Research Techniques Made Simple:How MicroRNAs Modify Protein Production

Jessica L. Feig PhD1, Keith M. Giles PhD2, Iman Osman MD2, Andrew G. Franks, Jr. MD2

1Medical Scientist Training Program, New York University School of Medicine, New York, New York USA 10016

2Ronald O. Perelman Department of Dermatology, New York University School of Medicine,New York, New York, USA

Introduction• The central dogma of molecular biology

includes DNA replication, mRNA transcription, and protein translation, stages at which regulation takes place.

• RNA interference defines a level of post-transcriptional control.

• MicroRNAs are examples of RNA interference.

aaaProtein

BProtein

B

Protein C

Protein C

Protein A

Protein A

Biological Pathway

aaa

aaa

aaa

a b

Mode of Action of MicroRNAs

microRNA Processing

Translation repression

Dicer

Exportin 5DROSHA

PASHA

DROSHAPASHA

Argonaute

mRNA destabilization

Rb

deAaaa

Methods for MicroRNA: Identification and Characterization

Whole Genome MicroRNA Array

microRNA identification

Phenotypic Confirmation

Western Blots

ConMiR + - - - MiR - + - +

Con Inh + - - - anti-miR - - + +

Target Prediction

Conservation of miR Site in the 3’UTR

Computational Biology TargetScan/MiRBase

3’ UTR Luciferase Assays

Target Validation

aaa

aaa

aaa

3’ UTR Luciferase AssayPromoter

Luciferase 3’UTR microRNA+cotransfection

transcriptionaaa

aaa

Translation repression

mRNA destabilization

Reduced or No Signal

aaatranslation

A)

B)

Luciferase

Breakthrough Discoveries in miRNA Biology: a special focus on the field of dermatology

2006: MiRNA biogenesis is required for skin development; epidermal specific deletion of Dicer

1993:First miRNA, lin4, discovered in C. elegans.

2009: epidermal –specific ablation of pasha shows an abnormal skin phenotype with evaginating hair follicles.

2008: MiR203 – targeting p63 identified as a master regulator of epidermal differentiation.

2010: miRNA 31 implicated in the hair cycle.

2011: miRNA 125b is a repressor of stem cell differentiation

2007: MiRNAs regulated in psoriasis and atopic Dermatitis

1993 2006 2007 2008 2009 2010 2011

2010: miRNAs first implicated in Sezary syndrome

2010: miRNAs expression in mycosis fungoides

2010: miRNA signature in malignant melanoma

2014:anti-miRNA 21 (increases TIMP) used to therapeutically modulate psoriatic pathogenesis with success.

2012 2013 2014

2014:Serum miRNAs identified in the prediction and detection of recurrence in melanoma patients

Shunsuke Noguchi(2014) 134, 461-469. doi:10.1038/jid.2013.310

Figure 4

e

f

MiR-203 Regulates Kinesin at the mRNA and Protein level

MiRNAs• miRNAs modulate transcription and repress

translation, providing a level of control before the production of protein can exert biological effects.

• A single miRNA can have multiple targets, potentially providing simultaneous regulation of many genes involved in a physiological pathway.

• Multiple miRNAs with the same target reinforce the developmental program via redundant effectors of regulation.

Limitations• Delivery limitations: Since miRNAs do not freely diffuse

into cells, special delivery approaches may be needed for both experimental and therapeutic design.

• Instability: Small RNAs are unstable and may be degraded upon entering a cell.

• Off-target effects: miRNA therapy can target an array of different gene products, with the possibility of undesired effects.