What is RNA?
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• Ribonucleic Acid (RNA), genetic material
of certain viruses and, in cellular
organisms, the molecule that directs the
middle steps of protein production.
• In RNA viruses, the RNA directs two
processes—protein synthesis
(production of the virus's protein coat)
and replication (the process by which
RNA copies itself).
• In cellular organisms, DNA cannot act
alone and relies upon RNA to transfer
this crucial information during protein
synthesis (production of the proteins
needed by the cell for its activities and
development).
FUNCTIONS AIMT
On the basis of functions RNA can be of following
types:
• rRNA (ribosomal RNA)
• tRNA (transfer RNA)
• mRNA (Messenger RNA)
• snRNA (including snoRNA) (Small nuclear RNA- splicing)
• Other ribozymes
• Telomerase RNA (telomerase maintenance)
• siRNA (small interfering RNA – RNA silencing)
• shRNA (short hairpin – RNA silencing)
• Pri-miRNA; miRNA ( (primary) micro RNA - regulation)
• sRNA (small RNA – bacteria)
• gRNA (guide RNA – RNA editing)
• Genomic RNA (viruses)
SMALL RNA(sRNA) AIMT
• Genomically encoded (intergenic regions)
• Frequently are complimentary to mRNA
– Usually imperfect complementary
• 80 – 120 (+) nts. in length
• Highly conserved between species
• Can have multiple unlinked targets
• Usually interact with the 5’ UTR
• Frequently require Hfq for function
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sRNA in prokaryotes:
1. Transcription inhibition
2. Translation Inhibition
3. mRNA degradation/stability
4. Translation stimulation
sRNA in eykaryotes:
5. Micro RNAs (miRNA)
– Similar to sRNAs in prokaryotes
6. Small interfering RNA (siRNA)
– Induces mRNA degradation
miRNA AIMT
• Genomically encoded
• Frequently are complimentary to mRNA
– Usually imperfect complementarily
• Can be encoded in clusters on the genome, or
individually
• Can have multiple unlinked targets
• miRNAs usually interact with the 3’ UTR
• Can alter mRNA stability, and translational initiation
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How do we know which siRNA are functional?
•Some ideal properties:
-GC content between 30-55%
-Low level of secondary structure
-Differential between thermodynamic stability of 5’
and 3’ ends: A/U content
-Specific positional nucleo
-tide preferences
-Avoid long GC stretches
RNAi AIMT
• It was first discovered in 1998 by
Andrew Fire and Craig Mello in
the nematode worm
Caenorhabditis elegans and
later found in a wide variety of
organisms, including mammals.
• RNAi in C. elegans-
– Silencing of a green
fluorescent protein (GFP)
reporter in C. elegans occurs
when animals feed on
bacteria expressing GFP
dsRNA (a)but not in animals
that are defective for RNAi(b)
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RNAi AIMT
• RNA interference (RNAi) is an evolutionally highly
conserved process of post-transcriptional gene
silencing (PTGS) by which double stranded RNA
(dsRNA), causes sequence-specific degradation of
mRNA sequences, forming siRNA.
• siRNA is similar to miRNA,but it,
– Typically induces mRNA degradation
– Requires exact base pairing for maximum effect
• Originally characterized in plants
– “Post transcriptional gene silencing”
– Plays a large role in plant “immune system”
– Subsequently found in a wide variety of
eukaryotes
HOW THE PROCESS OF SILENCING
WORKS? AIMT
MECHANISM OF SiRNA AIMT
• Two-step model to explain RNAi:
– I.) dsRNA is diced by an ATP-dependent ribonuclease
(Dicer) into short interfering RNAs (siRNAs).
• duplexes of 21 23 nucleotides bearing two-nucleotide 3'
overhanging ends.
– II.) siRNAs are transferred to a second enzyme
complex, designated RISC for RNAi-induced silencing
complex.
The siRNA guides RISC to the target mRNA, leading
to its destruction.
• the antisense strand of the siRNA is perfectly complementary
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• The complex becomes activated by unwinding
of the duplex.
• Activated RISC complexes can regulate gene
expression at many levels:
• promoting RNA degradation
• translational inhibition
• chromatin remodelling
• Amplification of the silencing signal in plants
may be accomplished by SiRNAs priming
RNA-directed RNA polymerase (RdRP)-
dependent synthesis of new dsRNA.
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USES AIMT
• RNAi is a universal, omnipresent conserved
mechanism in eukaryotic cells.
• The cellular mechanism of RNAi Predates
evolutionary divergence of plants and worms.
• key proteins involved in RNAi in disparate organisms
are highly conserved.
• Gene knockdowns
– Look at function/phenotype of a gene
• Therapeutic techniques
– Anti viral
– Anti cancer
– Anti neurological diseases
– Others
RESEARCH AIMT
Applying RNAi for the treatment of HIV
infection
• Several early reports demonstrating that
HIV was a highly susceptible target for
RNAi
• Can use RNAi in a gene therapy setting
by expressing short hairpin triggers.
Rationale for anti-HIV AIMT
Gene Therapy
• HIV infection is chronic and lifelong treatment
required.
• Increasing number of patients with drug resistant
virus.
• Toxicities of current chemotherapy agents (in
Highly Active Anti-Retroviral Therapy–cocktail).
• Difficult dosing regimens for patients
• Expense of HAART can exceed $20,000/yr
• Gene Therapy can replace or be used as an
adjuvant for chemotherapy
Isolated CD4+ Cells
T cell-based AIMT
CD4
CD4
CD4
gene therapy
CD4 CD4
CD4
CD4
Transduction of CD4+ cells
CD4
with genetic vectors CD4 CD4
CD4
CD4 CD4
CD4
CD4 Cell Selection CD4
and Expansion CD4
CD4
CD4
CD4
Relatively easy to obtain and CD4 CD4
genetically modify, but does
not capture all of viral Infusion
reservoirs such as monocytic
and dendritic cells.
REFERNCES AIMT
• Kevin Klein, PhD., Pathobiology 551
• Gene-IX, by Benjamin Lewin
• www.ansijournals.com
• http://www.sirna.com/about/index.html
• www.ncbi.nlm.nih.gov/pubmed
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