Biotechnology Engineering

1. RNA interference
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Lecture- 38

2. Roles of RNAi pathway in the cell
 Protects against RNA virus infections, especially in plants and
invertebrate animals
 Maintains genome stability by keeping mobile elements silent
 Represses protein synthesis and regulates the development of organisms
MicroRNA (miRNA), a class of endogenous RNA -- regulate gene
expression by base-pairing to mRNA, which results in either degradation of
the mRNA or suppression of translation.
 Keeps chromatin condensed and suppress transcription
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3. DICER
• Dicer is an endoribonuclease in the RNase III family that
cleaves double-stranded RNA (dsRNA) and pre-microRNA (miRNA)
into short double-stranded RNA fragments called small interfering
RNAs (siRNAs) about 20-25 nucleotides long, usually with a two-base
overhang on the 3' end.
• Dicer contains two RNase III domains and one PAZ domain; the
distance between these two regions of the molecule is determined
by the length and angle of the connector helix and may influence the
length of the siRNAs it produces.
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4. •Dicers interact with several partner proteins (TRBP in
humans, R2D2, Loqs in Drosophila).
•These partner proteins could play a role in dictating the
substrate specificity of dicer proteins.
•PAZ domain is important for protein-protein interaction.
•Dicer homologs exist in many organisms including
C.elegans, Drosophila, yeast and humans.
• Loss of dicer: loss of silencing
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5. RNA Induced Silencing Complex (RISC)
• RISC is a large (~500-kDa) RNA-multiprotein
complex which triggers mRNA degradation in
response to siRNA
• RNAi effector complex
Critical for target mRNA degradation or inhibition of translation
• Not well characterized: 4 subunits? More?
• Activities associated with RISC
– Helicase
– Endonuclease and exonuclease
– “homology seeking”/RNA binding
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6. Different classes of small RNA molecules
During dsRNA cleavage, different RNA
classes are produced:
– siRNA (short interfering RNA )
– miRNA (micro RNA)
– stRNA (small temporal RNA)
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7. siRNAs
• Small interfering RNAs that have an integral role in
the phenomenon of RNA interference (RNAi),
a form of post-transcriptional gene silencing
• 21-25 nt fragments which bind to the
complementary portion of the target mRNA
and tag it for degradation
• A single base pair difference between the siRNA
template and the target mRNA is enough to block
the process 7

8. miRNAs and stRNAs
• derived from ~70 nt ssRNA (single-stranded RNA)
which forms a stemloop; processed to 22nt RNAs
• found in:
Drosophila, C. elegans, HeLa cells
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9. Con….
•Role: the temporal regulation of development,
preventing translation of their target
miRNAs by binding to the target’s complementary 3’
untranslated regions (UTRs)
• Conservation: 15% of these miRNAs are conserved
with 1-2 mismatches across worm, fly, and
mammalian genomes
•Expression pattern: varies; some are expressed in all
cells and at all developmental stages and others have
a more restricted spatial and temporal expression
pattern 9

10. Overview of small RNA molecules
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11. Applications of RNAi
•Antiviral therapy
Potent and specific inhibition of human immunodeficiency
virus type 1 replication by RNA interference. An et al.(1999)
Modulation of HIV-1 replication by RNA interference.
Hannon (2002).
Selective silencing of viral gene expression in HPV-positive
human cervical carcinoma cells Jung et al. 2002.
•Biotechnology
Engineering of food plants that produce lower levels of natural
plant toxins.
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12. • Functional genomics: Identification of gene function
Analysis of unknown genes in sequenced genomes
Efforts are being made to target every human
gene via miRNAs
• Gene therapy: Down-regulation of certain
genes/mutated alleles
• Cancer treatments
• Agriculture
Cont…..
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