing
b). Chemistry of mRNA splicing
c). Spliceosome assembly and splice site recognition
i). Donor and acceptor splice sites
ii). Small nuclear RNAs
d). Mutations that disrupt splicing
Learning Objectives:
• Know the major steps in processing eukaryotic
mRNA
• Understand how the two transesterification
reactions remove an intron transcript and
ligate the exon transcripts
• Understand the nature of the donor and acceptor
splice sites
• Understand what a spliceosome is and how
splicing requires small nuclear RNAs
• Understand how splice sites are selected
• Understand how mutations in splice sites affect
mRNA production
Steps in mRNA processing (hnRNA is the precursor of mRNA)
• capping (occurs co-transcriptionally)
• cleavage and polyadenylation (forms the 3’ end)
• splicing (occurs in the nucleus prior to transport)
exon 1 intron 1 exon 2
Transcription of pre-mRNA and capping at the 5’ end
cap
Cleavage of the 3’ end and polyadenylation
cap
cap poly(A)
Splicing to remove intron sequences
cap poly(A)
Transport of mature mRNA to the cytoplasm
Capping occurs co-transcriptionally shortly after initiation
• guanylyltransferase (nuclear) transfers G residue to 5’ end
• methyltransferases (nuclear and cytoplasmic) add methyl
groups to 5’ terminal G and at two 2’ ribose positions on
the next two nucleotides
pppNpN
mGpppNmpNm
capping involves formation of a 5’- 5’ triphosphate bond
• cap function
• protects 5’ end of mRNA (increases mRNA stability)
• required for initiation of protein synthesis
Polyadenylation
• cleavage of the primary transcript occurs approximately
10-30 nucleotides 3’-ward of the AAUAAA consensus site
• polyadenylation catalyzed by poly(A) polymerase
• approximately 200 adenylate residues are added
cleavage
AAUAAA
mGpppNmpNm
AAUAAA A A
A
polyadenylation
mGpppNmpNm A
A
A
3’
• poly(A) is associated with poly(A) binding protein (PBP)
• function of poly(A) tail is to stabilize mRNA
Chemistry of mRNA splicing
• two cleavage-ligation reactions
• transesterification reactions - exchange of one
phosphodiester bond for another - not catalyzed by
traditional enzymes
• branch site adenosine forms 2’, 5’ phosphodiester bond
with guanosine at 5’ end of intron
intron 1
Pre-mRNA
2’OH-A branch site adenosine
exon 1 exon 2
5’ G-p-G-U
- A-G-p-G 3’
First clevage-ligation (transesterification) reaction
• ligation of exons releases lariat RNA (intron)
intron 1 Splicing
intermediate
U-G-5’-p-2’-A
A
exon 1 exon 2
5’ G-OH
O 3’ A-G-p-G
A - 3’
Second clevage-ligation reaction
intron 1
Lariat
U-G-5’-p-2’-A
A
3’ G-A
Spliced mRNA
exon 1 exon 2
5’ G-p-G 3’
Recognition of splice sites
• invariant GU and AG dinucleotides at intron ends
• donor (upstream) and acceptor (downstream) splice sites
are within conserved consensus sequences
donor (5’) splice site branch site acceptor (3’) splice site
G/GUAAGU..................…A.......…YYYYYNYAG/G
U1 U2
•small nuclear RNA (snRNA) U1 recognizes the
donor splice site sequence (base-pairing interaction)
• U2 snRNA binds to the branch site (base-pairing interaction)
Y= U or C for pyrimidine; N= any nucleotide
Spliceosome - assembly of the splicing apparatus
• snRNAs are associated with proteins (snRNPs or “snurps”)
• splicing snRNAs - U1, U2, U4, U5, U6
• antibodies to snRNPs are seen in the autoimmune
disease systemic lupus erythematosus (SLE)
= hnRNP proteins
Spliceosome assembly
intron 1
Step 1: binding of U1
and U2 snRNPs
U2
2’OH-A
exon 1 exon 2
5’ U1
G-p-G-U
- A-G-p-G 3’
intron 1 Step 2: binding of U4, U5, U6
U2 2’OH-A
exon 1
U4 U6 exon 2
5’
U5
G-p-G-U
- A-G-p-G 3’
U1
Step 3: U1 is released,
intron 1
then U4 is released
2’OH-A
U2
exon 1
U6 exon 2
5’ G-p-G-U
- U5 A-G-p-G 3’
Step 4: U6 binds the 5’ splice site and
the two splicing reactions occur,
catalyzed by U2 and U6 snRNPs
intron 1
2’OH-A
U6 U2
U-G-5’-p-2’-A
A
mRNA 3’ G-A U5
5’ G-p-G 3’
Frequency of bases in each position of the splice sites
Donor sequences
exon intron
%A 30 40 64 9 0 0 62 68 9 17 39 24
%U 20 7 13 12 0 100 6 12 5 63 22 26
%C 30 43 12 6 0 0 2 9 2 12 21 29
%G 19 9 12 73 100 0 29 12 84 9 18 20
A G G U A A G U
Acceptor sequences
intron exon
%A 15 10 10 15 6 15 11 19 12 3 10 25 4 100 0 22 17
%U 51 44 50 53 60 49 49 45 45 57 58 29 31 0 0 8 37
%C 19 25 31 21 24 30 33 28 36 36 28 22 65 0 0 18 22
%G 15 21 10 10 10 6 7 9 7 7 5 24 1 0 100 52 25
Y Y Y Y Y Y Y Y Y Y Y N Y A G G
Polypyrimidine track (Y = U or C; N = any nucleotide)
Mutations that disrupt splicing
• o-thalassemia - no -chain synthesis
• +-thalassemia - some -chain synthesis
Normal splice pattern:
Exon 1 Exon 2 Exon 3
Intron 1 Intron 2
Donor site: /GU Acceptor site: AG/
Intron 2 acceptor site mutation: no use of mutant site; use of cryptic splice site in intron 2
Translation of the retained
portion of intron 2 results
Exon 1 Exon 2 in premature termination
Intron 1 of translation due to a
stop codon within the
intron, 15 codons from
Intron 2 cryptic acceptor site: UUUCUUUCAG/G the cryptic splice site
mutant site: GG/
Intron 1 mutation creates a new acceptor splice site: use of both sites
Exon 1 Exon 2 Exon 3
Intron 2
Donor site: /GU AG/: Normal acceptor site (used 10% of the time in mutant)
CCUAUUAG/U: mutant site (used 90%of the time)
CCUAUUGG U: Normal intron sequence (never used because it does not conform to a splice site)
Translation of the retained portion of intron 1 results in termination at a stop codon in intron 1
Exon 1 (Hb E) mutation creates a new donor splice site: use of both sites
Exon 2 Exon 3
Intron 2
/GU: Normal donor site (used 60% of the time when exon 1 site is mutated)
GGUG/GUAAGGCC: mutant site (used 40%of the time)
GGUG GUGAGGCC: Normal sequence (never used because it does not conform to a splice site)
The GAG glutamate codon is mutated to an AAG lysine codon in Hb E
The incorrect splicing results in a frameshift and translation terminates at a stop codon in exon 2
