Biomedical Engineering Reference
In-Depth Information
(Darnell.et.al..2001;.Keene.and.Tenenbaum.2002;.Tenenbaum.et.al..2002;.Gerber.et.al..2004;.
Wang.et.al..2010)..In.RIPs,.the.antibody.to.the.desired.protein.is.immobilized.and.allowed.
to.interact.with.the.lysate.of.interest,.allowing.for.endogenous.protein-RNA.complexes.to.
form.and.be.captured..RNA.which.bind.to.the.protein.is.eluted.for.analysis.to.establish.
post-transcriptional.networks.
Another. method. for. studying. similar. interactions. is. cross-linking. and. immuno-
precipitation. (CLIP). (Ule. et. al.. 2003).. CLIP. uses. ultraviolet. light. to. covalently. cross-link.
speciic. protein-nucleic. acid. complexes,. following. immunoprecipitation. and. SDS-PAGE.
separation.to.isolate.them..This.method.has.successfully.isolated.intronic.RNA.(Ule.et.al..
2003). present. at. low. steady-state. levels. against. a. backdrop. of. the. abundant. ribosomal.
RNA..The.cross-linking.greatly.improves.the.protein-RNA.binding,.increasing.eficiency.
and. allowing. for. better. pull-down. of. bound. RNA,. but. at. the. cost. of. not. representing.
the. endogenous. binding.. iCLIP. (individual-nucleotide. resolution. CLIP). (König. et. al..
2010). and. PAR-CLIP. (photoactivatable-ribonucleoside-enhanced. cross-linking. and.
immunoprecipitation). (Hafner. et. al.. 2010). are. new. variations. that. improve. on. existing.
methods.and.increase.eficiency.
PAR-CLIP. is. a. particularly. useful. approach. that. enables. the. transcriptome-wide. isola-
tion.of.RNA-regulatory.elements.and.is.readily.applicable.to.any.RBP.or.RNP.complex.con-
tacting.RNA,.including.RNA.helicases,.polymerases,.or.nucleases,.expressed.in.cell.culture.
(Hafner.et.al..2010)..PAR-CLIP.provides.advancement.to.existing.RIP.(and.CLIP).methods.
by. allowing. improved. identiication. of. the. RNA. sequences. bound. by. targeted. RBPs.. As.
noted.earlier,.RIP.is.a.non-cross-linked.approach.that.is.ideal.for.identifying.endogenous.
RNA-RBP.associations.but.has.limitations.in.identifying.speciic.RNA.binding.sites.that.
require. cross-linking.. In. earlier. CLIP. methods,. RNA. was. cross-linked. with. low. eficacy.
to. proteins. by. 254.nm. UV. light.. Covalently. bound. protein-RNA. complexes. can. then. be.
fragmented.into.20-35.nucleotide.pieces.and.the.RNA.puriied,.ligated.to.adapters,.and,.
after. reverse. transcription,. sequenced. using. high-throughput. sequencing.. Since. cross-
linking.using.254.nm.UV.light.results.in.a.relatively.low.cross-linking.eficacy,.estimated.
to.range.from.1%.to.5%.by.using.puriied.protein.and.radiolabeled.RNA,.this.problem.was.
overcome.by.PAR-CLIP,.which.relies.on.metabolic.labeling.of.the.cells.with.photoreactive.
nucleoside.analogs,.4-thiouridine.(4-SU)..The.modiied.nucleoside.is.readily.taken.up.by.
mammalian. cells,. without. apparent. toxicity,. and. incorporated. into. nascent. RNA.. Cross-
linking.of.proteins.to.RNA.is.accomplished.by.irradiation.of.living.cells.with.UV.at.365.nm..
It.has.been.demonstrated.that.365.nm.cross-linking.coupled.with.4-SU.labeling.of.RNA.is.
substantially.more.effective.than.cross-linking.with.254.nm.UV.light.(Hafner.et.al..2010).
The.major.improvement.of.PAR-CLIP.compared.to.other.cross-linking.methods.is.the.
usage.of.photoactivatable.ribonucleosides..In.addition.to.increased.cross-linking.eficacy,.
the.incorporated.4-SU,.when.cross-linked,.frequently.leads.to.diagnostic.T.to.C.transitions,.
respectively,.in.the.cDNA.sequences,.providing.a.feature.to.ilter.cross-linked.from.non-
cross-linked.sequences..The.clusters.of.aligned.sequences,.representing.putative.binding.
sites,. can. be. ranked. according. to. the. number. of. the. T. to. C. transitions.. By. introducing.
photoreactive. nucleosides. that. generate. characteristic. sequence. changes. upon. cross-
linking,.PAR-CLIP.allows.one.to.separate.RNA.segments.bound.by.the.protein.of.interest.
from.the.background.non-cross-linked.RNAs..Further,.these.transitions.indicate.individual.
speciic.nucleotides.bound.to.the.protein,.allowing.for.accurate.determination.of.the.exact.
location.of.RNA-protein.interactions.in.the.complex.providing.footprinting.information..
A.comparison.of.the.techniques.is.illustrated.in.Figure.5.3..These.techniques.are.essential.
tools.for.studying.RNA-protein.interactions.for.both.understanding.biological.systems.as.
well.as.studying.artiicially.created.or.modiied.systems.
