Functional RNAs: RNA catalysts,

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Functional RNAs RNA catalysts, miRNA,
siRNA Pathobiology 551 Lecture 10 February 6,
2007
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Non-coding RNAs are very diverse in size and
functions
Costa FF. Gene. Jan 2007.
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RNAs have many functions

• tRNA (transfer)
• mRNA messenger(riboswitch regulation)
• snRNA (snoRNAsplicing)
• telomerase RNA (telomerase maintenance)
• siRNA (RNA silencing)
• shRNA (RNA silencing)
• pri-miRNA, miRNA (primary) micro RNA
  regulation
• sRNA (bacteria)
• gRNA (RNA editing)
• genomic RNAs (viruses)
• rRNA (ribosomal)
• ribozymes

Functions can be enzymatic, regulatory, or can
encode information
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Ribozymes Enzymatic RNAs

• Cleave RNA
• Able to regulate mRNA expression
• Can be associated with ribonucleoprotein
• complexes (RNPs)
• Often function in the presence of divalent
  cations
• Function in both prokaryotes and eukaryotes

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Ribozymes Ribonucleic acid enzymes

• Multiple Classes
• Group I Introns
• Group II Intons
• RNase P
• Hammerhead
• Hairpin
• Hepatitis Delta Virus and Varkud Satellite
• DNAzymes??

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Hammerhead Ribozyme
-- Smallest known ribozyme (40 nucleotides) --
Associated with plant viroid genome replication
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Hepatitis Delta Virus Ribozymes
-- Only known animal virus with ribozyme
activity -- Has two isomers and two functions
- Genomic RNA cleavage - Anti-genomic
cleavage -- One of the fastest known -
Cleavage rate 1/sec -- Divalent cation dependent
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DNAzymes??
-- NOT found in nature (as far as anyone has
found) -- created using in vitro selection --
use for antiviral agents (HIV-1, HCV, HBV)
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Riboswitches

• RNA genetic control elements found within 5UTRs
• RNA that binds metabolites/ions
• Purpose - regulate gene expression in response
  to
• binding small molecule metabolites
• Transcriptional termination
• Translation repression/initiation
• mRNA stability

• Present in both pro/eukaryotes

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Riboswitches
Two main criteria used to define the riboswitches
1) Direct (protein-free) binding of metabolite to
RNA 2) Regulation of genes is metabolite-dependen
t
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Movie Time
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Riboswitch Gene Regulation
Riboswitches are an important mechanism of gene
regulation. For example, nearly 2 of the genes
of the model organism Bacillus subtilis appear to
be controlled by riboswitches.
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Small RNAs (sRNA or sncRNA) in prokaryotes

• Genetically encoded (intergenic regions)
• Rho-independent transcription termination
• 80-100bp in length
• One sRNA can affect multiple target genes
• Many interact with RNA chaperone Hfq
• - Hfq binds to AU rich regions in mRNA
• Regulate by complementary base-pairing
• with mRNA (usu. Imperfect complementary) in
• 5 UTR and sometimes in 3UTR

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Transcript regulation

• By what mechanisms do sRNAs regulate mRNA?

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sRNA Outcomes in Prokaryotes

• Translation Inhibition
• mRNA degradation
• Translation stimulation
• mRNA Stabilization

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Many sRNAs still being discovered (E. coli)
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-- NIH Database
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How are sRNAs discovered?
Vogel and Sharma. 2005. Biol. Chem. v386.
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Small RNAs in Eukaryotes

• Micro RNAs (miRNA)
• Similar to sRNAs in prokaryotes
• Small interfering RNA (siRNA)
• Induces mRNA degradation

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miRNA

• Genomically encoded
• Frequently are complimentary to mRNA
• Usually imperfect complementarity
• Can be encoded in clusters on the genome, or
  individually
• Sequences are relatively conserved between
  species
• Can have multiple unlinked targets
• miRNAs always interact with the 3 UTR
• Can alter mRNA stability, and translational
  initiation

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miRNA structures

• Originate as a precursor RNA (primary miRNA
  pri-miRNA) of several hundred base pairs
• Pri-miRNA contain Caps and poly A tail
• Some contain introns while others do not
• Can be encoded by introns of other genes
• Transcribed by RNA Pol II
• Contain 80 bp imperfectstem loop

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miRNA Processing

• Drosha cleaves hairpin liberating a 60 bp
  hairpin (pre-miRNA)
• Pre-miRNA is exported to the cytoplasm
• In the cytoplasm, Dicer cleaves liberating a 22
  nucleotide RNA duplex with 2 bp 3overhangs.

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Drosha and Dicer

• Members of the RNase III family
• dsRNA-specific
• Cleave dsRNA leaving 2 bp 3 overhangs

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miRNA processing
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miRNA function

• Mechanism of action is unknown
• Interacts with the 3UTR of the target mRNA
• Inhibits translation from a mRNA without inducing
  mRNA degradation
• Can form a ribonucleoprotein complex (miRNP)
• Can be complexed with ribosomes and target mRNA
• Also important for silencing retrotransposons and
  endogenous retroviruses
• Important for development and differentiation

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siRNA...history

• Originally characterized in plants (1990)
• Post transcriptional gene silencing
• Plays a large role in plant immune system
• Fire and Mello successfully silenced genes in C.
  elegans 1998 (won Nobel in 2006)
• Triggered by dsRNA

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siRNA

• Similar to miRNA. BUT...
• Typically induces mRNA degradation
• Requires exact base pairing for maximum effect
• Dicer processes double stranded RNAs
• RISC (or other complexes) carry out the effector
  function
• RISC cleaves RNA that is complementary to dsRNA
  that activates pathway
• Dicer/RISC video clip

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GFP silencing in transgenic C. elegans
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RISC effector complex

• RNA-induced silencing complex
• Contains a member of the argonaute family
• Between 130 kDa and 500 kDa
• Other components are being characterized
• Cleaves RNA complementary to the siRNA, 12bp
  upstream of 3 terminus
• Assembling the RISC complex requires ATP, while
  RNA cleavage does not.

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siRNA designing the assay

• dsRNAs need to be lt30 bp in length
• Why?
• Well-designed siRNAs can result in gt90 reduction
  in target RNA
• 21nt dsRNAs most effective
• Sequence-specificity important
• Single bp-mismatches reduce silencing capability
• Many will make 3-4 siRNAs test all and go w/
  best
• Deliver by injection or transfection
• Vectors becoming more popular

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Uses of siRNA

• Gene knockdowns
• Look at function/phenotype of a gene
• Therapeutic techniques
• Anti viral
• Anti cancer
• Anti neurological diseases
• Others

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Caveats of siRNA

• Not found in apicomplexans
• Exceptions to the rule
• Scattered literature in P. falciparum but not
  reproducible
• T. brucei yes
• T. cruzi and Leishmania -- no

NOTE siRNA and other techniques are knockdowns
and do NOT give 100 knockouts. Nor do they work
on every gene or organism.
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Pubmed searches of siRNA
of citations
Year
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Lutz and Knaus 2002