بسم الله الرحمن الرحیم

1. تهیه کننده : علی قنبری دانشجوی کارشناسی ارشد بیوتکنولوژی کشاورزی استاد راهنما: جناب آقای دکتر باباییان بسم الله الرحمن الرحیم

2. RNA processing How do RNA processing events expand the vocabulary of mRNA? What steps are involved in changing the linear sequence of RNA between transcription and its ultimate use in translation or for other purposes? 3) What is the selective/evolutionary advantage to expanding the RNA vocabulary? 4) Why are RNAs modified so extensively?

3. 3 general types of RNA processing The biologically active forms of most RNAs are post-transcriptionally modified, especially in eukaryotes. 1) Remove nucleotides (splicing, site-specific endonucleases, deletional editing) 2) Addition of nucleotides (5' capping, 3' terminal transferases, insertional editing) 3) Covalent modification of bases or sugars (methyl caps, ADAR, tRNA/rRNA)

4. Processing of eukaryotic mRNAs Triose phosphate isomerase gene Site of cleavage and polyadenylation INTRONS stay IN the nucleus 5’ 3’ Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Exon 8 Exon 9 Transcription/capping Splicing/polyadenylation mRNA 5’ m7GTP AAAA … AAA-3’ Mature, spliced mRNA Export to cytoplasm Capping Polyadenylation Splicing = intron removal/exon ligation co-transcriptional: post-transcriptional:

5. The 5’-cap structure 7mG Cap of Euk. mRNAs • Can protect RNA from degradation • Identifies RNA as mRNA • Recognized by protein synthesis machinery • 2,2,7-trimethyl G cap is NOT used • by mRNA; it identifies snRNAs • (used in splicing), localizes them • to nucleus • tRNAs/rRNAs not capped Where have we seen 5’, 5’ linkages before?

6. Polyadenylation of pre-mRNA

7. Poly (A) tails • 100 - 250 Adenosines added to 3’ end of mRNA • Polyadenylation increases efficiency of translation, but is • not required for translation in vitro • Poly-A tail bound by “poly-A-binding protein” • PAB-II in nucleus, PAB-I in cytoplasm • protects message from exonucleases • • Tail length and stability is highly regulated---governs mRNA turnover

8. Formation of the spliceosome • The spliceosome is a large RNA-protein complex which catalyzes splicing reactions • It contains 45 proteins and 5 small nuclear RNA (snRNA) molecules (“snurps”) • Assembles at the appropriate 5’ splice site • “GU-AG” rule Exon Intron Exon 5’ … A G G U A/G A G U … … … N Y U R A Y … … Y Y Y Y Y Y Y Y Y N C A G G … 3’ 50 to >1000 nucleotides 10 to 40 nucleotides 5’ splice site consensus sequence Branch site consensus sequence Polypyrimidine tract 3’ splice site consensus sequence

9. Alternative splicing allows expansion of genetic information 4 5 6 7 8 16 17 1 2 3 9 10 11 12 13 14 15 18 Alternativepre-mRNA splicing combination of exons 4-8 1-3 9-15 18 16 or 17

10. -G- Group I intron self-splicing G 3’ G- G-OH AG 5’ 5’ Step 1 Step 2 OH + AG 3’ 5’ Differs from pre-mRNA splicing in several ways: all information regarding splice site recognition and catalysis is internal to RNA sequence 1st step of splicing uses exogenous guanosine as first nucleophile found in rRNAs of certain organisms such as Tetrahymena thermophila Engineered forms are being used in gene therapy approaches.

11. III 3’ 5’ 5’ 3’ Stem II 5’ 5’ Stem I I II III 5’ Catalytic core Stem III 5’ I II Murray et al,Mol Cell. 2000; 5 Hammerhead: a small self-cleaving ribozme

12. A G G 5’ A U U G U U A C 5’ C A G G 5’ U A G G U C G A U A A U A G A G G G C C C A C C A G C U A C G A C C G G A U G G C A G A G U A 5’ G G U C G G A G G C C A C C A G C C G A C C G G U G G A G G U A 5’ Hammerhead: a small self-cleaving ribozyme Internal guide sequences bind substrate, determine sequence specificity of cleavage Cleavage products + Conserved catalytic core