lecture10 - RNA Processing and Export 1001 The 5 Cap(m7Gppp...

1001
RNA Processing and Export

1002
5'-->5' linkage
The 5' Cap (m
7
Gppp): Structure and function
Several enzymatic reactions add the m7Gppp cap
to pre-mRNA :
1. Step 1: γ -phosphate removal from 5 '-end of RNA
2. Step 2: Transfer of GM P from GTP to the
5 ' diphosphate.
3. Step 3: Transfer of methyl groups from
S-adenosylmethionine to the N7 position and the
2 ' -oxygens of ribose
(In animals and plants, not yeast)
(In vertebrates)
Functions of the cap:
1. Splicing
2. RNA stability
3. Export
4. Translation
Enzyme involved: RNA triphosphatase, capping
enzyme, guanine-7-methyl transfrase, 2 ’ -O-
methyltransferase

1003
AAAAA
(100-250)
Polyadenylation of mRNA
1
2
3
4
1
2
3
4
Factors co-transcriptionally bind two sequences in the RNA:
the upstream AAUAAA signal
the downstream GU- or U-rich sequence
Poly(A) polymerase binds and facilitates cleavage
at the poly(A) site.
Bound poly(A) polymerase, in a slow phase, adds
about 12 A ’ s to the 3' end of the RNA.
Poly(A)-binding protein accelerates poly(A) addition
by poly(A) polymerase.
Functions of polyadenylation :
1. Protect 3'-ends of RNAs
2. Alternative processing generates different messages
3. RNA export
4. Translation (see p. 131 fig. 4-31)

1004
TBP
mRNA
sequence

1005
mRNA Splicing
maturing the message
diversifying the message

1006

1007
exons introns
3’ UTR 5’ UTR cap poly(A) tail promoter ORF
eukaryotic class II gene structure

1008
mRNA Splicing and the Human Genome
(~2.9 x 10
9
bp)
[ ]
Introns (~30%)
Exons (~1.2%)
Avg. exon length 145 nt
Avg. intron length 3365 nt
Avg. of 10-12 introns/gene

1009
Splice Site Consensus Sequences
Yeast
Mammalian
5' Splice
site
3' Splice
site
Branch
point
Exon 1
Exon 2
GUAUGU
UACUAAC PyAG
Exon 1 Exon 2
GU
A
G
AGN NNCUNAC PyAG
Exon 1 Exon 2
Splicing

1010
Splice sites sequences are conserved
Three conserved sequence elements are essential for splicing
The 5 ’ -splice site (ss), the 3 ’ -splice site (ss) and the branch
point site (BPS)
20-50 nts upstream of the 3 ’ ss a conserved adenine
surrounded by a 11 nt polypyrimidine tract serves as a branch point; The
entire conserved sequence is known as the BPS

1011
The splicing reaction
An intron is removed in a