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
e). Alternative splicing
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
• βo-thalassemia - no β-chain synthesis
• β+-thalassemia - some β-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 βο mutation: no use of mutant site; use of cryptic splice site in intron 2
Translation of the retained
portion of intron 2 results in
Exon 1 Exon 2 premature termination of
Intron 1 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 β+ 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 β+ mutant)

CCUAUUAG/U: β+ 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 β+ 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: β+ 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
Patterns of alternative exon usage
• one gene can produce several (or numerous) different
but related protein species (isoforms)

Cassette

Mutually exclusive

Internal acceptor site

Alternative promoters
The Troponin T (muscle protein) pre-mRNA
is alternatively spliced to give rise to
64 different isoforms of the protein

Constitutively spliced exons (exons 1-3, 9-15, and 18)

Mutually exclusive exons (exons 16 and 17)

Alternatively spliced exons (exons 4-8)

Exons 4-8 are spliced in every possible way

giving rise to 32 different possibilities

Exons 16 and 17, which are mutually exclusive,

double the possibilities; hence 64 isoforms