6' RNA Processing

1
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
2

• 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
3

• 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
• capping involves formation of a 5- 5
  triphosphate bond
• cap function
• protects 5 end of mRNA (increases mRNA
  stability)

pppNpN
mGpppNmpNm
4

• 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
• poly(A) is associated with poly(A) binding
  protein (PBP)
• function of poly(A) tail is to stabilize mRNA

cleavage
AAUAAA
mGpppNmpNm
polyadenylation
AAUAAA
A
A
A
A
mGpppNmpNm
A
A
3
5

• 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
branch site adenosine
2OH-A
exon 1
exon 2
G-p-G-U A-G-p-G
-
5
3
First clevage-ligation (transesterification)
reaction
6

• ligation of exons releases lariat RNA (intron)

intron 1
Splicing intermediate
U-G-5-p-2-A
A
exon 1
exon 2
G-OH 3 A-G-p-G
5
3
A
O
-
Second clevage-ligation reaction
intron 1
Lariat
U-G-5-p-2-A
A
3 G-A
Spliced mRNA
exon 1
exon 2
G-p-G
5
3
7

• Recognition of splice sites
• invariant GU and AG dinucleotides at intron ends
• donor (upstream) and acceptor (downstream)
  splice sites
• are within conserved consensus sequences
• 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

donor (5) splice site
acceptor (3) splice site
branch site
G/GUAAGU..................A.......YYYYYNYAG/G
U1
U2
8

• 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 Step 1 binding of U1 and
U2 snRNPs
intron 1
2OH-A
exon 1
exon 2
G-p-G-U A-G-p-G
-
5
3
9
intron 1
Step 2 binding of U4, U5, U6
2OH-A
U4 U6
exon 1
exon 2
U5
G-p-G-U A-G-p-G
-
5
3
U1
Step 3 U1 is released, then U4 is released
intron 1
2OH-A
U6
exon 1
exon 2
G-p-G-U A-G-p-G
-
5
3
10
Step 4 U6 binds the 5 splice site and the two
splicing reactions occur, catalyzed by U2 and U6
snRNPs
intron 1
2OH-A
U2
U-G-5-p-2-A
A
3 G-A
mRNA
G-p-G
5
3
11
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 2
9 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)
12

• Mutations that disrupt splicing
• bo-thalassemia - no b-chain synthesis
• b-thalassemia - some b-chain synthesis

Normal splice pattern
Donor site /GU
Acceptor site AG/
Intron 2 acceptor site bo mutation no use of
mutant site use of cryptic splice site in intron
2
Translation of the retained portion of intron 2
results in premature termination of translation
due to a stop codon within the intron, 15 codons
from the cryptic splice site
Exon 1
Exon 2
Intron 1
Intron 2 cryptic acceptor site UUUCUUUCAG/G
mutant site GG/
13
Intron 1 b mutation creates a new acceptor
splice site use of both sites
AG/ Normal acceptor site (used 10 of the time
in b mutant)
Donor site /GU
CCUAUUAG/U b mutant site (used 90of 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 b 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 b mutant site (used 40of 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
14

• 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
15
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