molecular biology lecture 10

8/12/2019 Molecular Biology Lecture 10

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BIOL 321 - MicroRNAs

Department of Biochemistry

St. George’s University



OBJECTIVES

- History and Discovery of microRNAs

- Biogenesis of microRNAs

- Function of microRNAs- MicroRNA targeting and mRNA regulation (degradation vs.

translational silencing)

- RNAi and medicine

- MicroRNAs and Disease

- Disease treatment with RNAi Therapy

Small RNAs in Regulation of Eukaryotic Gene Expression

1. Silencing by RNA interference (RNAi)

Historical context

2. Regulation by microRNA (miRNA)

Molecular pathway (common elements w/ miRNA)

Outcomes and functions

Historical context

Molecular pathway for miRNA productionOutcomes and functions



CRASH COURSE: mRNA Regulation

Post-transcription regulationCytoplasm

Deadenylation

Pol II

Nucleus

AA

AA

P bodies

AA

eIF3

A

A A A A A

AA AA AA AA

Pol IIPol II

**Fine-tuning by microRNAs



22 12 29 56 74 220

418

704

1073

1359

1959

0

500

1000

1500

2000

2500

N u m b e r o f m i R P u b l i c a t i o

n s

Discovery of microRNAs

Lin-4: anti-sense

RNA C. elegans

development

miRNAs

identified in

viral genomicsmiRNAsimplicated

in leukemia

miRNAs discovered in

human, mouse, and

Drosophila

miRNAs

Proposed to be

involved MRD

IMPICATED IN MANY CELLULAR FUNCTIONS

Metabolism

Cell cycle

Development

Apoptosis

Telomerase Activity

Autophagy

Oxidative Stres

T-cell Activation



All the excitement is

about this small blobof low molecular

weight RNA found at

the bottom of these

examples of RNA gelelectrophoresis.

Initially discarded

as degraded RNA



Historical discoveries in the field of microRNAs:

1990:Two independent research groups

introduced a pigment producing

transgene into petunias with the hope of

deepening the color of the flowers.

Surprisingly, the flowers were

variegated or sometimes completelywhite.

Napoli C. et al.; Plant Cell 2:279-289 (1990)

Van der Krol et al.; Plant Cell 2:291-299 (1990)

** Initially called co-suppression or

post-transcriptional gene silencing(PTGS)



1998:

Injection of small amounts of dsRNA into C. elegans produced

strong knockdown of gene function (loss of protein).

Fig. 16.29

No probe control Wild type

Antisense dsRNA

• Injection in gonads of dsRNA

for mex-3 (abundant RNA)

gave much more efficient

inhibition in embryos than

antisense RNA

• dsRNA had to include exons;

introns and promoter didn‟t work

• Effect was incredibly potent

and even spread to other cells

within the worm

• Termed „RNA Interference‟

• Incredibly useful as a tool for

molecular biology



RNAi occurs in a diverse groups of eukaryotes including:

- Protozoa - Fungi

- Plants (~600 miRNA) - Insects (~200 miRNA)

- Worms (100 - 200 miRNA) - Fish

- Birds - Mammals (~1500 miRNA)

**** 1/3 of human RNAs may be targets of miRNA

**** A complex set of biochemical mechanisms has been conserved by

evolution to facilitate microRNA mediated gene expression control



EVOLUTION of microRNA-1

Mus musculus

C. elegans

Xenopus tropicalis

Gallus gallus

Homo sapiens

100%

100%

95%

5 ’ - U G G A A U G U A A A G A A G U A U G U A U - 3 ’

T.s. elegans

*One nucleotide

change



QUICK STATs ABOUT MICRORNA



MiRNA Function: Eukaryotes process several hundred

different small RNAs

a) Cell regulation

- Embryonic development (brain)

- Cell proliferation

- Cell death

- Fat metabolism- Cell differentiation

b) Human disease **

- Chronic lympocytic leukemia- Colonic adenocarcinoma

- Burkitt‟s lymphoma

- Viral infections



Several types of naturally-forming small RNAs have been

discovered:

1) siRNA - short interfering RNA

-derived from long dsRNAs and „random‟ processing

-regulates expression by mRNA degradation

2) miRNA - micro RNA-derived from specific pre-miRNA species

-regulates expression by repressing mRNA translation

*** Additional small interfering RNAs have been identified recently having various

functions*** Artificial microRNAs are used in the laboratory to study gene function



Silencing RNA:siRNA vs. miRNA

siRNAs and microRNAs are NOT the same thing and they differ in many

ways.

siRNA are usually used in MEDICAL APPLICATIONS to cl eaves its target

mRNA or generated „randomly‟ from naturally transcri bed dsRNA.

microRNA is generated within the cell, highly conserved, rarely have perfect

complementarity with mRNA sequences. THIS IS A NATURALLY OCCURING

SYSTEM

siRNA imperfect complementary = simi lar to microRNA

microRNA perfect complementary = similar to siRNA

Origin: microRNAs often derive from conserved genomic

loci, whereas siRNAs often derive from random dsRNA or

synthetic dsRNA.

“So they are very different but also very similar.”



Biogenesis of microRNAs

Exportin

5’ 3’

PRE-miRNA

(60-80nt)

PRIMARY-miRNA

Cytoplasm

Nucleus

PRE-miRNA

Cleavage

miRNA Duplex

miRISC

Loading

Mature-miRNA

(18-22nt)

Target

mRNAmRNA Cleavage/

Translational

Repression

RNA

Pol II

Drosha

Dicer

miRISC

MicroRNA processing:

a) Transcription of a 5‟ capped, poly-A tailed primary-miRNA transcript.

b) Hair-pin structure directs DsRNA-specific nuclease Drosha. Cleavage

results in formation of 70 nt pre-miRNA.c) Nuclear export of pre-miRNA.



DicerDicerDicer

RISC

RISC

RISC

RISCRISC

RISC RISC

Dicer cleaves

dsRNA into

siRNA (20-25ntds.)

RNA-induced silencing

complex (RISC) (or other protein

complex) binds to short dsRNAsiRNA

unwinds

Short RNA directs binding of the

RISC complex to a specific

target mRNA

Dicer cleaves long

dsRNAs

RISC complex removed one

strand from the short dsRNA

RNA Interference (RNAi) / microRNA Processing

dsRNAs



Fig. 16.45

• With perfect or near -perfect

complementarity, miRNA can act like

siRNA, giving cleavage of target

• This pathway operates extensively in

plants, less so in animals

Extent of Complementarity Determines Whether

miRNA Gives Cleavage or Repression of Target

mRNA



MicroRNAs regulate

gene expression by

binding to partially

complimentary sites inthe 3‟UTR of a mRNA

and subsequent

inhibition their

translation by

interfering with

ribosome assembly

In C. elegans, lin-4 encodes a 22 nt

non-coding RNA that binds to 7

conserved sites located in the 3‟ UTR

of the lin-14 gene.



Small RNAs can form a complex with chromatin remodeling enzymes

(eg. DNA methyltransferase or histone deacetylase) to induce the

formation of transcriptional repressed chromatin.

rasiRNA



Mechanisms of siRNA Silencing

Carthew and Sontheimer, Cell (2009) 136, 642-655.

• siRNA pathway

can act at the level

of DNA also

• Imperfect base-

pairing between

guide strand andtarget can give

non-degradative

silencing (miRNA

pathway)

• Off -target

responses of

synthetic siRNA

often use this

miRNA pathway



Possible Mechanisms of Translation

Inhibition by miRNA

Carthew and Sontheimer, Cell (2009) 136, 642-655.

Translation elongation block

Does Translational Repression Involve



Does Translational Repression Involve

Competition For Cap Binding?

Mathonnet et al , Science (2007) 317, 1764-1767

• Inhibitory effect on translation reversed by

increased eIF4F concentration (at right)

• Luciferase mRNA with miRNA

binding sites for endogenous let-7

miRNA (RL-6xB)

• Decrease inferred to reflect

decreased translation initiation

• Translation also inhibited, and inhibition

eliminated with inclusion of IRES in mRNA

•

Decreased 80S peak monitored byglycerol gradient centrifugation,

rescued by competitor oligo to let-7



Antisense vs. RNAi

Antisense • Single stranded DNA oligo-

nucleotides of variablelength.

• Does not depend onspecific cellular machinery(requires dsRNA).

• Not very efficient.

• Works in all species.

RNAi • Large dsRNAs or small (21-

25 bp) siRNAs.

• Depends on presence ofparticular cellular machinery(DICER, RISC, RdRP).

• Efficiency depends on

availability of cellularmachinery in vivo.

• Full potential is limited toparticular species.

A Molecular Arms Race Between



A Molecular Arms Race Between

Virus and Host

Ding and Voinnet, Cell (2007) 130, 413-422

• Viruses could use RNAi to compromise host

• Why not mutate so that a viral sequence

corresponds to an important host sequence?

• Viruses have most likely done this, and there

is likely a continual arms race

• Even amplification and spread of an siRNA

signal could be hijacked by viruses to „prime‟

new cells for easy infection



ANTISENSE RNA in cancer and gene therapy

a) siRNAs directed against transcripts of Hepatitis B virus in

cell culture resulted in a decrease in viral levels.

b) IV injection of apolipoprotein B siRNA into mice trail veins

resulted in gene knockdown and reduced cholesterol.

c) Electropulsation of siRNAs into mouse muscle results in

gene knockdown.

d) Nasal administration of siRNAs against parainfluenza virusin mice resulted in decreased viral replication

Antisense RNA as a mode of Treatment = FUTURE?



RNAi (dsRNA) Therapy



RNAi in medicine – Knocking down the

expression of harmful genes

dsRNAs directed against

transcripts of Bcl-2 in two

studies:1) Human Melanoma MB001

cell culture

2) Human Breast Carcinoma

MCF-7 cell culture

RNAi Bcl-2 Caspase Apoptosis

(cell death)

Both studies

resulted in an

increase of

apoptotic activity

(cell death)



Human Melanoma MB 001 Cells A-Untreated B-Mock C - Bcl-2 siRNA (160 nM) D - 1600nM

A B

C D

Experiments conducted at BIOGEN

Decrease in cell number

Effect of Bcl 2 gene silencing on the induction of



Effect of Bcl-2 genesilencing on MCF-7

cell growth0

20

40

60

80

100

Untreated Mock Scrambled 200 1000 R e l a t i v e g r o w t h ( % )

siRNA (nM)

Effect of Bcl-2 gene silencing on the induction of

apoptosis in MCF-7 cells

0

2000

4000

6000

8000

10000

Unterated Mock 100 500 2000

C a s p a s e 3 / 7 a c t i v i t y

R F L U

siRNA (nM)

0

20

40

60

80

10 0

Untreated Mock 500 L e v e l o f B c l - 2 p r o t e i n

siRNA (nM)

Effect of Bcl-2 gene

silencing on Bcl-2

protein level

Effect of Bcl-2 gene

silencing on Caspase

Activity



MicroRNAs in Disease



Mir-35

5‟ 3‟

Mir-36

Mir-37

Mir-38

Mir-39

Mir-40

Mir-41

Some miRNAs in the genomeare amplified or down-

regulated in cancer tumors.

These miRNAs are typically

found to be involved in

regulating cell proliferation.

MicroRNA dysregulation and Cancer

EXAMPLE

MicroRNAs -15a, -16 and -21 are frequently found to be down-regulated in

cancer cells. These microRNAs are responsible to arresting cell

proliferation (miR-15a and -16) as well as inducing apoptosis (miR-21),

therefore promoting tumour growth.



MicroRNAs as biomarkers of Disease

i RNA i fil



microRNA expression profiles

classify human cancers

• 334 human tumor samples

• Analyzed array of 217 mammalian miRNA

• miRNA demonstrated distinct profiles in various types of cancer

• miRNA profiles better discriminated between tumor tissue of origin,compared with mRNA profiles

• miRNA „signature‟ was able to correctly classify 7/12 “carcinoma ofunknown primary” where morphology/routine pathology wasunrevealing (but the tissue source was known)

Lu et al. Nature 435: 834, 2005



microRNA expression profiles classify human

cancers

Lu et al. Nature 435: 834, 2005Samples (patients)

m i R s



NEJM 353:1793, 2005

MicroRNAs



MicroRNAs

as

Biomarkers



MicroRNAs

and RNAi have an extremely

promising future in both

disease treatment anddisease prognosis

molecular biology lecture 10

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