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Post-transcriptional RNA Modifications

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Post-transcriptional RNA Modifications

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  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 5¢ 3¢ 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 OH OH Attaches GMP to the 5’ end Figure 12.19

  9. Capping – Step 3 OH O 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 Exon Intron Exon UACUUAUCC 3¢ Py12N PyAGG 5¢ A/CGGU Pu AGUA 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 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 2 Exon 1 Exon 1 Exon 2 cat/gtaagt Exon 1 Exon 2 Exon 1 Exon 2