Post-transcriptional RNA Regulons Affecting Cell Cycle and Proliferation

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Please citethisarticle inpressas:Blackinton JG,KeeneJD.Post-transcriptional RNAregulonsaffectingcell cycle andproliferation.Semin

Cell DevBiol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.05.014

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2 J.G. Blackinton, J.D. Keene / Seminars in Cell & Developmental Biology xxx (2014) xxxxxx

are to respond to endogenous and exogenous cell signals effi-

ciently and economically in order to help coordinate production of

components of biochemical pathways or macromolecular protein

complexes; for example, biochemical pathways, subunits of cellu-

lar pumps, organelles, or other machines that function together

in cellular compartments. RNA regulons consisting of RNAs and

RNA-bindingproteinshave beenwidelydocumented to coordinate

post-transcriptional processes in many species; the general topic

has been broadly reviewed in recent years [317]. Therefore, this

article will focus onRNAregulons/PTROs identified todate that are

involved in coordinating the production of proteins regulating cell

cycle initiation and progression.

The concept of post-transcriptional RNAoperons and regulons,

generally referred to as PTROs or RNA regulons, invokes the clas-

sical bacterial operons discovered by Jacob,Monod andcoworkers

and Monod [18] as a didactic tool to make it easily understood.

However, there are significant differences between the classical

operons and regulons of bacteria and RNA regulons. These differ-

ences are not based on bacterial versus eukaryotic mechanisms,

as RNA regulons have recently been described in archaea and

bacteria [1924]. Instead, they are due to the fundamental differ-

ences in the organization of DNA and RNA in any cell. While DNA

genes in bacterialoperons areconstrainedby covalentarchitecture

and physical location, RNAs are diverse in quantity and location,affording them vast combinatorial opportunities for RNAprotein

and RNARNA interactions and transiently dynamic regulation of

mRNAsubsetsencodingfunctionally relatedproteins.This variabil-

ity in quantityandlocalization ofRNAs affords RNAregulonsmany

additional properties. First, multiple copies of each mRNA species

arecapableof joiningmany differentRNAregulonssimultaneously

based on different recognition elements in the RNA (RREs or USER

codes). Second, RNA regulons are agile with reversible interac-

tions between RNA binding proteins (RBPs) and RNAs. Therefore,

RNA regulons have highly dynamic RNA targeting and the RNPs

can be remodeled rapidly because the coordinated RNAs them-

selvesare combinatorial. Third, RNAregulons cangovern localized

dynamic regulation of functionally related mRNAs. This is partic-

ularly important in eukaryotes, as cellular compartmentalizationrequires greater control of localizationamong differentorganelles.

In sum,multiple copies of eachmRNA can use their multiple RREs

or USERs to join groups of functionally related mRNAs in time

and space in order to coordinate multiple functions in combina-

tion. These overlapping mRNA networks have modular coherence

because the RNPs that form the RNA regulons can be remodeled

withdifferent functional statesdependingon signal inputs [2529].

Physically compartmentalized nuclear and cytoplasmic RNP

regulonsthat coordinate theproductionof functionallyrelatedpro-

teins have also been termed quasi-genomes to describe their oft

remote locations in cells [1,2]. However, the combinatorial use of

functionally related mRNAs, whether physically clustered or not

is highly plastic in real-time and that plasticity is not available to

classical operons and regulons in bacteria. Perhaps a better RNAregulon analogy may be that made to music, where each mRNA

equates to individual notes from each instrument that must har-

monizein differentcoordinatedcombinations in time andspace to

produceamelodically orchestratedsymphony.Moreover,imagina-

tivecomposers canrearrange thenotes andinstruments toproduce

different musical scores just as RNA regulons provide an avenue

of evolutionary opportunity for proteins to gain new functions by

rewiring existing RNA recognition elements or proteins in real-

time, orevolvingnew interactingpartners[3032]. Agoodexample

of RNAprotein rewiring have been observed with PUF3 family

RBPs, with changes in classes of regulatory targets within fungal

species [31,32] as well as across many other metazoan species

[30,33,34]. Presumably as a consequence of these myriad advan-

tages, RNA regulons have evolved to play functionally important

rolesin nearlyallspeciesacross the threedomainsof life [3]. Indeed,

the dynamic plasticity of RNA regulons are just beginning to be

understood [1,2,7,3538].

Itwas previously assumed that everymRNA species functioned

independently in the eukaryotic cell and was regulated indepen-

dently by distinct RBPs. But, as noted above, we now understand

thatwith few exceptions every mRNA is a member of one ormore

RNA regulon(s) and most RBPs regulate large numbers of mRNAs

[3,3942]. This is because each mRNA has multiple copies and

each one of those copies has the potential to associate with other

mRNAsthatencodeproteins thatare functionallyrelated[17]. Thus,

becausemRNAsarethemselves combinatorial,meaningthat differ-

ent combinations ofmRNAs canform differentRNAregulons, their

formation depends on the needs of the cell in time and space [2].

The first studies to indicate that RBPs can target multiple growth

regulatory mRNAs used in vitro RNA binding methods and Selex

procedures withrandomRNAs [4347] andwithmRNAs extracted

from humanbrain [39].

The first in vivo evidence indicating global coordination by

endogenous RNA regulons was derived using RNA immunopre-

cipitation(RIP) followedbymicroarray withmammalianneuronal

ELAV/Hu proteins known to be involved in cell growth and dif-

ferentiation [25]. This method was developed to avoid sonication

or other harsh lysing to maintain physiological interactions, andis therefore entirely different from that used by Mili and Steitz

when they observed mRNA reassortment [48]. To date, we have

not observed reassortment when using the original Tenenbaum

RIP procedure [49]. The Tenenbaum study used both DNA arrays

and multichannel RNase protection assays to verify that mRNAs

encodingcell cyclefactorswere among themRNA targetarraysub-

sets of ELAV/HuB and ELAV/HuR including n-myc, l-myc, b-myc,

max, and cyclins A2, B1, C, D1 and D2, but not cyclin D3, cyclin

B2 [25]. (Fig. 1) These findings were consistent with concomitant

findings by Gorospe and colleagues [50] showing that cyclin A and

B mRNAs were bound and stabilized by HuR during cell prolifera-

tion. These findingswere later confirmed in the Gorospe Labusing

theglobalRIP-chipprocedureofTenenbaumet al.withmicroarrays

[51].Since then, many global post-transcriptional procedures like

RIP-chip/seq [25,52], temporal RNA decay and global nuclear run-

on (reviewed in [13]), polysome profiling, RNA interference, and

cell synchronization have been used to identify cellular anddevel-

opmental RNA regulons [315]. RNA regulons have been shown

to coordinate cellular processes during cellular homeostasis and

normal replicative events such as cell cycle, as well as during

developmental programs, biological activations, or environmen-

tal perturbations. Many RNA regulons have also been discovered

using genetic approaches while others were observed as dynamic

patterns of mRNA co-regulation during a regulatory event. The

trypanosomelifecyclefieldhas employedthis lattermethodexten-

sively, followed by identifying the key RBPs or global coordinators

[5,16,28]. Through these techniques, many examples of RNA reg-ulons have emerged over the past decade at nearly every step

of post-transcriptional RNA processing, including splicing, export,

translation, RNA stability and localization [6,10].

An unresolved question, however, iswhether RNAregulons are

directly or indirectly coupled to one another at each step along

the path of RNA processing and how mRNA coordination via RNA

regulons maintains a consistent mRNA target set as the informa-

tion flows from the chromatin to the ribosome [6]. In other words,

howismRNAtrafficdirectedthrough theRNAprocessingpathway?

It would seem efficient for mRNAs encoding functionally related

proteins to flow together from step to step in a pathway when

they are destined to function together at the final steps of gene

expression(Fig.2).Muchof the information regarding thecoupling

of each of these processes is based upon single mRNAs. However,
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J.G. Blackinton, J.D. Keene / Seminars in Cell & Developmental Biology xxx (2014) xxxxxx 3

Fig. 1. ELAV/Hu RBPs interact with multiplemRNA targets encoding cell cycle reg-

ulators. Following the RIP procedure, bound mRNA targets were identified using

a multi component RNAase protection assay. Note that control mRNAs encoding

ribosomal protein L32 and GAPDH that did not bind to the RBP.

Reproduced fromTenenbaumetal.,ProcNatlAcadSciU SA97:1408514090 (2000)

[25].

evidence for coupling of post-transcriptional interactions globallyis beginning to accumulate, and among the most compelling early

examplesare fromexperimentswithSaccharomyces cerevisiaeRNA

regulons.

2. Global RNA regulons in budding yeast

Silver and colleagues [53] presented the first solid confir-

mation of the RNA regulon model in 2003. They demonstrated

that functionally related mRNAs associated with two yeast RNA

export factors, Yra1 andMex67 coordinately exportmultiple func-

tionally related mRNAs to the cytoplasm [53,54]. For example,

Yra1 associated largely with cell-cycle regulatory factors andpro-

teinsinvolvedin carbohydratemetabolism,Mex67 associatedwith

mRNAs encoding membrane proteins, cell-wall components, and

RNA-regulatory factors (Fig. 3A). This latter set ofMex67 exported

mRNAs continues to be a consistent finding thatmany RBPs inter-

actwith mRNAs encoding regulatory RBPs, translation factors, and

transcription factors. This concept of broad regulation of regula-

tory factors has been termed regulators of regulators as RNA

regulons [25,49,55]. While Yra1 associated with approximately

1000mRNAs, some encoding proteins that repressed S phase and

induced G1 phase of the cell cycle, and Mex67 associated with

approximately 1150 mRNAs, some of which encoded membrane

proteins and translation factors, they both shared349overlapping

transcriptsencodingcellwallcomponentsandheat-shockproteins.

This finding of mRNA target overlap was consistent with results

from Tenenbaum et al. [25] showing that RBPs such as HuR and

eIF4E each associated with unique as well as overlapping tran-scripts. This would be expected if each mRNA has more than one

RRE or USER code, thus, demonstrating that networks of mRNAs

could be modular and overlapping [2]. An exciting aspect of the

work by Hieronymus and Silver [53] was the connection between

theYra1mRNA export factorand theAbf1 transcription factor. Not

only does theAbf1 transcription factor regulate theYra1 gene, but

the Abf1 mRNA is exported by Yra1 protein. This was confirmed

genetically suggesting regulatory crosstalk between transcription

and export.

A longstanding curiosity has been whether mRNAs exit the

nucleus randomly as they are passed from the RNA processing

apparatus through nuclear pores or whether they are organized

intofunctionally coherentsubpopulations. An earlystudy fromthe

Cole lab demonstrated mRNA-specific regulation in RNA export.Using yeast mutants of the nucleoporin Rip1p, mRNAs encoding

certainheatshockproteinswereefficientlyexporteddespiteglobal

polyA+ mRNA export suppression under stress conditions [56]. A

Fig.2. Couplingof RNARegulonsfromtranscriptionto translation.A model oftwo distinctRNA processing pathwaysare depictedfor a setofmRNAs.The functionallyrelated

set of mRNAs in blue are spliced, exported and translated through regulatory events directed by splicing factor A, export factor B and translation factor C in RNA regulons

that are coupled together through interactionsbetween A, B andC. The functionallyrelated set of mRNAs in redare likewise spliced, exported and translated by D, E and F,

respectively, through similar coupling. (For interpretation of thereferences to color in this figure legend, thereader is referred to theweb version of thearticle.)


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Fig. 3. Early examplesof RNAregulons.(A) CoordinatedmRNA exportshowingtwo yeast RBPexport factorsMex67 andYra1 as they transportgroups of functionallyrelated

mRNAs out of the nucleus from derived from the data of Hieronymus and Silver (2003) [53]. Reproduced from Keene,Nat Genet 33: 111112 (2003) [54]. (B) Coordinated

regulation of theyeastPUF3 RBPtransporting nuclear mRNAs that encodemitochondrial proteins tomitochondria fortranslation anduptakefromGerberet al. (2004) [35].

Reproduced from Keene,Nat RevGenet 8:533543 (2007) [3].

unique nuclear pore complex containing Rip1p alongwith several

nucleoporinsexportsheatshockmRNAs tothecytoplasm.Differen-

tial mRNA export of this type likely involves distinct RNA-binding

surfaces, although it is possible that some of the Nup proteins

themselves either possess RNA-binding properties or are coupledwith specific RBPs, possibly coupled to the splicing apparatus [57].

While several examples of pre-messenger RNA splicing regulons

andRNAexport regulonshavebeenpublished, demonstrating cou-

plingof theseRNAregulonsatthesestepsalongthepathwayof RNA

processing has remained challenging. As discussed below, a sub-

sequent study from the Silver lab using Drosophila cells [58], also

showed export of heat-shock factors whose mRNAs are uniquely

exported like those of Saavedra et al. [56] but have known roles in

splicingvia thePCIdomain-containingprotein-2that subsequently

associateswithpolysomes.However,it isnotknownfromthiswork

whetherthesubsequenteventsdownstreamof theheat-shock spe-

cific exportmechanism are coordinated as an RNAregulon(s).

Brown and colleagues discovered that nuclear-encoded mito-

chondrial mRNAs bound to the PUF3 RNA-binding protein are

exportedoutof thenucleus tomitochondria,undergoderepression

and translation, allowing the new functionally related proteins to

enterthemitochondrion[35]. Derepression of factors alreadyposi-

tioned for activation at a remote site is an efficient and accurate

means to coordinate a group function (Fig. 3B). Several subsequentstudies confirmed and extended these findings using genetic and

biochemicalmethods[5961].Moreover,the Brown laboratoryfol-

lowed up this initial discovery by identifying a total of 46 RBPs of

S. cerevisiae that also bound to unique modular subsets of mRNAs

encodingfunctionallyrelated proteins[62]. Anumberof theseRBPs

bound similar messages at different stages in the RNA life cycle,

hinting at a role for coupling of RBPs.

In addition to these early confirmations of the RNA regulon

model, many additional distinct biological processes, pathways

and macromolecular complexes across the three domains of life

have been shown to be regulated together at the mRNA level,

and in nearly every case regulatory RBPs involved in coordinat-

ing thesefunctionswereidentified[3,10,13,63] (seereview articles

noted above). Below,we focus on cell cycle functions in yeast and


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Fig. 4. Coordination ofmRNAs associated with NUPs andRBPs that are subsequently translated into cell cycle regulators. Several RBPs have been genetically demonstrated

to influence cell cycle events, includingexport factorNUP98and translationfactorsPUM1,DND1 andGLD-1as describedin thetext. Interestingly, theseRBPs viatheirmRNA

targets actas repressors of cell growthand have been characterized as tumor suppressors.As describedin thetext, loss of function oftheseRBPscauses increasedcellgrowth

through defects at cell cycle checkpoints.

mammalian cells that arepotentially coordinatedas RNAregulons

at the levels of RNAexport and translation/stability.

3. Nuclear export RNA regulons during the cell cycle

Many published examples of splicing events functioning as

RNA regulons have suggested that discrete subsets of mRNAs

are coordinately spliced, associated with RBP complexes such as

the exon junction complex and eventually exported to the cyto-

plasm[6466]. Silverandcoworkers observedupregulationof RNA

splice variants encoding prodeath functions as directed by the

ASF/SF2 RBP [64]. Among thekeyalternativelyspliced targetswere

the apoptosis regulators Bcl2, Mcl1 and caspase-9. Using a high-

throughputRNAinterferencescreen, theyfound thatmanyof thesecell cycle regulatorsweregeneticallylinkedto themitoticregulator

aurora kinase A. Overall their data indicated that these alternative

splicing regulators coordinate amechanistic balance between pro-

and anti- apoptosis and cell cycle. Thus, the data suggest that cou-

pling between the cell cycleandalternative splicing is coordinated

viaoverlapping RNAregulonsandhas implicationsforoncogenesis

[64]. Figs. 2 and 4 In addition, there aredefinitiveexamplesof very

early nuclear RNA imprinting such as the exon-junction complex

that helps determine the final fate of a given mRNA, aswell as the

fate of functionally related subsets of mRNAs localized for trans-

lation in the cytoplasm [15,67,68]. Several studies demonstrated

the association of export proteins with mRNA subsets in organ-

isms fromyeast toflies tomammals, someofwhichincludemRNAs

encoding cell cycle and proliferation factors [9,53,54,58,69].

For example, Guthrie and colleagues [69] demonstrated that

hnRNP-type RNA export factors Nab2, Nab4 and Npl3 in S. cere-

visiae each bind to their own unique specific subset of mRNAs.

Using the RIP-chip procedure, they found that Npl3 preferentially

binds to ribosomal protein mRNAs, while Nab4 strongly prefers

mRNAs encoding factors involved in amino acid metabolism. The

latter observation was consistent with genetic mutants of Nab4

that also showed a desensitized growthphenotype when exposed

to aminoacid stress. Aswith other RIP-chip studies, they identified

a common set ofmotifs: AAAAG prevalent in Nab2 and UAUAUAA

or ACACTACA in Nab4 mRNA targets. The authors concluded that

there are multiple routes of mRNA export for functionally related

mRNA subsets, and hypothesized that Npl3maybe responsible for

the bulk mRNA export given that many hnRNP proteins associatewith it, but that these balances may shift during stress conditions.

In addition, the authors suggest coupling of RNA export with 3

end processing via interactions between Nab2 and polyA-binding

protein. It was not clear whether the 3 ends of mRNAs encoding

the functionally related proteinswould be coordinately cleaved in

a simultaneous or sequential order by cleavage factor 1 in concert

with their coordinated export to the cytoplasm via these hnRNP

proteins. In any case, this landmark paper presents evidence that

theseimportantproteinscanexportdistinctcombinationsofmRNA

cargos.

As noted above, using a genome-wide RNA interference screen,

Silver andcolleagues identified72 factors,somerequired fordiffer-

ential nuclear export in Drosophila, and examined specific mRNAs

encoding heat shock factors that are in turn, coupled to polysomes


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and translation via a PCI domain protein that associates with RNA

[58]. These findings, while not global, have strong implications for

coupling of RNA regulons from splicing to translation.

4. Nuclear export RNA regulons inmammalian cell cycle

4.1. eIF4E RNA regulon of cell cycle and proliferation

Specific nuclear pore components have been shown in mam-malian cells to be unique for export of mRNA subsets encoding

proteinsregulating cellulargrowth,DNAdamageandcellcycle reg-

ulation[7076]. For example, thewell-studiedcapbindingprotein,

eIF4E, is a major regulator of protein synthesis and has mitogenic

and oncogenic propertiesdiscovered by Sonenberg and colleagues

and confirmed in many laboratories [15,7780]. The overexpres-

sion of eIF4E increased Ras activation and it could be reversed by

expression ofGTPaseactivating protein, a knownnegative antago-

nist [77]. More recently,Bordenand colleagues reported that eIF4E

associates in thenucleuswith RNPs that mediate nuclear exportof

discrete mRNA subsets as a RNA regulon via a CRM1-dependent

mechanism [70]. The mRNA targets of this RNA export regulon

were identified by a RIP-chip/seq type immunoprecipitation and

included several cyclins,mdm2, c-myc andother cell cycle-relatedfactors. ThesemRNAswere found to have a specific stem-loopRRE

they termed 4E-SE functioning as a USER. The authors examined

the 3Dstructural featuresof thestem-loop and confirmed itsstruc-

ture using nuclease probes, and demonstrated that the 4E-SE was

required to form stable RNP complexes using cyclin D1 as their

model. They concluded that eIF4E, acting as a RNA regulon, could

modulate the coordinated expression of these cell cycle proteins

efficiently, thereby impacting proliferation and cell survival.

4.2. Nup96 mRNA subsets encoding cell cycle regulators

A study in the Blobel and Matunis labs revealed that a poly-

cistronic cDNA encodes a precursor protein containing both the

Nup98 and Nup96 nucleoporins [81]. Further analysis indicatedthat this precursor protein is cleaved by a protease to yield these

two nucleoporins that were later found to have related functional

roles in nucleocytoplasmic transport. Interestingly, both proteins

have roles in cell cycle, p53 pathways and carcinogenesis inmany

species [71,73,75,82,83] . In acute myeloid leukemia, a transloca-

tion creates a fusion protein between Nup98 and a homeodomain

DNA binding domain that blocks thenormal functioningof Nup96,

and thereby, disrupting its putative tumor suppressor function as

discussed below in Section 4.3 from the data of Singer et al. [75].

Nup98 can shuttle into andout of thenucleus andappears to inter-

act directly with the chromatin to affect transcription of cell cycle

genes inDrosophila [73,83]. In any case, bothNup96 andNup98 are

suggested to function as part of post-transcriptional RNAregulons

coordinatingtransportofmRNAs encodingcell cycle, developmentand oncogenesis.

Nup96hasbeen showntohave several importantfunctions and

is a componentof theNup107160 complex that is cell cycle regu-

lated. In some cases, each component of the complex hasdifferent

stoichiometry in the nuclear pore during stages of the cell cycle

[71]. Case in point, Fontoura and coworkers demonstrated that at

mitosis the Nup96 subunit is downregulated preferentially as a

requirement for T cells to exit G1/S phase of the cell cycle and in

heterozygous mutant Nup96 mouse T cells that express lowered

levels of Nup96, cell cycle progression increased. Moreover, levels

of cyclin Awere also downregulated early inmitosis,whilecyclin B

was increased intheseT cells.In theNup96+/ heterozygousmouse

cells, decreasedNup96 increased the rate of cell cycle progression

that correlated with failure to export specific mRNAs that encode

cell cycleregulators.ThesemRNA targets ofNup96associatedwith

cell cycledysfunction includedcyclinD3, CDK6andIB thataccu-

mulated in the cytoplasm while the nuclear/cytoplasmic ratio of

GAPDH, ICAM-1 and -tubulin did not change when Nup96 levels

were lowered [71,82]. Thecytoplasmic predominance of cyclinD3

andCDK6mRNAs resulted in increasedproductionof their proteins

andpremature activation of S phase.

While it appears that Nup96 suppresses export of these mRNA

specific subsets, these investigatorssuggestedthatNup96 couldbe

a potentialRBP. However, further studies areneededto definehow

these cell cycle-specific mRNAs are selected from the up-stream

processing apparatusandmoved through the pipeline. These find-

ingsledWozniakandGoldfarb[82] to suggest that thesefindingsfit

thecriteriafor aRNAregulonand speculated thatNup96itself could

be the transacting RBP that controls the RNA regulon. (Fig. 2) How

discrete subsets of functionally related mRNAs are able to locate

andassociatewiththenuclearpore subunits isnot knownbutcould

involve a coupling of export to thenascent splicing apparatus that

are themselves function as an early organizational step in the RNA

regulon pathway of RNA coordination [6,64].

4.3. Nup98 mRNA subsets encoding cell cycle regulators

Nup98is another astounding example ofa potentialRNA exportregulon that is involved in cell cycle and p53 regulation in mam-

maliancells. Privesand coworkers reported thatNup98 is required

for the expression of the p21 (CDKN1A) oncogenic protein on the

post-transcriptional levelandtherebyaffectsseveralproteinsin the

p53 pathway [75]. In an RNA interference screen, Nup98 appeared

tobe involved in targetingof p53regulated genes.Previous studies

demonstrateda linkbetweenNup98andleukemiaviaanunrelated

mechanism[83]. However, it is exceedingly interesting tonotethat

Nup98andNup96 (discussed above in Section 4.2.) areproducts of

a cleavage event of a precursor protein [8183]. The p21 regula-

tion reported in the Prives Lab showed that Nup98 forms an RNP

complexwithp21mRNAvia its 3 UTRandpreventsdegradationof

the p21message and can be rescued byblocking the RNA degrada-

tion exosome system. Using a RIPprocedure, the 3 UTR ofp21wasshown to directly bind to Nup98 in HepG2 cells, and further anal-

ysis suggested a C-rich RRE thatwas also present in other mRNAs.

For example, their data based on computational analysis indicated

that mRNA encoding the 14-3-3 protein, another p53 pathway

factor, is similarly bound to Nup98 and protected from degrada-

tion.Moreover, theauthors confirmedusingRIP-seq and qPCR that

the14-3-3proteinhasa similar C-richRRE forbinding. Theywent

on to demonstrate that Nup98 is required for cells to undergo p53

mediated senescence and could partially protect cells from DNA

damage-mediated apoptosis. Nup98 as well as 14-3-3 levels of

expression were both decreased in concert followingDNA damage

as demonstrated usingmurinehepatocellular carcinomas and in a

cohort of patient samples [75]. One important conclusion is that if

Nup98 is a tumor suppressor, it would be advantageous for cancercells to inactivateNup98 during their evolution. It will be interest-

ing to see how the functions of Nup98 are compromised among

diverse collections of carcinoma or leukemia cells.

In summary, the findings thatNup98 can function as a RBP that

binds to a subset of p53 target genes and regulate the stability of

their mRNAs is both exciting and unique (Fig. 4). It is a poten-

tially separate oncogenic pathway whereby Nup98 can regulate

p53 genes as a tumor suppressor. The authors suggest that this

may representa p53post-transcriptional RNAregulon thatcouples

with p53 transcriptional regulation to coordinate otherwise nor-

mal functions whichare potentially dysregulated at several stages

along the gene expression pathway. It will be interesting to use

genomewideRIP-chip/seqand PAR-CLIPmethodstodeterminethe

globalset ofmRNAsthat interactwithNup98undervariousgrowth


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conditions.OthermorediverseexamplesofRNAregulonsandRBPs

affectingcell cycle and DNAdamageresponsesarediscussedin the

next section.

5. Coordinated RNA targeting in cell cycle, p53 and DNA

damage

5.1. RNA regulons responding to the DNA damage response

Among the most prevalent mutational events in cancer is that

of thecheckpoint kinase, ataxia telangiectasiamutated (ATM)[84].

TheATMresponseactivateswhenDNA undergoesdouble stranded

breaksby irradiation, oxidative stress or other forms of physical or

chemical assault causingthe kinaseto phosphorylatea seriesof cell

cyclecheckpoint regulators includingcheckpoint kinase2 or Chk2

and otherproteins in thep53pathwayinvolved intheDNAdamage

response and repair. This response helps tominimize the effects of

DNA damage by delaying the cell cycle or inducing apoptosis to

eliminate damaged cells. Several RBPs including HuR, AUF1, TIAR,

RPL26,nucleolin andothers aredocumented to respond to induced

stressandDNAdamage[72,74,76,8587], in some cases, becoming

relocalized to stress granules. The role of stress granules resulting

fromoxidative stress isbelievedtotemporarilystoreRBPs andtheirRNA cargo in order to protect the mRNAs until the DNA is repaired

[88]. Forexample, after exposingmammalian cells toUVClight the

levels of p53 mRNA did not change, but translation of its mRNA

to protein was increased. The increased production of p53 under

these conditions was shown to involve the HuR RBP acting on the

3 UTR of the p53 mRNA. These results were confirmed using RNA

interference against HuR to demonstrate the expected inhibition

of p53 production. It is logical to expect thatwhen the synthesis of

p53protein is altered, thedownstream transcription targets of p53

would be affected accordingly.

Gartenhaus and coworkers took advantage of these observa-

tions to examinewhetherHuRparticipates in the ATMresponseon

a global level via RNA operons and regulons [29]. The Chk2 kinase

has been shown to phosphorylate HuR at residues 88, 100, and118, and thereby alter its specificity of binding to the SIRT1 mRNA

followingoxidative stress induction. A previousmodel was gener-

ated in the Gorospe lab proposing thatATM and Chk2 cooperate to

regulatethebindingof specificmRNA subsets byHuR[89]. UsingB-

lymphocytecelllinesanda RIP-chipribonomicsapproach, followed

byconfirmationof individual target effects,Gartenhaus andcollab-

orators concluded thatmodular networksofHuRregulatedtargets

encoding functionally related proteinsof the p53and other growth

pathways are coordinated globally in responses to DNA damage

andstress[29]. Forexample, this study identified numerousmRNA

targets of HuR that encoded proteins involved in cell proliferation,

cell cycle, cell arrest, MAP kinase regulation and stress responses.

Among them was p21, FOXO3, MEK1, MEK2 and DUSP10 whose

mRNA binding was directly assessed for downstream ATM/Chk2signaling using HuRmutants at Chk2 phosphorylation sites S88A,

S100A and T118A, as well as testing the effects of Chk2 inhibitors

in the presence or absence of irradiation. Overall, this network

of mRNA targets represented an ATM/Chk2 cascade affecting the

phosphorylation state of HuR thatsubsequently targeted function-

allyrelatedmRNAsubpopulations encodingfactors involvedin cell

proliferation and cell cycle regulation.

These dynamic datasetswere similar to earlier studies inwhich

the Gartenhaus group compared HuR and AUF1 RBPs and demon-

strated changes in overlapping functionally related mRNA subsets

using MCT-1 oncogene-mediated transformation of immortalized

breast epithelial MCF10A cells [90]. This work demonstrated the

combinatorial binding of multiple RBPs to some of the same

mRNAs as suggested in other studies [25,27,35,76], and others),

but also showed that overlapping mRNA targets of HuR and AUF1

involved in cell cycle and cell signaling pathways were differen-

tially expressed during tumorigenesis [90]. In sum, these global

mRNA targeting studies with ELAV/Hu proteins have consistently

identified many of the samemRNA targets encoding cell prolifer-

ation and cell cycle proteins in support of the RNA regulon model

and many of its dynamic properties (Fig. 2).

5.2. RNA regulons targeting p53 and DNA damage at translation

A striking study from the Tofilon laboratory comparing tran-

scriptional with translational regulation of genes expressed in

glioblastoma cell lines following gamma irradiation revealed that

a response at the level of the ribosome determines the changes

and obviously the differences in protein production [91,92]. The

initial study compared three independently derived cell glioma

lines before and after 7 Grays of irradiation using mRNA analy-

sis by microarray [91]. By quantifying mRNAs that increased or

decreased in each of these cell lines after irradiation, the authors

definitively demonstrated that about 500600 RNAs shifted on

polysome gradients from the inactive to the translationally active

regions,whileonlyabout100mRNAs changedin thetranscriptome

(totalmRNA)data.Moreover,comparingthe threecelllinesshowed

zero overlap among the transcriptome mRNAs that changed, butapproximately 300 RNAs that changed consistently among the

translatome datasets. These dramatic results are compelling and

unexpected, and were confirmed and extended in a subsequent

paper that compared other types of cancer by these criteria

[92].

L et al. [91] used western blotting to confirm that the protein

products of the mRNAs found to increase or decrease their posi-

tions on polysome gradients corresponded to the observed up or

down shifts of the mRNAs. As one might expect, the identifica-

tion of these mRNAs and their encoded proteins showed that they

were functionally related. For example, the key proteins included

primarily cell cycle regulators, cell death, repair and cell prolifer-

ation factors [91]. As with other studies of this type, many DNA

andRNA regulatory proteinswere included as well reminiscent ofthe regulators of regulators findings. The authors went on to sug-

gest that these results are consistent with thepost-transcriptional

RNAregulonmodel,anddiscussed translation factors such aseIF4E

and4E-binding proteins aswell as RBPs like fragile Xmental retar-

dation and CUGBP that could be involved in globally coordinating

these growth regulatorymRNAs.

5.3. Global translational dynamics during the cell cycle

While many RNA regulons have been described in the litera-

ture to date, those coordinating cell cycle and cell proliferation,

and those implicated in oncogenesis are compelling.Moreover, the

global role of translational RNA regulons are particularly exciting

because the coordinated production of functionally related pro-teins is themost important endgame forbiological systems [15]. A

recent study by Ruggero and colleagues provided strong evidence

for translational events determining the expression of cell cycle

progression, lipid metabolism and the DNA damage/repair path-

ways at each stage of the cell cycle [93]. Several previous reports

characterizing changes in mRNA populations throughout the cell

cycle have identified responsive gene sets.

Toexaminetranslational regulationduringthecell cycle,Stumpf

and coworkers [93] used ribosomal profiling/occupancy and high-

throughput sequencing of RNA from synchronized HeLa cells to

identify mRNAs unique to G1, S phase, and mitosis. The authors

found that 12% of the transcripts in the cell change ribosome occu-

pancy (higher or lower)at some stageof thecell cycle. As expected,

duringmitosis there were fewer changes inmRNA levels in either


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G1orSphasestogetheror separately.They identified coregroups

of mRNAs that change in all phases of the cell cycle. The patterns

of clustering transcripts with ribosome occupancy were distinctly

differentthan themRNAs expressedby thetranscriptome, suggest-

ing to theauthors that the two processes areuncoupledduringthe

cell cycle.While there were features of the 5 UTR such as GC con-

tent of certain transcripts noted these were not deemed to be the

chiefdeterminants of cell cycle translational changes.On theother

hand, specific mRNAs encoding known regulators of cell cycling

were identified. These included increases in cyclin CCNE2 mRNA

translation in G1 and S phases, and decreases in occupancy of the

E2F1transcriptionfactormRNA, possibly resulting frommicroRNA

functions.

The authors noted distinct changes in ribosome occupancy of

mRNAs encoding mitotic checkpoint factors and strong increase

in translation of the RICTOR subunit of the mTOR complex 2 from

G1 to S phase that was shown to be dependent upon its 5 UTR.

They noted a total of 353 differentially expressedmRNAs between

the three phases of G1, S and M. There was clear evidence that

these transcripts encoded functionally related cell cycle regulatory

proteins. Thus, the authors constructed modular network models

that coordinate the translation of nuclear transport, metabolism,

cohesins, condensins andDNA repair components. In sum, this is a

very interesting study that demonstrates dynamic changes in thetranslatomeduring thecell cycle consistent with our own concept

of RNAregulons [15].

5.4. Developmental RNA regulons of p53 and cell cycle pathways

There have been various examples of RNA regulons found in

cell lines and several that appear to coordinate various processes

in tissues and organs[3,10,13]. Some of the clearest examples have

come from genetic studies in mice and C. elegans. With respect to

cell cycle regulation in developing systems, we will consider a C.

elegansGLD1 RBP that coordinates cell cyclemRNAs in developing

embryos, a mouse Pumilio 1 RBP that coordinates components of

the p53/MAP kinase/cell cycle pathways during spermatogenesis,

and theDND1 (deadendhomlog1)RBPthat affects cell cyclearrestand differentiation of testicular teratomas inmouse.

The C. elegans GLD1 RBP is a member of the STAR family of

RBPs that are regulators of germ cell development and can func-

tionas tumor suppressors. GLD1mutantsaffect sex determination,

mitosis-meiosis decisions and germ cell identity and have phe-

notypes involving cell division, cell cycle and DNA replication,

and appear to function as translational repressors. Rafal Ciosk and

coworkers used RIP-chip to quantify GLD1RBP-mRNA targetbind-

ing strengths in whole worms and by confirming with cell lines

to identifymRNAtargets andestablishapproximate bindingaffini-

ties [94]. Using cellstransfectedwithepitopetaggedGLD1, extracts

of transgenic adult worms were used for the RIP procedure and

mRNA targets quantified by microarray. These experiments took

advantage of the earlier methods that allow one to use cell type-specific RBPs to RIP mRNAs away from endogenous RBPs in the

same tissues [25,49]. The data were highly reproducible (r=0.96)

and nontagged controls were used, and the previously demon-

strated heptanucleotide repeat UACUAAC/U with minor sequence

variants.

Approximately,950mRNAtargetswereidentifiedthatencoded

functionally related proteins that participate in cytokinesis, cell

division, reproduction, cell cycle and DNA replication in a man-

ner consistent with the RNA regulon model. Among the targets, it

seems that GLD1may relay its tumor suppression by translation-

ally repressing cyclin E mRNA, and possibly B-type cyclins as well

[95]. Overall, these functional groups were deemed to be consis-

tentwithmeiotic cell cycleprogression, sexdetermination andcell

fate specification phenotypes observed in earlier studies [94,96].

(Fig.4) Notonlywere theauthors able tomonitor dynamic changes

in individual targets in the embryos, theywere also able toquantify

apparent binding affinities of the targets in the extracts based on

bindingmotifsusingfluorescence polarization, and tomatch them

with the in vitro binding and translational repression of the tar-

gets byGLD1. Interestingly, a GLD1 PAR-CLIP study in C. elegansby

Rajewsky and coworkers [97] found an approximate 70% identity

with theGLD1RIP-chipmRNA targets ofWright et al. [94] and they

represented thesamefunctionallyrelated proteinsconsistentwith

theRNAregulon coordination model.

TheCapel laboratory investigated the functions of DND1 RBP in

formation of mouse testicular teratomas, and its role in cell cycle

arrest as well as thedifferentiationof primordial germ cells (PGC).

The DND1 RBP is necessary for preventing cell death of primor-

dial germ cells in gonads, as demonstrated using PGC defects in

mouse embryos with DND1 Termutations [98,99]. These investi-

gatorsshow that DND1regulatesmitotic arrest inmale PGCcellsat

the level of translation acting principally on cell cycle genes. They

hypothesize that the balance of positive and negative cell cycle

regulators, consistent with the observation that cell cycle arrest

by DND1, is able to prevent tumor formation. Using the RIP-chip

procedure with DND1, they identified functionally related mRNA

subsets that regulate the cell cycle and tumor-suppressor signal-

ing pathways in mouse germ cells at stage E14.5. In particular,mRNAs encoding p21, p27, Lats2, p53 and Pten, representing cell

cycle inhibitors were highlyenriched in the RIP-chip study. On the

other hand, Akt2, Akt3, cyclins E2, cyclinE1 and Akt1 representing

cellcycleactivatorswerenot enriched(Fig.4).Theauthorsconclude

that these results support the notion thatDND1regulatescell cycle

events that areconsistentwith itsestablishedphenotypesas a RNA

regulon [99].

Another related example of a cell cycle regulatory RNA regu-

lon was discovered in the Lin laboratory while investigating loss

of spermatocytes in the mouse germline [74]. The Pumilio 1 mam-

malian gene is a member of the PUF RBP family that is known to

suppress expression ofmRNAs towhichthey bind [30,33,35]. Dur-

ingspermatogenesis in thetestes anoverwhelminglylargenumber

of sperm cells is generated by mitosis and must be eliminatedthrough apoptosis to keep the organ healthy. Genetic deletion of

Pumilio1 inmicecauses increasedactivationofp53-mediatedapo-

ptosis, resulting in reduction in sperm numbers and infertility. On

theother hand,deletionofp53in the Pumilio 1 nullmice decreased

apoptosisandrescuedthistesticularhypotrophyphenotype. In this

study, RIP-chip studies of Pumilio 1 targeting in testicular lysates

revealedthatit interactswithmRNAsthatencodep53pathway,cell

cycle and MAP kinase-signaling components, and represses their

translation[74]. Usinga variety of cell imaging andmoleculartech-

niques, the authorsconfirmedthesetargets invitroand invivo, and

report that a distinct subset of these mRNAs encode key members

of the p53 and cell cycle pathways, thus, functioning as a Pumilio 1

RNA operon/regulon (Fig. 4).

6. Conclusions and common themes

A theme of this article is the challenging problem of under-

standinghow each step of the RNA processingpathway is coupled

physically to the next step, and whether RNA regulons at each

step are maintained and/or transferred down the pathway. The

traditional assumption of many biologists in the past has been

that transcription is the sole coordinator of cellular events and

that protein production is generally a more passive process. But

while transcription is the initial process that starts gene expres-

sion,accumulating evidencesupportsa viewthat transcriptionand

translation are intertwined and co-dependent [1,6,13,15,100,101] .

Both processes appear to be coordinated independently, but


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Post-transcriptional RNA Regulons Affecting Cell Cycle and Proliferation

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Transcript of Post-transcriptional RNA Regulons Affecting Cell Cycle and Proliferation