Post-transcriptional RNA Modifications

1
Post-transcriptional RNA Modifications

• Gene Expression Part 2

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Post-transcriptional Modifications

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

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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
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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
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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

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Capping Step 1
Removes the ?-phosphate
Figure 12.19
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Capping Step 2
Attaches GMP to the 5 end
Figure 12.19
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Capping Step 3
OH
OH
Adds methyl groups to guanine riboses
CH3
Figure 12.19
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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
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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
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Splicing of Pre-mRNAs
Intron loops out and exons brought closer together
Figure 12.22
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Splicing of Pre-mRNA
This is a splicosome
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Splicing of Pre-mRNA
Intron will be degraded and the snRNPs used again
Figure 12.22
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Spliceosomal Cycle
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Association of snRNAs with Pre-mRNA
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Spliceosomal Complex
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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
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Alternative Splicing of Tropomyosin


Alternative pA


Alternative pA
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Alternative Splicing Regulates Drosophila Sex
Determination
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Myostatin Mutations in Cattle, Dogs, Mice
Belgian Blue
Whippet
Mouse
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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