Course Code: MPL104T

Lecture No. 7
Importance of siRNA and micro RNA

Course Leader
Dr. Mohammad Azamthulla / Dr Anita Murali

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 Content

• Importance of siRNA
• Importance of micro RNA

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 Lecture objectives

By the end of this lecture, students will be able to:

• Explain the importance of siRNA
• Explain the importance of micro RNA

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 Introduction
• Over the last decade, ~20 - 30 nucleotide RNA molecules have
emerged as critical regulators in the expression and function of
eukaryotic genomes
• Two primary categories of these RNAs - short interfering RNAs
(siRNAs) and microRNAs (miRNAs) act in both somatic and germline
lineages in a broad range of eukaryotic species to regulate
endogenous genes and to defend the genome from invasive nucleic
acids
• Understanding of siRNA and miRNA based regulation has direct
implications for fundamental biology as well as disease etiology and
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 What is RNA?

• Ribonucleic acid
– 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)

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 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

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 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)

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 Illustration
of miRNA
processing

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 Another View

Microproces
sor Complex

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

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 Players

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 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?)

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 Differences in miRNA Mode of Action

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 miRNA Registry

• http://www.sanger.ac.uk/Software/Rfam/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

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 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

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 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

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 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

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 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

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 siRNA Delivery

• For cell culture

– Lipid-based transfection
– Electroporation
• In vivo
– Lipid-based
– Conjugations
• Bacterial phage RNA
• Cholesterol
• Atelocollagen
– Viral systems (ie retrovirus & adenovirus)

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 siRNA Delivery & Processing

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 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

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 Common aspects of all miRNA and
siRNA pathways
Double-stranded RNA precursors of
various kinds are processed by a
Dicer protein into short (~20–30)
fragments. One strand of the
processed duplex is loaded into an
Argonaute protein, enabling target
RNA recognition through Watson-
Crick base pairing. Once the target
is recognized, its expression is
modulated by one of several distinct
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mechanisms, © Ramaiah
depending on the
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 (B) Dicer proteins cleave dsRNA precursors into
characteristic lengths through the action of two RNase III
domains. The domain arrangement of most Dicer enzymes
is shown at the top. Processing occurs most readily at
dsRNA ends, which associate with the PAZ domain present
in most Dicer enzymes. The substrate is then positioned
within the active sites of the RNase III domains, which
cleave the ~20–30 nt miRNA/siRNA duplex from its
precursor. This model is supported by the crystal structure
of Giardia Dicer, shown with a dsRNA modeled into the
structure. In addition to the canonical PAZ and RNase III
domains, the structure shows active-site metal ions
(purple) and a ‘‘ruler’’ helix (red) that helps to specify the
length of the siRNA product.

(C) Argonaute proteins are RNA silencing effectors that are

guided to their targets by short single-stranded nucleic
acids. The canonical arrangement of Ago domains is given
at the top. Below is a crystal structure of the Thermus
thermophilus Ago protein, with a bound DNA guide strand
base paired to an RNA target. The 50 end of the guide
strand associates with a binding pocket in the Mid domain,
and the 30 end binds the PAZ domain. The target cleavage
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site is juxtaposed with active-site residues in the PIWI
© Ramaiah University of Applied Sciences
 Several different categories of
transcripts can adopt dsRNA structures
that can be processed by Dicer into
siRNAs. These duplexes can be intraor
intermolecular, and although most are
perfectly base paired, some (e.g.,
hairpin RNAs and gene/ pseudogene
duplexes) are not. An siRNA consists of
a guide strand (red), which assembles
into functional siRISC, and a passenger
strand (blue), which is ejected and
degraded. All forms of siRISC contain
the siRNA bound to an Ago protein, and
many if not most forms of siRISC
contain additional factors. Target RNAs
are then recognized by base pairing,
and silencing ensues through one of
several mechanisms. In many species,
the siRNA populations that engage a
target can be amplified by the action of
RNA-dependent RNA polymerase
(RdRP) enzymes, strengthening 24and
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siRNAs
Sources of siRNA Precursors
The canonical inducer of RNAi is long, linear, perfectly basepaired
dsRNA, introduced directly into the cytoplasm or taken up from the
environment
These dsRNAs are processed by Dicer into the siRNAs that direct
silencing
siRNAs were originally observed during transgene and virus induced
silencing in plants, consistent with a natural role in genome defense
(Figure)
In 2002 and 2003, centromeres, transposons and other repetitive
sequences were uncovered as another wellspring of siRNAs 25
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 siRNAs cont…
More recently, other sources of endogenous siRNAs (endo-siRNAs)
have been identified, include convergent mRNA transcripts and other
natural sense-antisense pairs, duplexes involving pseudogene-derived
antisense transcripts and the sense mRNAs from their cognate genes and
hairpin RNAs (hpRNAs)
Thus, it has become clear that siRNAs are not solely the products of
foreign nucleic acid but arise from endogenous genomic loci as well
(Figure)
As such, they differ from many exogenous siRNAs (exo-siRNAs) in that
they or their precursors have an obligate nuclear phase
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 Figure 3. Mechanisms of siRNA Silencing
During canonical RNAi (lower right), siRISC recognizes
perfectly complementary mRNA, leading to Ago-catalyze
mRNA cleavage at a single site within the duplex. Aft
cleavage, functional siRISC
is regenerated, whereas the mRNA fragments are furth
degraded. siRNAs are also capable of recognizing targe
with imperfect complementarity (upper right).
In some cases, they can silence targets by miRNA-lik
mechanisms involving translational repression an
exonucleolytic degradation, though the frequency wi
which natural siRNAs use these pathways is not clear. Final
siRISC can direct heterochromatin formation (left) b
associating with nascent transcripts and RNA polymeras
(RNA Pol II in S. pombe and RNA
Pol IV/V in A. thaliana). In plants, target engagement leads
the association or activation of a DNA methyltransfera
(DMT) that methylates the DNA (lower left), leading
heterochromatin formation.
In S. pombe and probably in animals (upper left),th
pathway involves a histone methyltransferase (HMT) th
methylates Lys9 of histone H3 (data not shown), thereb
inducing heterochromatinization.
In most eukaryotes other than insects and mammals, targ
recognition by siRISC induces the synthesis of seconda
dsRNAs and siRNAs by RdRP enzymes (lower middle). Th
secondary dsRNAsare processed by Dicer into siRNAs, whic
add to the pool of siRISC. In nematodes, many of th
secondary siRNAs arise as single-stranded, unprime
transcripts with 50-triphosphates and
do not require Dicer processing. 27
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MicroRNAs
• Although we had learned about miRNAs, the past few years have
seen some surprising new discoveries from following a few simple
rules of production and action, miRNAs show diverse features that
are defying simple classification
• Diversification is a key feature of life processes and miRNAs are no
exception.

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 Biogenesis of miRNAs and Assembly into
miRISC in Plants and Animals

Nuclear transcription leads tocappedand

polyadenylated pri-miRNAs.

In plants, Dcl1 processes the RNA in

succession. The order of processing is not
certain. The terminal loop may first be
excised or it might be the flanking
segments that are cleaved first. The second
processing step by Dcl1 yields a mature
miRNA/miRNA* duplex that becomes
methylated and exported from the nucleus

In animals, the pri-miRNA is processed by

Drosha with the aid of DGCR8 to generate a
pre-miRNA species. This is exported from
the nucleus and processed by Dicer to
formthe mature miRNA/miRNA* duplex.
After processing, miRNAs are assembled
into miRISC. Only one strand of the duplex
is stably associated with an miRISC complex
—the miRNA strand is usually 29 more
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than University
the miRISC* strand,
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 Possible Mechanisms of miRISC-
Mediated Repression
Nonrepressed mRNAs recruit
initiation factors and ribosomal
subunits and form circularized
structures that enhance translation
(top). When miRISCs bind to mRNAs,
they can repress initiation at the cap
recognition stage (upper left) or the
60S recruitment stage (lower left).
Alternatively, they can induce
deadenylation of the mRNA and
thereby inhibit circularization of the
mRNA (bottom). They can also
repress a postinitiation stage of
translation by inducing ribosomes to
drop off prematurely (lower right).
Finally, they can promote mRNA
degradation by inducing
deadenylation followed by
decapping
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 • Most unwavering distinction between miRNAs and siRNAs has been
whether they silence their own expression
• Almost all siRNAs, silence the same locus from which they were
derived, sometimes have the ability to silence other loci as well
• But their capacity to strike at their encoding DNA and RNA makes
their production a continual struggle against themselves
• It also means that they are highly adaptable, allowing for little or no
selective pressure to maintain their sequence conservation
• In contrast, most miRNAs do not silence their own loci but silence
other genes

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 • This is attributable to the nuclear processing of miRNA precursor
RNAs coupled with nuclear exclusion of mature effector miRNAs.
Even this rule, however, is not absolute; human miR-29b is a nuclear
miRNA and Imp8 stimulates nuclear localization of Ago2 in cells
• Nevertheless, the overwhelming number of miRNAs that exert
strictly heterotypic silencing are more severely constrained in the
precision of their sequence structure because of the necessary
coupling of their sequence to heterologous targets
• This feature makes miRNAs less adaptable for functions such as
biological
• defense
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 • The leap of small RNAs from laboratory into the marketplace is
ongoing, and much of what has been learned in the past few years is
acting as a springboard
• Biotechnology will have to contend with issues such as specificity
that directly relate to mechanistic studies pursued in the lab. Other
hurdles such as delivery remain, and one hopes that in the next few
years as we learn more about the cell biology of RNAi, these and
other issues will be addressed as rapidly as we have witnessed to
date

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 Summary
• Two primary categories of these RNAs - short interfering RNAs
(siRNAs) and microRNAs (miRNAs) act in both somatic and
germline lineages in a broad range of eukaryotic species to
regulate endogenous genes and to defend the genome from
invasive nucleic acids
• Double-stranded RNA precursors of various kinds are processed
by a Dicer protein into short (~20–30) fragments
• Most unwavering distinction between miRNAs and siRNAs has
been whether they silence their own expression

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