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Page 1: 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

Page 2: RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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AUUUA repeatsARE (A/U Rich Element).

ARE are bound by proteins that stimulate deadenylation

Two Pathways for RNA decay

Page 3: RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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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.

Page 4: RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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Deletion analysis of Iron Responsive Region

Iron Responsive Region

Page 5: RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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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

Page 6: RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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Deletion of IRE like elements

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

Nuclease Action

Page 7: RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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Transferrin Receptor Regulation The Model

Non-sense Mediate Decay(NDM)

Page 8: RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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NDM requires an intron

Exon Junction Complex

Alberts

Page 9: RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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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

Page 10: RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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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

Page 11: RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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RISC and Argonaute

RNAi and Translation

Page 12: RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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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

Page 13: RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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Overview of RNAi

Rad21 = cohesine

Chromodomain promotes spread of silencing

RNAi Transcriptional Silencing

Page 14: RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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miRNA’s

Microprocessor Complex

Illustration of miRNA

processing

Page 15: RNA Stability - Buffalo State Collegefaculty.buffalostate.edu/wadswogj/courses/305/Lectures/RNA Stability.pdfRNA Stability • Differential Stability • Regulation of Stability •

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Differences in miRNA Mode of Action


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