This seminar discusses small interfering RNA (siRNA) and microRNA (miRNA). siRNA and miRNA are types of small non-coding RNA molecules that play important roles in RNA interference and post-transcriptional gene regulation. The seminar covers the production, mechanisms of action, functions, and differences between siRNA and miRNA. It also discusses the potential roles of miRNA in disease and the nervous system, as well as the importance and therapeutic applications of siRNA and miRNA.

1. Seminar topic:
siRNA and miRNA
PRESENTED BY: SIRAJUDDIN MOLLA
M.Phram, 1st Semester
DEPARTMENT OF PHARMACOLOGY
SPER, JAMIA HAMDARD
1

2. Index
• RNA interference (RNAi)
• siRNA (Small interfering RNA)
• Function of siRNA
• siRNA Production and Mechanism of action
• miRNA (microRNA)
• miRNA Production and Mechanism of action
• Functions of miRNAs
• Differences between miRNA and siRNA
• miRNA and disease
• miRNA and the nervous system
• Importance of miRNA
2

3. • RNA interference (RNAi), the biological mechanism by
which double stranded RNA (dsRNA) induces gene
silencing by targeting complementary mRNA for
degradation
• Also called post transcriptional gene silencing (PTSG).
• Play important role in post translational gene
transposon regulation, defending cells against viruses.
• 2 types of small silencing RNA molecules
a) siRNA
b) miRNA
RNA interference (RNAi)
3

4. • Small interfering RNA (siRNA), sometimes known as short
interfering RNA or silencing RNA.
• It is a class of double-stranded, non-coding RNA molecules.
• 20-25 base pairs in length
• It is similar to miRNA
• Operating within the RNA interference (RNAi) pathway by the
enzyme Dicer.
• It interferes with the expression of specific genes with
complementary nucleotide sequences by degrading mRNA after
transcription, resulting no translation.
siRNA
(Small interfering RNA)
4

5.  Small (or short) interfering RNA (siRNA) is the
commonly used RNA interference (RNAi) tool for
inducing short-term silencing of protein coding
genes.
 It is a double stranded RNA molecule which
interferes with the expression of specific genes by
degrading mRNA after transcription & preventing
translation.
Function of siRNA
5

6. • The first step of RNAi involves processing and cleavage of longer
double-stranded RNA into siRNAs generally bearing a 2
nucleotide overhang on the 3' end of each strand.
• The enzyme responsible for this processing is an RNase III-like
enzyme termed Dicer.
• Dicer domain includes an ATPase/RNA helicase domain,
catalytic RNAase lll family enzyme.
• Dicer and dsRNA binding protein  form RISC loading complex
• RISC (RNA induced silencing complex).
• After that load the RNA duplex into RISC.
siRNA Production
and
Mechanism of action
6

7. Long dsRNA
RISC-loading
complex
RISC
recycling
Guide-strand
selection,
passenger-
strand
cleavage/ejecti
on
Passenger-
strand
Guide-
strand
Dicer
processing of
long dsRNA
Target
mRNA
slicin
g
5’ 3’
5’
3’
Dicer
siRNA
3’
TRBP (transactivation response element RNA-binding protein)
7

8. siRNA Production
and
Mechanism of action
• The first step of RNAi involves processing and cleavage of longer
double-stranded RNA into siRNAs generally bearing a 2
nucleotide overhang on the 3' end of each strand.
• The enzyme responsible for this processing is an RNase III-like
enzyme termed Dicer.
• Dicer domain includes an ATPase/RNA helicase domain,
catalytic RNAase lll family enzyme.
• Dicer and dsRNA binding protein  form RISC loading complex
• RISC (RNA induced silencing complex).
• After that load the RNA duplex into RISC.
8

9. • Within the RISC complex, siRNA strands are separated and the
strand with the more stable 5' end is typically integrated to
the active RISC complex (guide RNA).
• The antisense single-stranded siRNA component then
guides and aligns the RIS complex on the target mRNA.
• The action of catalytic RISC protein a member of the Argonaut
family (Ago-2) having endonuclease activity  mRNA is
cleaved which is complementary to their bound siRNA.
stop
translation
When siRNA
completely forms
base pairing with
target mRNA
mRNA degrade
otherwise inhibits
mRNA to bind
with ribosome
Contd..
9

10. miRNA
(microRNA)
A miRNA is small non-coding RNAs molecule
Found only in eukaryotic cells (plants, animals) and sometimes in
viruses.
miRNAs are defined as 21-25 (avg. 22) nucleotide single-stranded
RNAs (ssRNAs), which are produced from hairpin shaped precursors
Transcribed by RNA polymerase II from independent genes or introns
of protein-coding genes
 miRNA functions in RNA silencing and post-transcriptional
regulation of gene expression via base-pairing with complementary
sequences within mRNA molecules.
They play important gene-regulatory roles in both plants and animals.
The first miRNA (lin-4) was discovered in C.elegans in the year 1993.
10

11. The pri-miRNA is processed within the nucleus to a precursor
miRNA (pre-miRNA) by Drosha, a class 2 RNase III enzyme.
The transport of pre-miRNAs to the cytoplasm is mediated by
exportin-5 (EXP-5).
In the cytoplasm, they are further processed to become mature
miRNAs by Dicer, an RNase III type protein and loaded onto the
Argonaute (ago) protein to produce the effector RNA-induced
silencing complex (RISC).
After that Followed the same pathway as siRNA
miRNA Production
and
Mechanism of action
11

12. Chromosome
having specific
gene to produce
miRNA
Transcription
RNA Pol II or III
Primary or pri-miRNA Having complementary base
pair itself forms hairpin
structure (imperfect)
Drosha
(endonuclease)
Pasha (droshophila)
DGCR8 (mammals)
Pre miRNA
70bp
cytoplasm
nucleu
s
Exportin
5
miRN
A
12

13. The pri-miRNA is processed within the nucleus to a precursor
miRNA (pre-miRNA) by Drosha (endonuclease), a class-2
Rnase-III family enzyme.
Pasha (in Droshophila) and DGCR8 (in mammals) act as RNA
binding protein
The transport of pre-miRNAs to the cytoplasm is mediated
by exportin-5 (EXP-5).
In the cytoplasm, they are further processed to become
mature miRNAs by Dicer, an RNase III type protein and
loaded onto the Argonaute (ago) protein to produce the
effector RNA-induced silencing complex (RISC).
After that Followed the same pathway as siRNA
miRNA Production
and
Mechanism of action
13

14. About 50% of the annotated human miRNAs map within fragile
sites of chromosomes, which are areas of the genome that are
associated with various human cancers.
Recent evidence indicates that miRNAs can function as tumour
suppressors and oncogenes, and they are therefore referred to
'oncomirs’.
Gene therapies that use miRNAs might be an effective approach
to blocking tumour progression.
miR-15 and miR-16, which negatively regulate BCL2, are
promising candidates for cancer treatment.
Functions of miRNAs
14

15. Properties miRNA siRNA
Origin found in Animals, plants, protists found in Ainmals, fungi, plants,
protists
Biogenesis (nature of
precursor)
Cleavage of Single-stranded
RNA molecules that forms short
hairpin (imperfect stem-loop
secondary structure)
Cleavage of long bimolecular
RNA duplexes or single
stranded RNA that forms long
extended hairpins
Nature of regulatory
target
Regulate different genes or
Genes other than those from
which they were transcribed
Mediate the silencing of the
same (or very similar) genes
from which they were
originated or transcribed
Action
Some trigger degradation of
mRNA, others inhibit translation
Some trigger degradation of
mRNA, others inhibit
transcription
endonuclease Dicer/drosha dependent Dicer dependent
Differences between
miRNA and siRNA
15

16.  Just as miRNA is involved in the normal functioning of
eukaryotic cell, so has dysregulation of miRNA been
associated with disease.
 miRNA and inherited diseases:
• A mutation in the seed region of miR-96 causes
hereditary progressive hearing loss.
• A mutation in the seed region of miR-184 causes
hereditary keratoconus with anterior polar cataract.
• Deletion of the miR-17-92 cluster causes skeletal and
growth defects.
miRNA and disease
The seed sequence of a
miRNA is defined as the
first 2–8 nucleotides
starting at the 5′ end and
counting toward the 3′ end
16

17.  miRNAs appear to regulate the nervous systems.
Neural miRNAs are involved at various stages of
synaptic development, including
• dendritogenesis (involving miR-134)
• synapse formation
• synapse maturation (where miR-134 and
138 are thought to be involved).
miRNA and the nervous system
17

18. miRNAs represent small RNA molecules encoded in
the genomes of plants and animals.
These highly conserved 22 nudeotides long RNA
sequences regulate the expression of genes by
binding to the 3'untranslated regions (3’UTR) of
specific mRNAs.
A growing body of evidence shows that miRNAs are
one of the key players in cell differentiation and
growth, mobility and apoptosis (programmed cell
death).
Importance of miRNA
18

19. • Discovered a little over two decades ago, small interfering
RNAs (siRNAs) and microRNAs (miRNAs) are noncoding
RNAs with important roles in gene regulation.
• They have recently been investigated as novel classes of
therapeutic agents for the treatment of a wide range of
disorders including cancers and infections.
• Clinical trials of siRNA and miRNA-based drugs have
been initiated.
• The therapeutic approaches of siRNAs and miRNAs are
different as well as physicochemical properties, delivery,
and clinical applications.
Novel siRNA and miRNA
19

20. • Therapeutic approaches based on siRNA involve the introduction of a
synthetic siRNA into the target cells to elicit RNA interference (RNAi),
thereby inhibiting the expression of a specific messenger RNA (mRNA)
to produce a gene silencing effect.9
• By contrast, miRNA-based therapeutics comprise two approaches:
miRNA inhibition and miRNA replacement.
• Inhibition approach resembles antisense therapy,10 with synthetic
stranded RNAs acting as miRNA antagonists (also known as
or anti-miRs) to inhibit the action of the endogenous miRNAs.
• In the replacement approach, synthetic miRNAs (also known as miRNA
mimics) are used to mimic the function of the endogenous miRNAs.11
• It thus leads to mRNA degradation/inhibition, and produces a gene
silencing effect.
Therapeutic approaches
20

21. siRNA and miRNA as
therapeutic agents
• The specific gene silencing effect of siRNAs makes them useful
tools for target identification and validation in drug discovery
and development.38,39
• Since miRNAs have multiple mRNA targets and the disruption of
their functions contributes to the development of many diseases
including cancers, neurodegenerative disorders and
cardiovascular diseases, their clinical use as biomarkers and in
diagnostics is rapidly developing.40
• Furthermore, both siRNAs and miRNAs have huge potential as
therapeutic agents. They can overcome the major limitation of
traditional small drug molecules, which can only target certain
classes of proteins.
21

22. • Even for protein-based drugs including monoclonal antibodies
that are highly specific, their targets are mainly limited to cell-
surface receptors or circulating proteins.
• By contrast, siRNAs and miRNAs can downregulate the expression
of virtually all genes and their mRNA transcripts.
• Since many diseases result from the expression of undesired or
mutated genes, or from overexpression of certain normal genes,
discovery of siRNA and miRNA opens up a whole new therapeutic
approach for the treatment of diseases by targeting genes that are
involved in the pathological process.
Contd..
22

23. Design of therapeutic siRNA
• The first essential step for successful siRNA therapy is
the design of a siRNA sequence that is potent and
specific to the intended mRNA to minimize any off-
target effect.
• A conventional siRNA consists of 19–21 nucleotides
with two nucleotide overhangs at the 3′ end, usually TT
and UU, which are important for recognition by the
RNAi machinery.41
• Increasing the length of the dsRNA may enhance its
potency, as demonstrated by an in vitro study that
dsRNAs with 27 nucleotides were up to 100 times more
potent than the conventional siRNAs with 21
nucleotides.42
• On the other hand, dsRNAs longer than 30 nucleotides
20
The interferon (IFN) pathway
plays a critical role in the
human immune response.
Following viral infection, the
human body triggers a
complex regulatory system of
innate and adaptive immune
responses designed to defend
against the virus.
23

24. • Compared with siRNAs, miRNAs have a broader therapeutic
application.
• Since more than 60% of the human protein-coding genes contain at
least one conserved miRNA-binding site, together with the
of numerous non-conserved sites, the majority of protein-coding
genes are under the control of miRNAs.29
• The goal of miRNA replacement therapy using synthetic miRNAs (or
miRNA mimics) is to achieve the same biological functions as the
endogenous miRNAs.
• Therefore the synthetic miRNAs should possess the ability to be
loaded to RISC and silence the target mRNAs through the natural
miRNA signaling pathway.
Design of therapeutic miRNA
24

25. • A single-stranded RNA molecule containing the sequence
that is identical to the guide strand of the mature miRNA
could be functioned as miRNA mimic.
• However, the double stranded miRNA containing both guide
guide and passenger strands was found to be 100 to 1,000
times more potent than the single stranded one.4,14
• The double stranded structure can facilitate the proper
loading of the RNA molecule into the RISC, thereby
enhancing the gene silencing effect.
25
Contd..

26. • Therefore, designing miRNA mimics with a duplex
structure has become the direction of therapeutic
development.
• Synthetic miRNA precursors with longer sequences
a few extra nucleotides to a full-length pri-miRNA) have
also been proposed as therapeutic agents.78
• Since pri-miRNAs require processing in the nucleus,
whereas pre-miRNA do not, different strategies are
required for the delivery of different types of miRNA
mimics to their cellular targets.79
26
Contd..

27. • The first clinical trial of siRNA therapeutics was initiated in
2004,175 merely 6 years after the discovery of RNAi.
• The rapid progress of siRNA advancing into clinical trials is
perhaps due to the experience gained during the
development of antisense and other nucleic acid-based
therapies.
• To date, around 30 siRNA candidates have reached various
stages of clinical trials for the treatment of different diseases.
• In comparison, the clinical development of miRNA as
therapeutics is lagging behind, with only two miRNA
therapeutics, both of which are indicated for the treatment of
cancers, being registered in clinical trial to date.
siRNA AND miRNA
therapeutics in clinical studies
27

28. • The first miRNA therapeutic trial began in 2013 with the
second one starting in early 2015.
• Although siRNAs share many similarities with miRNAs, the
relatively slow progress of miRNA therapeutics could
due to their uncertain mechanism of action and specificity.
• The diverse potential applications of miRNAs (e.g., as
drug target and biomarkers) may also have distracted
from their development as therapeutic agents.
28
Contd..

29. Therapeutic indications of siRNA
and miRNA therapeutics.
29

30. • Synthetic siRNAs and miRNAs hold great promises as new classes
of therapeutic agents by silencing the gene(s) of interest.
• They have been studied for the treatment of various human diseases
including cancers, viral infections, ocular conditions, genetic
disorders, and cardiovascular diseases.
• The most attractive aspect of siRNA and miRNA therapeutics is
their ability to target virtually any gene(s), which may not be
possible with small molecules or protein-based drugs.
• While the therapeutic efficacy of siRNAs and miRNAs has been
successfully demonstrated in vivo, several technical barriers still
need to be overcome in order for these RNA molecules to be used
clinically.
• The experience from antisense and gene therapy has contributed
to the rapid progress of siRNAs and miRNAs into clinical
Conclusions and future prospects
30

31. • Currently, the development of siRNAs is advancing
ahead of miRNAs, with a larger number of candidates
already entered clinical trials, possibly due to the
uncertainties of the complex roles of miRNAs during the
early years of their discovery.
• With the recent surge in intensive research concerning
miRNAs, it can be expected that significant advance will be
made for their future role in therapeutics.
31
Contd..

32. 1. The Cell, A Molecular Approach. Geoffrey M Cooper.
2. Pharmacogenomics: The Search for Individualized Therapies. Edited by J.
Licinio and M -L. Wong
4. Molecular Pharmacology: From DNA to Drug Discovery. John Dickenson et.al
5. Basic Cell Culture protocols by Cheril D.Helgason and Cindy L.Miller
6. Basic Cell Culture (Practical Approach ) by J. M. Davis (Editor)
7. Animal Cell Culture: A Practical Approach by John R. Masters (Editor)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877448/#:~:text=Discovered%2
0a%20little%20over%20two,disorders%20including%20cancers%20and%20infect
ions.
References
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33. 33