From RNA Structures to RNA Nanomachines - The Nanobiotechnology - page 119

Culture cells normally Culture cells normally Culture cells in

4-thiouridine

UV light to cross-link RNA to protein UV light to cross-link RNA to protein

Lyse cells

Bound RNA

Magnetic
bead

Protein A/G coat
Antibody

Digest and reverse transcribe sample

Sequence and compare to existing
databases to determine binding
regions

Reverse transcription of cross-linked
4-thiouridine introduces T-C transitions

Rinse/proteinase to
free RNA

Sequence and compare to existing
databases to determine binding
regions (a) (b) (c)

Figure 5.3
(See companion CD for color igure.) . RIP/CLIP/PAR-CLIP.. RIP,. CLIP,. and. PAR-CLIP. represent. different.
methods. of. investigating. RNA-protein. interactions.. Although. the. basic. rinse. and. elution. steps. are. largely.
similar,. the. cell. culture. and. sample. preparation. for. sequencing. vary. between. techniques.. (a). Standard. RIP,.
(b).Clip,.and.(c).Par-clip.

5.3.2 Noncoding rNA regulation

The.ability.for.two.trans-acting.RNAs.to.structurally.interact.with.each.other.is.an.attrac-
tive.and.exciting.hypothesis.(George.and.Tenenbaum.2006)..Most.RNA-RNA.interactions.
are.typically.visualized.as.linear.base.pairing.between.two.sets.of.sequences.or.abutting.
sequences.such.as.kissing.complexes..The.effect.of.trans.interactions.on.RNA.structure.is.
often.not.considered..It.should.not.be.surprising.that.eukaryotic.cells.would.have.evolved.
to. also. use. structural. changes. in. RNA. ( cis . as. well. as. trans . interactions). to. regulate. gene.
expression..Some.possible.means.of.interactions.are.shown.in.Figure.5.4..The.illustration.
depicts.versatility.of.the.miRNA's.control.over.the.message..It.can.either.enhance.or.reduce.
protein.binding,.or.in.some.cases.create.even.alternate.binding.sites.in.the.same.message..
The. biochemical. outcome. inside. the. cell. will. be. different. in. each. case.. MicroRNAs. and.
RNA-binding. proteins. (RBPs). can. regulate. gene. expression,. most. notably. through. their.
actions.with.the.untranslated.regions.(UTRs).of.mRNA..miRNAs.modulate.RBP-binding.
sites.in.a.dynamic.manner,.targeting.and.sharing.a.common.regulatory.code..This.would.
provide. a. mechanism. to. inluence. the. structure. (shape). of. the. mRNA. to. ensure. that. the.
appropriate. regulatory. elements. are. utilized. for. the. optimal. expression. of. a. multifunc-
tional.mRNA.transcript.
Our.work.and.other.studies.previously.noted.examples.of.signiicant.parallel.target-
ing.by.miRNAs.and.RBP.regulatory.elements.(Pillai.et.al..2005;.Bhattacharyya.et.al..2006;.