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Post-transcriptional RNA Modifications

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Title: Post-transcriptional RNA Modifications


1
Post-transcriptional RNA Modifications
  • Gene Expression Part 2

2
Post-transcriptional Modifications
  • Processing cleavage of 45S rRNA precursor,
    base modifications of tRNAs, rRNAs
  • Capping mRNAs snRNAs
  • Polyadenylation - mRNAs
  • Splicing mRNAs, some tRNAs

3
Processing of 45S rRNA Precursor
Promoter
18S
5.8S
28S
Transcription
45S transcript
18S
5.8S
28S
5
3
Cleavage (the light pink regions are degraded)
18S
5.8S
28S
rRNA
rRNA
rRNA
4
Processing of pre-tRNAs
Endonuclease
3
5
A
C
Exonuclease (RNase D)
RNase P
C
T
T
mG
P
mG Methylguanosine
P
P Pseudouridine
I
T Thiouridine
I Inosine
Anticodon
5
snoRNAs
D box
C box
Yeast C/D box snoRNA
6
Post-transcriptional Modifications Regulate Gene
Expression
  • Capping
  • Stability
  • Translation efficiency
  • Polyadenylation
  • Stability
  • Translation efficiency
  • Timing of translation/storage of mRNA
  • Splicing
  • Production of varied proteins from same gene
  • Developmental regulation tissue specificity,
    sex determination

7
Capping Step 1
Removes the ?-phosphate
Figure 12.19
8
Capping Step 2
Attaches GMP to the 5 end
Figure 12.19
9
Capping Step 3
OH
OH
Adds methyl groups to guanine riboses
CH3
Figure 12.19
10
Polyadenylation
Polyadenylation sequence
G/U
5
3
AAUAAA
Endonuclease cleavage occurs about 20 nucleotides
downstream from the AAUAAA sequence.
5
AAUAAA
PolyA-polymerase adds adenine nucleotides to the
3 end.
AAAAAAAAAAAA....
5
3
AAUAAA
PolyA tail
11
Splicing Intron Sequences
Intron
Exon
Exon
UACUUAUCC
3
5
Py12N PyAGG
A/CGGU Pu AGUA
5 splice site
3 splice site
Branch site
Branch Point
Exon n A G G U A A G U Intron Y
N Y Y R A Y ....Y12 N C A G N ..
Exon n1 64 73 100 100 62
68 84 63 80 80 87 75 100
95 65 100 100
Yeast consensus
12
Splicing of Pre-mRNAs
Intron loops out and exons brought closer together
Figure 12.22
13
Splicing of Pre-mRNA
This is a splicosome
14
Splicing of Pre-mRNA
Intron will be degraded and the snRNPs used again
Figure 12.22
15
Spliceosomal Cycle
16
Association of snRNAs with Pre-mRNA
17
Spliceosomal Complex
18
U2 snRNA interaction with branch point shown by
compensatory mutagenesis
Potential interaction based on sequence
complementarity
Mutations in branch points prevent
splicing Compensatory mutations in U2 snRNA
restore splicing
U2 snRNA
19
Alternative Splicing of Tropomyosin


Alternative pA


Alternative pA
20
Alternative Splicing Regulates Drosophila Sex
Determination
21
Myostatin Mutations in Cattle, Dogs, Mice
Belgian Blue
Whippet
Mouse
22
Myostatin Splice Site Mutation
Wildtype myostatin
Mutant myostatin
Exon 1
Exon 2
Exon 1
Exon 2
AGTgtaagt
AGTgtaaat
Exon 1
Exon 2
Exon 1
Exon 2
cat/gtaagt
Exon 1
Exon 2
Exon 1
Exon 2
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