RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA...

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1 RNA Stability Differential Stability Regulation of Stability Iron Regulation Nonsense Mediated Decay • RNAi mRNA half life Steady State mRNA Half-Life in Hours Β-Actin 60 Dihydrofolate reductase 97 G3P dehydrogenase 75-130 Hsp70 2 Ornithine decarboxylase 0.5 C-myc 0.1

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Transcript of RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA...

  • 1

    RNA Stability

    • Differential Stability• Regulation of Stability

    • Iron Regulation

    • Nonsense Mediated Decay• RNAi

    mRNA half lifeSteady State

    mRNA Half-Life in HoursΒ-Actin 60Dihydrofolate reductase 97G3P dehydrogenase 75-130Hsp70 2Ornithine decarboxylase 0.5C-myc 0.1

  • 2

    AUUUA repeatsARE (A/U Rich Element).

    ARE are bound by proteins that stimulate deadenylation

    Two Pathways for RNA decay

  • 3

    Regulated turnover

    Transferrin Receptor

    Northern Blot for fibroblast transferrin receptor mRNA over time as a function of two different concentrations of iron.

    Hemin treatment of the cells leads to increased intracellular iron concentrations = Shorter half-life of ~45 min.

    Desferrioxamine treatment of cells leads to lower ntracellular iron concentrations = Longer half-life of >180 min.

  • 4

    Deletion analysis of Iron Responsive Region

    Iron Responsive Region

  • 5

    Comparison to ferretin IRE’s

    IRE of FerretinFerretinG

    IRE

    IBP IBP+ Fe

    High Affinity Form Low Affinity Form- Fe

    Low IronFerritinG

    IRE

    IBP

    Prevents Scanning

    High Iron

    FerritinGIRE

    Scan to start codon

    IBP Secondary structure denatured

    Scanning Blocked

    Aconitase apoprotein

  • 6

    Deletion of IRE like elements

    H – Heme treated cellsD – desferrioxamine treated cells (iron chelator)

    Nuclease Action

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    Transferrin Receptor Regulation The Model

    Non-sense Mediate Decay(NDM)

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    NDM requires an intron

    Exon Junction Complex

    Alberts

  • 9

    Deadenylation independent decapping

    RNA Interference(RNAi)

    – Gene silencing with dsRNA– Generation of miRNA from dsRNA– RISC targeted mRNA destruction– Translational Inhibition– Amplification of RNAi– Transcriptional Inactivation

  • 10

    Fire and Mello• GFP Transgenic Worms• Injected Sense RNA, Antisense RNA and Mixture of antisense and

    sense RNA into ovaries• Observe GFP expression in offspring

    Double Stranded RNA and Dicer

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    RISC and Argonaute

    RNAi and Translation

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    Translational Inhibition

    RNAi Amplification by RNA dependent RNA Polymerase

    AAAAAAA

    siRNA primer generated by dicer

    AAAAAAA

    RdRP

    AAAAAAA

    RdRP

    AAAAAAA

    ds RNA generated which is a new target for dicer

    RNA dependent RNA Polymerase

  • 13

    Overview of RNAi

    Rad21 = cohesine

    Chromodomain promotes spread of silencing

    RNAi Transcriptional Silencing

  • 14

    miRNA’s

    Microprocessor Complex

    Illustration of miRNA

    processing

  • 15

    Differences in miRNA Mode of Action

    RNA StabilitymRNA half life�Steady StateAUUUA repeats�ARE (A/U Rich Element).Two Pathways for RNA decayRegulated turnoverTransferrin ReceptorDeletion analysis of Iron Responsive RegionIron Responsive RegionComparison to ferretin IRE’sIRE of FerretinDeletion of IRE like elementsNuclease ActionTransferrin Receptor Regulation The ModelNon-sense Mediate Decay�(NDM) NDM requires an intronExon Junction ComplexRNA Interference�(RNAi)Fire and MelloDouble Stranded RNA and DicerRISC and ArgonauteRNAi and TranslationTranslational InhibitionRNAi Amplification by RNA dependent RNA PolymeraseRNAi Transcriptional SilencingmiRNA’sIllustration of miRNA processingDifferences in miRNA Mode of Action