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. Promoter Transcription Cleavage (the light pink regions are degraded) 45S transcript 18S5.8S28S 53 18S5.8S28S 18S5.8S28S rRNA Processing of 45S rRNA Precursor

4. 35 Endonuclease Exonuclease (RNase D) P = Pseudouridine T = Thiouridine I = Inosine m G = Methylguanosine RNase P mGmG T T A P P I C C Anticodon Processing of pre-tRNAs

5. snoRNAs Yeast C/D box snoRNA D’ box C’ box

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. Figure 12.19 Removes the  -phosphate Capping – Step 1

8. Capping –Step 2 Attaches GMP to the 5’ end OH Figure 12.19

9. OH Figure 12.19 Adds methyl groups to guanine & riboses Capping – Step 3 OH O CH 3

10. 5 3 5 5 3 Endonuclease cleavage occurs about 20 nucleotides downstream from the AAUAAA sequence. PolyA-polymerase adds adenine nucleotides to the 3  end. Polyadenylation sequence PolyA tail AAAAAAAAAAAA.... AAUAAA Polyadenylation G/U

11. Splicing – Intron Sequences 3 5 5 splice site3 splice site Branch site IntronExon Py 12 N PyAGG A / C GGU Pu AGUA UACUUAUCC Exon n ……A G G U A A G U …Intron …Y N Y Y R A Y …....Y 12 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 Branch Point Yeast consensus

12. Intron loops out and exons brought closer together Figure 12.22 Splicing of Pre-mRNAs

13. Splicing of Pre-mRNA This is a splicosome

14. Figure 12.22 Intron will be degraded and the snRNPs used again Splicing of Pre-mRNA

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 U2 snRNA Mutations in branch points prevent splicing Compensatory mutations in U2 snRNA restore splicing

19. Alternative Splicing of Tropomyosin *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 AGTgtaaat AGTgtaagt cat/gtaagt Wildtype myostatinMutant myostatin Exon 1 Exon 2