RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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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

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AUUUA repeatsARE (A/U Rich Element).

ARE are bound by proteins that stimulate deadenylation

Two Pathways for RNA decay

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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.

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Deletion analysis of Iron Responsive Region

Iron Responsive Region

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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

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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

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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

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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

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Overview of RNAi

Rad21 = cohesine

Chromodomain promotes spread of silencing

RNAi Transcriptional Silencing

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miRNA’s

Microprocessor Complex

Illustration of miRNA

processing

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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