RNA interference

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Lecture- 38

Roles of RNAi pathway in the cell

Protects against RNA virus infections, especially in plants andinvertebrate 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 geneexpression by base-pairing to mRNA, which results in either degradation ofthe mRNA or suppression of translation.

Keeps chromatin condensed and suppress transcription

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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 interferingRNAs (siRNAs) about 20-25 nucleotides long, usually with a two-baseoverhang on the 3' end.

• Dicer contains two RNase III domains and one PAZ domain; thedistance between these two regions of the molecule is determinedby the length and angle of the connector helix and may influence thelength of the siRNAs it produces.

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•Dicers interact with several partner proteins (TRBP inhumans, R2D2, Loqs in Drosophila).

•These partner proteins could play a role in dictating thesubstrate 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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RNA Induced Silencing Complex (RISC)

• RISC is a large (~500-kDa) RNA-multiprotein

complex which triggers mRNA degradation in

response to siRNA

• RNAi effector complexCritical 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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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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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

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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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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

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Overview of small RNA molecules

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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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• 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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