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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 5¢ 18S 5.8S 28S 3¢ Cleavage (the light pink regions are degraded) 18S 5.8S 28S rRNA rRNA rRNA
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Processing of pre-tRNAs
Endonuclease 5¢ 3¢ A C RNase P Exonuclease (RNase D) C T T mG P mG = Methylguanosine P P = Pseudouridine I T = Thiouridine I = Inosine Anticodon
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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 Timing of translation/storage of mRNA Splicing Production of varied proteins from same gene Developmental regulation – tissue specificity, sex determination
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Removes the -phosphate
Capping – Step 1 Removes the -phosphate Figure 12.19
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Attaches GMP to the 5’ end
Capping –Step 2 Attaches GMP to the 5’ end OH Figure 12.19
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Adds methyl groups to guanine & riboses
Capping – Step 3 OH OH Adds methyl groups to guanine & riboses OH O CH3 Figure 12.19
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Polyadenylation Polyadenylation sequence 5¢ 3¢
G/U 5¢ AAUAAA 3¢ Endonuclease cleavage occurs about 20 nucleotides downstream from the AAUAAA sequence. 5¢ AAUAAA PolyA-polymerase adds adenine nucleotides to the 3¢ end. 5¢ AAAAAAAAAAAA.... AAUAAA 3¢ PolyA tail
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Splicing – Intron Sequences
Exon Intron Exon 5¢ UACUUAUCC A/CGGU Pu AGUA Py12N PyAGG 3¢ 5¢ splice site Branch site 3¢ splice 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 n+1 Yeast consensus
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Intron loops out and exons brought closer together
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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Intron will be degraded and the snRNPs used again
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
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