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RNA Switches Genetic Research Tools

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Title: RNA Switches Genetic Research Tools


1
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
2
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
3
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)

4
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.

5
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.

6
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.

7
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.

8
Anti-term.
Term.
Anti-Anti-Term.
RNA poly.
DNA
LIGAND
RNA poly.
Regulation by Transcriptional Termination
9
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.

10
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.

11
Anti-anti-SD
Anti-SD
SD
AUG
Ribosome
LIGAND
Anti-SD
SD
Anti-anti-SD
AUG
Regulation by Translational Initiation Inhibition
12
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.

13
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.

14
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.

15
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.

16
Secondary Structure of self cleavage RNA molecule
17
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.

18
Translation regulation by an mRNA Thermo sensor
Ribosome
mRNA
SD
AUG
Temperature Increase
SD
AUG
19
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.

20
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.

21
STRUCTURE OF RNA SWITCHES AND THEIR LIGANDS
22
Natural small molecule-regulated RNA switches
                                               
                                                  
                                        
23
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.

24
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)

25
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.

26
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.

27
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)

28
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.

29
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.

30
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.

31
RNA Biochips An array of RNA molecular switches
constructed using seven distinct
effector-modulated ribozymes based on the
hammerhead self-cleaving RNA.                     
                                                  
   
32
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.

33
  • 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.

34
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.

35
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.

36
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 .

37
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.

38
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39
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.

40
  • 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.
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