1. Last modified 3/24/05Burke - C485 Lecture 29/30 - RNA Processing بسم الله الرحمن الرحیم تهیه کننده : علی قنبری دانشجوی کارشناسی ارشد بیوتکنولوژی کشاورزی استاد راهنما : جناب آقای دکتر باباییان

2. Last modified 3/24/05Burke - C485 Lecture 29/30 - RNA Processing RNA processing 1)How do RNA processing events expand the vocabulary of mRNA? 2)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. Last modified 3/24/05Burke - C485 Lecture 29/30 - RNA Processing 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. Last modified 3/24/05Burke - C485 Lecture 29/30 - RNA Processing Processing of eukaryotic mRNAs Capping Polyadenylation Splicing = intron removal/exon ligation post-transcriptional: co-transcriptional: Triose phosphate isomerase gene Site of cleavage and polyadenylation 5’3’ Transcription/capping Splicing/polyadenylation mRNA 5’ m 7 GTPAAAA … AAA-3’ INTRONS stay IN the nucleus Exon 1Exon 2Exon 3Exon 4Exon 5Exon 6 Exon 7 Exon 8Exon 9 Mature, spliced mRNA Export to cytoplasm

5. Last modified 3/24/05Burke - C485 Lecture 29/30 - RNA Processing The 5’-cap structure 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? 7mG Cap of Euk. mRNAs

6. Last modified 3/24/05Burke - C485 Lecture 29/30 - RNA Processing Polyadenylation of pre-mRNA

7. Last modified 3/24/05Burke - C485 Lecture 29/30 - RNA Processing 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. Last modified 3/24/05Burke - C485 Lecture 29/30 - RNA Processing 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 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’ Exon Intron 50 to >1000 nucleotides 5’ splice site consensus sequence Branch site consensus sequence 3’ splice site consensus sequence 10 to 40 nucleotides Polypyrimidine tract

9. Last modified 3/24/05Burke - C485 Lecture 29/30 - RNA Processing Alternative splicing allows expansion of genetic information 123 45678 9 17 1110 16 1412131518 1-3 combination of exons 4-8 9-15 16 or 17 18 Alternative pre-mRNA splicing

10. Last modified 3/24/05Burke - C485 Lecture 29/30 - RNA Processing Group I intron self-splicing G G -OH G- AGAG OH -G--G- AGAG Step 1 Step 2 + 5’ 3’ 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. Last modified 3/24/05Burke - C485 Lecture 29/30 - RNA Processing 5’ I II III Stem II Stem III Murray et al, Mol Cell. 2000; 5 Stem I Catalytic core 3’5’ 3’ 5’ I II III Hammerhead: a small self-cleaving ribozme

12. Last modified 3/24/05Burke - C485 Lecture 29/30 - RNA Processing Hammerhead: a small self-cleaving ribozyme C U G AUG A GGCC G A A A GGCC G A A A C A U U AGG A U G U C 5’ G G CGACC GCUGG C U G AUG A GGCC G A A A GGCC G A A A C A U U AGG A U G U C G G CGACC GCUGG Internal guide sequences bind substrate, determine sequence specificity of cleavage Conserved catalytic core + Cleavage products