miRNA & siRNA

gooseliverBiotechnology

Oct 22, 2013 (3 years and 7 months ago)

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miRNA & siRNA

Regulation of Gene
Expression by RNA


Brian Reinert

Traditional RNAs

What is RNA?

R
ibo
n
ucleic
a
cid


Ribonucleotides (Ribose, base, & phosphate)

Types


Coding: messenger RNA (mRNA)


Non
-
coding:

Ribosomal RNA (rRNA)

Transfer RNA (tRNA)

Small nuclear RNA (snRNA)

Small nucleolar RNA (snoRNA)

Interference RNA (RNAi)

Short interfering RNA (siRNA)

Micro RNA (miRNA)

mRNA Structure

Coding region

Untranslated regions


5’ UTR

7methyl
-
G cap


Bound by cap binding proteins

Translation regulation


3’ UTR

Stability elements

Subcellular localization (zip codes)

poly(A) tail



mRNA

Timeline for RNAi Dicsoveries

Nature Biotechnology


21
, 1441
-

1446 (2003)


Hot Topics

As of today a PubMed search for siRNA
retrieved 4617 journal articles since
2001

A search for miRNA retrieved 530
journal articles since 2001

RNAi = Big
Money?

Nature Biotechnology


21
, 1441
-

1446 (2003)


What is the Difference between
miRNA and siRNA?

Function of both species is regulation of gene
expression

Difference is in where they originate

siRNA originates with dsRNA

siRNA is most commonly a response to foreign RNA
(usually viral) and is often 100% complementary to
the target

miRNA originates with ssRNA that forms a hairpin
secondary structure

miRNA regulates post
-
transcriptional gene expression
and is often not 100% complementary to the target


miRNA Details

Originate from capped & polyadenylated
full length precursors (pri
-
miRNA)

Hairpin precursor ~70 nt (pre
-
miRNA)

Mature miRNA ~22 nt (miRNA)

First discovered in 1993 by Victor
Ambros at Harvard (
lin
-
4
)

Let
-
7

discovered in 2000 by Frank
Slack as a postdoc at Harvard (Ruvkun
lab)


Illustration
of miRNA
processing

Another View

Microprocessor
Complex

Processing
bodies are
sites of
storage
and/or
degradation
of mRNA

Summary of Players

Drosha and Pasha are part of the
“Microprocessor” protein complex (~600
-
650kDa)

Drosha and Dicer are RNase III enzymes

Pasha is a dsRNA binding protein

Exportin 5 is a member of the karyopherin
nucleocytoplasmic transport factors that
requires Ran and GTP

Argonautes are RNase H enzymes

Players

What are the functions of
miRNA?

Involved in the post
-
transcriptional
regulation of gene expression

Important in development

Metabolic regulation (miR
-
375 & insulin
secretion)

Multiple genomic loci (different
expression patterns?)

Differences in miRNA Mode of
Action

miRNA Registry

http://www.sanger.ac.uk/Software/Rfa
m/mirna/index.shtml

Latest release contains 1620 2909
predicted and verified miRNAs

227 321 predicted and 131 223
experimentally verified in
Homo sapiens

Mouse and human are highly conserved

Human is not conserved with plants

siRNA

Cellular response to foreign RNA

Modification of histones/DNA*

New tool for researchers


Can knock down gene expression


Transient or stable expression


Several different methods of expression


Several different methods of delivery

Many companies sell predesigned siRNA
guaranteed to knockdown gene expression

Design your own

siRNA Design

Initial use of longer dsRNA lead to a non
-
specific Type I interferon response
(widespread changes in protein
expression

apoptosis)

Dr. Thomas Tuschl’s lab discovered that RNAi
is mediated by 21 and 22 nt RNAs

Also discovered the important characteristics
needed by the RNAs

Worked with Dharmacon to offer technology
to the public

Further Improvements

Modified nuclease resistant RNAs

Integrated DNA Technologies (IDT)
discovered that Dicer substrates
increase siRNA potency by up to 100
fold

Better methods of delivery and
expression

siRNA Expression

For transient expression: duplex RNA
can be delivered to the cell

For a stable expression: a vector
containing the DNA to produce a hairpin
RNA

The vector may be plasmid, retrovirus,
adenovirus

siRNA Delivery

For cell culture


Lipid
-
based transfection


Electroporation

In vivo


Lipid
-
based


Conjugations

Bacterial phage RNA

Cholesterol

Atelocollagen


Viral systems (ie retrovirus & adenovirus)

siRNA Delivery & Processing

Applications for siRNA

Basic research


Determining protein function


Easier than a knockout and may be used for
partial knockdowns

Clinical research


You name it


Cancer, hypercholesterolemia, infections,
developmental defects

Nature

Web Feature

References

Ambros, V. (2001). "microRNAs: tiny regulators with great potential."
Cell

107
(7): 823
-
6.

Bartel, B. (2005). "MicroRNAs directing siRNA biogenesis."
Nat Struct Mol Biol

12
(7): 569
-
71.

Cullen, B. R. (2004). "Transcription and processing of human microRNA precursors."
Mol Cell

16
(6): 861
-
5.

Elbashir, S. M., W. Lendeckel, et al. (2001). "RNA interference is mediated by 21
-

and 22
-
nucleotide RNAs."
Genes Dev

15
(2):
188
-
200.Griffiths
-
Jones, S. (2004). "The microRNA Registry."
Nucleic Acids Res

32
(Database issue): D109
-
11.

Kim, V. N. (2005). "Small RNAs: classification, biogenesis, and function."
Mol Cells

19
(1): 1
-
15.

Lee, Y., K. Jeon, et al. (2002). "MicroRNA maturation: stepwise processing and subcellular localization."
Embo J

21
(17): 4663
-
70.

Lorenz, C., P. Hadwiger, et al. (2004). "Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing

in

liver cells."
Bioorg Med Chem Lett

14
(19): 4975
-
7.

Mattick, J. S. and I. V. Makunin (2005). "Small regulatory RNAs in mammals."
Hum Mol Genet

14 Suppl 1
: R121
-
32.

Matzke, M. A. and J. A. Birchler (2005). "RNAi
-
mediated pathways in the nucleus."
Nat Rev Genet

6
(1): 24
-
35.

McManus, M. T. (2003). "MicroRNAs and cancer."
Semin Cancer Biol

13
(4): 253
-
8.

Pasquinelli, A. E., S. Hunter, et al. (2005). "MicroRNAs: a developing story."
Curr Opin Genet Dev

15
(2): 200
-
5.

Rossi, J. J. (2005). "RNAi and the P
-
body connection."
7
(7): 643
-
644.

Sontheimer, E. J. and R. W. Carthew (2005). "Silence from within: endogenous siRNAs and miRNAs."
Cell

122
(1): 9
-
12.

Soutschek, J., A. Akinc, et al. (2004). "Therapeutic silencing of an endogenous gene by systemic administration of modified
siRNAs."
Nature

432
(7014): 173
-
8.

Takeshita, F., Y. Minakuchi, et al. (2005). "Efficient delivery of small interfering RNA to bone
-
metastatic tumors by using
atelocollagen in vivo."
Proc Natl Acad Sci U S A

102
(34): 12177
-
82.

Tomari, Y. and P. D. Zamore (2005). "MicroRNA biogenesis: drosha can't cut it without a partner."
Curr Biol

15
(2): R61
-
4.

Vermeulen, A., L. Behlen, et al. (2005). "The contributions of dsRNA structure to Dicer specificity and efficiency."
Rna
.

www.ambion.com

www.darmacon.com

www.idtdna.com

http://www.nature.com/focus/rnai/animations/index.html