BIO 344 1st Edition Lecture 14 Outline of Last Lecture I. Post transcriptional regulation of gene expressiona. Alternative splicing, capping, poly adenylationb. Export and translationII. 5’ capa. Why cap—effect on exportIII. 3’ poly A taila. Why adenylation—effect on export and translationIV. Capping, splicing, and poly adenylation for efficient exporta. Coordination of processing eventsV. RecyclingOutline of Current Lecture I. RNA editinga. Glutamate receptori. NMDA and AMPAb. Modification by ADARc. Other RNA directed modificationsCurrent LectureRNA editing- RNA editing by ADAR proteinso Modification guided by small RNAs- Glutamate receptoro Homozygous null mutant mice—non functioning glutamate receptor (AMPA) Mice die within several weeks (20 days) after birth Develop seizures, agitation, neural defectso RNA editing role Hippocampuslearning, short term memoryThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Atrophies without glutamate receptor NMDA and AMPA receptor in post synaptic cell I normal neurons- Weak stimulation activates AMPA depolarization- Can depolarize to expel magnesium from NMDA receptor and thusactivate NMDA to take up glutamate and increase AMPA in the membrane GluR AMPA forms heterotetromer of Glu 1, 2, 3, 4 or Glu A, B, C, D- GluB mutated in the mice- GluR B—CAG codon on DNA codes for glutamine but protein contains argening at that site in wild typeo Argenine is critical for preventing ion flux through Glu R-Bo How can argenine be there? Possibilities: something at the translational level, post-translational level, or RNA level- Discussing the RNA level, how could you determine if the CAGCGG is encoded by RNA?o mRNA with DNA primer reverse transcriptase cDNA- how did AG?o ADAR= adenosine deaminase association with RNA Adenosine modified amino group inosine Inosine= pairs with C just like G does ADAR enzyme edits double stranded RNA- Guide RNA for editing GluRo QR site—downstream introno Take GluR-B -/- (null mouse) Add GluR-B gene lives Add cDNA (no introns) GluR-B dieso The editing site is in a region that’s predicted to fold into double stranded RNAo [EXON—QR editing site]------------------ (intron) Intron folds back and base pairs making double stranded RNA Without the intron, no basepairing with QR site ADAR binds and changes AI Cotranscriptional because depends on the intron- ADAR2 -/- mice also die within 20 days of birth, just as GluR-Bo Does this prove correlation? Yes. Does this prove causation? No.o But ADAR2 is the enzyme required for GluR-B editing, so how can we prove causation? In vitro?- Take a GluR-B pre mRNA and add ADAR 2 to see if A changes to I- No—does not prove causation because, in vitro, telomerase can use its RNA component as a template Look at an ADAR -/- mutant’s RNA before death?- See that CAG does not change to I in cDNA sequence- Yes, shows that ADART is required for AIo Or, what happens if mutant cDNA already encoding the argenine codon is put into a GluR-B mutant mouse? ADAR is bypassed because it’s job has been done for it These mice live- Other RNA directed RNA modificationso rRNA is highly modified pseudouridineo cotranscriptional modification as rRNa is transcribed, it’s modified at dozens of sites by 2 enzymes how do they recognize so many targets? - Guide RNAs= noncoding small RNAs (ncRNA)o snoRNAs and microRNAs direct the 2 enzymes to a numberof targets RNA guided RNA modificationo snoRNA can also target a codon and modify it to correct mutations ex: modify a stop codon in a mutant to pseudouridine because RNA will not recognize it or stop at
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