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RNA-ligand interactions and
control of gene expression
Outline
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RNA interference (microRNAs and siRNAs)
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Ribosomal RNA, tRNA, and mRNA interactions
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RNA interactions affecting translation and
transcription (outside the ribosome)
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Naked RNA
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RNA-metabolite interactions (riboswitches)
RNA interference
RNA interference (RNAi)
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RNAi is the process whereby double-stranded RNA
(dsRNA) induces homology-dependent degradation
of cognate RNA (i.e. gene silencing)
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RNAi is central to cellular mechanisms of posttranscriptional gene silencing (PTGS) and can also
effect transcriptional gene silencing (TGS)
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RNAi is highly conserved among eukaryotes (fungi,
protozoans, plants, nematodes, invertebrates,
mammals)
Overview of RNAi
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Double-stranded RNA (dsRNA)
is processed by Dicer, an RNase
III family member, to produce
21-23nt small interfering RNAs
(siRNAs)
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siRNAs are manipulated by a
multi-component nuclease
called the RNA-induced
silencing complex (RISC).
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RISC specifically cleaves
mRNAs that have perfect
complementarity to an siRNA
strand
Components of the RNAi pathway
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Dicer
Dicer belongs to the RNase III family of dsRNA-specific ribonucleases.
Contains dsRNA-binding motifs and a PAZ domain believed to
mediate protein-protein interactions.
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Argonaute homologs (Dicer/RISC associated)
Argonaute family members are highly basic proteins that contain PAZ
and PIWI domains. Argonaute is the only identified component of
RISC.
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RNA-dependent RNA polymerase (RdRP)
Endogenous RdRPs influence RNAi in certain eukaryotes including
fungi, plants, C. elegans. Function to amplify the dsRNA signal.
Discovery of RNAi
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RNAi was initially discovered and characterized in
the C. elegans
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It was observed that antisense or sense RNA could
equally effect silencing (knock-down) of target gene
expression
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A later study demonstrated that antisense and sense
RNA combined (dsRNA) is 10-times more effective in
silencing target gene expression
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Genetic studies in C. elegans have helped to identify
genes encoding RNAi machinery
The bottom line
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Any means by which siRNAs can be generated and
delivered to a cell can elicit RNAi and gene-specific
silencing
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siRNAs are powerful tools for manipulating gene
expression and determining gene function
Nature did not exhaust billions
of years of evolution on RNAi
solely for the benefit of modern
day biologists!
Biological significance of RNAi
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Cellular immune response to viruses
In certain organism (especially plants), RNAi serves as a first line of
defense against viral infection, as viral replication typically includes
dsRNA species
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Genetic stability
RNAi repress the mobility of transposable genetic elements in C.
elegans and S. pombe which requires the formation of dsRNA
Revealed that RNAi can also effect transcriptional gene silencing by
promoting heterochromatin formation (histone and DNA
methylation)
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Organismal development and germline fate
Developmental processes are affected by endogenous non-coding
RNAs that function through the RNAi pathway (microRNAs)
MicroRNAs (miRNAs)
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miRNAs are products of endogenous genes and function
through the RNA interference (RNAi) pathway to posttranscriptionally regulate the expression of other genes.
miRNA genes
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To date, 1185 miRNA genes have been identified in various
organisms, including approximately 200 in each human,
mouse, and rat genomes.
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Certain miRNAs are
highly conserved among
eukaryotic species.
miRNAs in development
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miRNAs are differentially expressed among various tissue
types and at various stages in cellular differentiation. e.g.
there exist stem cell and neuronal cell specific miRNAs.
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Disruption of RNAi pathway results in early embryonic
lethality due to depletion of stem cells.
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Certain miRNAs are known to control developmental
timing and fate specification in C. elegans, leaf
morphogenesis in plants, and hematopoetic lineage
differentiation in mice.