RNA Switches Genetic Research Tools

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RNA Switches Genetic Research Tools
RAVINDER NAGPAL1, MALVIKA MALIK1, MONICA PUNIYA2,
AARTI BHARDWAJ3, SHALINI JAIN4 and HARIOM
YADAV4 1Dairy Microbiology, 2Dairy Cattle
Nutrition, 4Animal Biochemistry, National Dairy
Research Institute, Karnal 132001, Haryana, 3
Meerut Institute of Engineering and Technology,
Meerut-250002, U.P., India. Email
yadavhariom_at_gmail.com
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Role of RNA inside the Cell

• According to traditional central dogma of
  genetics RNA is the intermediate carrier of
  genetic information between DNA and Protein.
• DNA RNA
  PROTEIN

Transcription
Translation
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New roles discovered for RNA

• Certain RNA molecules, ribozymes, could catalyze
  biochemical reactions, a job previously thought
  to be the exclusive province of enzymes, which
  are proteins. (Kruger et al, 1980)
• Certain RNA molecules could behave like another
  type of protein antibodies. Researchers
  synthesized RNA molecules - now known as
  aptamers- that, like antibodies, can latch on
  tightly to specific target molecules.
  
• (Ellington and Szostak, 1990)

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contd...

• Certain RNA sequences can directly sense
  environmental factors and small molecule
  metabolites, this allow the associated mRNA to
  regulate their own transcription or translation.
  (Breaker, 2002)
• These self regulatory messages are called as RNA
  sensors and RNA switches.

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RNA Switches

• RNA switches are mRNAs that sense the environment
  directly, shutting themselves down in response to
  particular chemical clues.
• Breaker, Nudler, Yura and Cossart laboratories
  report that specific RNA sequences can act as
  environmental sensors of vitamin cofactors
  (including vitamins B1, B2 and B12) and
  temperature, which allow them to directly
  regulate the transcription or translation of
  associated mRNAs.

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contd...

• Distinct RNA molecules directly perform or
  mediate enzymatic processes such as RNA cleavage,
  splicing and translation.
• Non-coding RNAs are involved in a tremendous
  variety of gene regulatory mechanisms that
  operate at both the DNA and mRNA level .
• Seven types of RNA switches have been found in
  bacteria so far. These include switches
  controlling the manufacture of the vitamins B1
  and B2 and the nucleotide guanine.

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Mode of action of RNA Switches

• RNA Switches can regulate gene expression at both
  transcription as well as translation level.
• At transcription level they can induce premature
  termination of mRNA transcript.
• At translation level the initiation of protein
  synthesis is stopped by these switches.
• In certain cases RNA switches have self-cleavage
  property, thus regulating their own expression.
• RNA switches can also act as thermo sensors,
  regulating gene expression according to the
  temperature change.

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Anti-term.
Term.
Anti-Anti-Term.
RNA poly.
DNA
LIGAND
RNA poly.
Regulation by Transcriptional Termination
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Examples of Transcription Termination Mechanism

• Transcription termination mechanism has been
  reported functional in the regulation of rib and
  thi operon in Bacillus subtilis.
  (Mironova et al, 2002)
• rib operon - riboflavin biosynthesis.
• thi operon - thiamine biosynthesis.
• In case of rib operon the 5 Untranslated Region
  of the mRNA transcript can fold into two
  confirmations, depending upon the presence or
  absence of FMN.

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contd...

• In low concentration/absence of FMN the nascent
  transcript come out of RNA polymerase and attain
  a confirmation that allow the transcription to be
  completed.
• In presence of FMN, it binds to the nascent
  transcript and transcript folds in such a way
  that the transcription is terminated , resulting
  in premature transcript which leads to no gene
  expression.

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Anti-anti-SD
Anti-SD
SD
AUG
Ribosome
LIGAND
Anti-SD
SD
Anti-anti-SD
AUG
Regulation by Translational Initiation Inhibition
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Example of Translation Inhibition Mechanism

• Translation initiation inhibition mechanism has
  been proposed for regulation of thiM and thiC
  genes required for biosynthesis of thiamin.
  (Mandal and Breaker, 2004)
• The translation initiation for these genes is
  sensitive to the presence of thiamin.
• Binding of thiamin to the mRNA precludes the
  Ribosome Binding Site from ribosome subunit
  access.

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Observations

• Gram-positive bacteria are more likely to couple
  these elements to a terminator/anti-terminator
  system, that is at the level of transcription.
• Gram-negative bacteria more typically link these
  elements to a SD-sequestration mechanism, that is
  at the level of translation.

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contd...

• This type of regulation is found to be economic
  for the bacterial system as-
• In Gram positive bacteria the genes for one
  biosynthetic pathway are arranged in a cluster,
  therefore they are regulated at the transcription
  level.
• In Gram negative bacteria the genes for one
  biosynthetic pathway are scattered along the
  whole genome and are therefore regulated at the
  translation level.

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Self-cleavage of the RNA molecules

• The regulation of glmS gene(glutamine-fructose-6-p
  hosphate amidotransferase)in B. subtilis is by
  associated activity of ribozyme and riboswitches.
• In this case the 5 UTR of glmS binds with
  gluosamin-6-phosphate, product of GlmS activity
  and start acting as ribozyme, due to
  conformational change.
• This results in the self cleavage of the mRNA by
  an internal phosphoester transfer.

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Secondary Structure of self cleavage RNA molecule
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Activation of gene expression by RNA Thermo
sensors

• There are two well-studied examples of
  temperature-dependent regulation of gene
  expression and/or activity-
• during the heat shock response
• during pathogenic invasion
• Almost all the known thermo sensors regulate the
  gene expression at the translational level.

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Translation regulation by an mRNA Thermo sensor
Ribosome
mRNA
SD
AUG
Temperature Increase
SD
AUG
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Examples of RNA Thermo sensors

• Translation of LcrF, a general activator of
  virulence-related gene expression in Yersinia
  pestis, was found to be thermally regulated.
  
  (Hoe and Goguen, 1993)
• A new report from the Cossart lab (2002) now
  provides strong evidence for an RNA thermo sensor
  that regulates translation of PrfA, a general
  activator of virulence genes in a pathogenic
  variety of Listeria, L. monocytogenes.

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Regulation of virulence gene in L. monocytogenes

• In L. monocytogenes prf A gene is responsible for
  the virulence of the organism.
• The prfA gene is transcribed at both 30C and
  37C.
• But it is translated only at 37C.
• sequences in the 5' UTR of the prfA mRNA could
  form an extended hairpin that includes the SD
  sequence.
• At 37C, when the pathogen enters into the host
  system, this structure is destabilized the SD
  sequence is free initiation of translation.

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STRUCTURE OF RNA SWITCHES AND THEIR LIGANDS
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Natural small molecule-regulated RNA switches
                                               
                                                  
                                        
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Advantages of RNA switches for the organisms
possessing them

• Stringent control RNA switches are very specific
  to their substrate. eg. recognition of TPP by THI
  boxes is 1000 times greater than for either
  thiamine or thiamine monophosphate.
  
  ( Winkler et. al. 2004)
• Ligand can be reused Binding of the substrate to
  the RNA molecule is required for a short time,
  after this the ligand can be reused for the same
  or different purpose.
• Only one gene is involved There is no
  involvement of another gene product, which
  reduces the effect of mutation that will
  deregulate the mechanism.

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Gene expression regulation by RNA in Eukaryotes

• De-repressor RNA
• Recent data indicate that small non-coding RNA
  species  function as co-activators of eukaryotic
  gene transcription .
• Small activator or de-repressor RNA molecules
  play a role in initiating and stabilizing
  transcription bubbles, mRNA synthesis by RNA
  polymerase. (Frenster, 1965)

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dsRNA as a regulator of gene expression
                                               
             

• ds RNA has role in several chromatin and/ or
  genomic DNA modifications, which lead in the
  regulation of specific genes.
• ds RNA dependent mechanism can act at both
  transcriptional as well as post transcriptional
  levels.
• This type of gene expression is given different
  names in different organisms.
• RNA interference (RNAi) , in case of animals.
• Post transcriptional gene silencing (PTGS) , in
  case of plants.
• Quelling, in case of filamentous fungi.

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Other RNAs involved in regulation of gene
expression

• micro RNA (mi RNA)- a class of noncoding small
  RNA identified for their role in translational
  repression in some animals.
• short hairpin RNA (sh RNA)- a special class of
  RNA that becomes double stranded by folding of
  the RNA strand over itself, has been shown to be
  responsible for the transcriptional regulation.

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Applications of RNA switches in genetic research

• RNA switches can be used to create small
  molecule-regulated transgenes, which may allow
  researchers to manipulate expression of any
  individual construct within a battery of
  simultaneously introduced experimental
  constructs.
  (Werstuck and Green,
  1998)
• We can imagine exploiting RNA thermoregulation to
  create heat-inducible transgenes.
  (Brand and Perrimon,
  1993)

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contd...

• Aptamers have been placed within 5' UTRs and
  shown to inhibit gene expression upon
  introduction of the appropriate ligand.
  
  (Werstuck and Green, 1998)
• Self-cleaving RNAs can be placed under allosteric
  control by various small molecules, which can
  then be used to analyze the composition of
  chemical and biological mixtures .
  (Seetharaman et al, 2001)
• RNA switches can be used in gene therapy,
  allowing the patient to take pills to switch
  introduced gene on or off.

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contd...

• It may be possible to design drugs using RNA
  switches cognate ligands that would
  constitutively repress associated gene activity.
• Such compounds would efficiently inhibit
  bacterial growth by simultaneously repressing
  multiple components in a given biosynthetic/metabo
  lic/transport pathway.
• Such compounds might be likely to have relatively
  low toxicity, as they would be designed to target
  RNA, not protein.

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RNA on a Chip

• In 1995, Breaker successfully engineered
  RNA-based molecular switches.
• A molecular switch is a molecule that turns on or
  off by another molecule or compound.
• With dozens of these switches on hand, Breaker
  created an array of biosensors that use RNA to
  measure or detect compounds.

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RNA Biochips An array of RNA molecular switches
constructed using seven distinct
effector-modulated ribozymes based on the
hammerhead self-cleaving RNA.                     
                                                  
   
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Array for RNA

• Breaker placed the RNA switches on a gold-coated
  silicon surface and arranged them in clusters.
• Each switch was designed to bind only to a
  specific molecule its target and then to
  release a signal that identifies the target
  molecule. (In the prototype, the switches
  released a radioactive signal.)
• This array of RNA switches was tested on a
  variety of complex mixtures. In one experiment
  different strains of E. coli found in bacterial
  cultures were successfully identified.

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• The array's ability to simultaneously identify a
  potentially large number of compounds, combined
  with the precise exclusivity of each switch, adds
  up to a recipe for a powerful and wide-ranging
  laboratory on a dime-sized slice of silicon.

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RNA Super chip

• Future RNA chips, capable of revealing the
  molecular composition of complex mixtures-like
  blood serum and industrial waste-far more
  comprehensively than current biochips can be
  synthesized.
• Advanced versions of RNA biochip could be used
  for many different targets like drugs, toxins and
  metabolites, as well as proteins and nucleic
  acids.

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Benefit of using RNA biochip

• Benefit of RNA switches is their ability to
  withstand the sometimes unpredictable and harsh
  environment outside the lab as compared to a
  protein biochip.
• Breaker's RNA switches have been engineered to
  refold back to their original form after heating,
  this snap-back character will give RNA biochips a
  considerable advantage for use in more exotic
  test environments.

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Obstacles

• Manufacturing costs .
• The chemical stability of the switches .RNA is
  vulnerable to certain chemicals often found in
  test situations that can disintegrate a switch.
• The finer points of molecular recognition .
• This technology is so new that it is unclear just
  how many different compounds it will prove
  possible to recognize .

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Future Prospects

• RNA switches must be engineered to release a
  fluorescent, rather than a radioactive signal.
• Use of an RNA chip in diverse fields like
  chemical engineering, environmental science, and
  even biological and chemical warfare defense.
• Use of RNA chip in the detection and
  identification of pathogens.

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Conclusion

• Apart from being the carrier of genetic
  information , RNA can also act as the regulator
  of expression this information. These regulators
  are called as RNA switches.
• RNA switches have been discovered to be involved
  in the regulation of gene expression in
  prokaryotes.
• RNA switches can be used in genetic research to
  study the effect of environmental changes on
  particular gene expression.

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• RNA switches can be used to in gene therapy and
  as drugs.
• Researchers are trying to synthesize artificial
  RNA switches and we can hope that in future we
  will be able to use RNA switches not only in
  genetic research but also in antimicrobial
  therapy, as biosensors for different chemicals
  and in detection of pathogenic microorganisms.