from cis-regulatory elements to complex rnps and...

From Cis-Regulatory Elements to ComplexRNPs and Back

Fatima Gebauer1, Thomas Preiss2, and Matthias W. Hentze3

1Gene Regulation Programme, Centre for Genomic Regulation (CRG) and UPF, 08003-Barcelona, Spain2Genome Biology Department, The John Curtin School of Medical Research, The Australian NationalUniversity, Acton (Canberra) ACT 0200, Australia

3European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany

Correspondence: [email protected]; [email protected]; [email protected]

Messenger RNAs (mRNAs), the templates for translation, have evolved to harbor abundantcis-acting sequences that affect their posttranscriptional fates. These elements are frequentlylocated in the untranslated regions and serve as binding sites for trans-acting factors, RNA-binding proteins, and/or small non-coding RNAs. This article provides a systematic synopsisof cis-acting elements, trans-acting factors, and the mechanisms by which they affect trans-lation. It also highlights recent technical advances that have ushered in the era of transcrip-tome-wide studies of the ribonucleoprotein complexes formed by mRNAs and their trans-acting factors.

Translational regulatory mechanisms arebased on two key principles: signal-depen-

dent covalent modifications of general transla-tion (initiation) factors and trans-acting RNA-binding factors (RNA-binding proteins [RBPs]and miRNAs) to alter the translational fateof mRNAs. The first cis-regulatory elements tobe found in eukaryotic mRNAs were the up-stream open reading frames (uORFs) of theyeast GCN4 mRNA (Mueller and Hinnebusch1986) and the iron-responsive elements of mam-malian ferritin mRNAs (Hentze et al. 1987;Leibold and Munro 1987). Coincidentally, theyprovided one example of each: cis-acting ele-ments that function in the context of modifiedinitiation factor activity (GCN4), or that serve asbinding sites for the first translational regulatory

proteins, the iron regulatory proteins (IRPs)(Hinnebusch 2005; Hentze et al. 2010), respec-tively. More than two decades later, translationalcontrol is recognized as a major control pointfor the flux from genetic information to shapingproteomes, and is even reported to be the pre-dominant mechanism for the control of geneexpression (Schwanhausser et al. 2011).

Initially not anticipated, mRNAs now haveto be seen as linear yet structured arrays of nu-merous cis-acting elements, mostly in the 50 and30 untranslated regions (UTRs) but probablyspreading across the whole message. This situa-tion is mirrored with regard to mRNA engage-ment with trans-acting factors, and mRNAsmust therefore be examined as messenger ribo-nucleoprotein particles (mRNPs).

Editors: John W.B. Hershey, Nahum Sonenberg, and Michael B. Mathews

Additional Perspectives on Protein Synthesis and Translational Control available at www.cshperspectives.org

Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a012245

Cite this article as Cold Spring Harb Perspect Biol 2012;4:a012245

1

on September 14, 2020 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from

http://cshperspectives.cshlp.org/

Cis-acting elements come in different flavors(Fig. 1). Hairpin or higher-order (e.g., pseudo-knot) intramolecular mRNA structures caninfluence translation in their own right (i.e.,without binding factors). It has been well docu-mented that they can affect initiation, particu-larly when positioned in the 50 UTR close to thecap structure (Pelletier and Sonenberg 1985;Kozak 1986), and elongation, being involvedin numerous frameshifting events (Namy et al.2004). Internal ribosome entry sequences (IRES)represent another important cis-acting elementthat typically occurs in 50UTRs but has also beenreported to occur within the coding region ofmRNAs (Holcik et al. 2000). In cellular mRNAs,IRES coexist with the 50-cap structure andendow mRNAs with the potential to be trans-lated under conditions in which cap-depen-dent translation is compromised (e.g., differentforms of cell stress, apoptosis). A third catego-ry of common cis-acting elements comprisesuORFs. Theyoccur singularlyor multiply withinthe 50 UTRs of numerous mRNAs and influencethe translation of the downstream major ORF,usually negatively. This effect can be exertedby the element itself, but appears to be usedalso for RBP-mediated translational regulation(see below). A notable exception is the GCN4mRNA in yeast and ATF4 mRNA in mammals,where uORFs serve to promote the translation ofthe downstream major ORF under conditions ofincreased eIF2 phosphorylation (Hinnebusch2005). Binding sites for regulatory RBPs ormiRNAs can be combined on given mRNAs

to yield translational outcomes that integratemultiple signals via the respective trans-actingfactors. In a complementary concept, nearlyall trans-acting factors bind to a multitude ofmRNAs that frequently encode functionallyrelated proteins, subjecting these families ofmRNAs to coordinated, operon-like regulation(Keene 2007). Finally, RNA editing or modifica-tion (e.g., methylation) can provide an addition-al layer of regulatory intervention for cis-act-ing elements (Li et al. 2009; Squires et al. 2012;Zhang et al. 2012).

The arrival of technologies to examinemRNAs and RBPs in a highly parallel, transcrip-tome-wide fashion places translational controlat the heart of modern systems biology. Below,we discuss how system-wide studies and reduc-tionist mechanistic experimentation must con-verge and complement each other for a deeperunderstanding of translational control.

THE mRNP AS A TEMPLATE FORTRANSLATION AND TRANSLATIONALCONTROL

Eukaryotic mRNAs pass through all stages oftheir life cycle from transcription, to processing,transport (including specific subcellular mRNAlocalization), translation, and decay, as assem-blies of dynamic mRNPs rather than as “naked”nucleic acids (Martin and Ephrussi 2009; Mooreand Proudfoot 2009; Sonenberg and Hinne-busch 2009). The implications of this notionare particularly profound for the interaction

Hairpin

5′ (A)n 3′

IRES

uORF ORF

Figure 1. Cis-acting elements that influence mRNA translation. The nearly ubiquitous 50 m7GpppN cap structure(black circle) and 30 poly(A) tail ((A)n) strongly stimulate translation. Secondary structures (e.g., hairpin) andupstream open reading frames (uORFs) in the 50 UTR usually inhibit translation. Internal ribosome entrysequences (IRES) stimulate translation independently of the cap structure. Binding sites for regulatory RNA-binding proteins or microRNAs (ovals) can provide positive or (more frequently) negative regulation. (Adaptedand modified from Gebauer and Hentze 2004.)

F. Gebauer et al.

2 Cite this article as Cold Spring Harb Perspect Biol 2012;4:a012245



of the mRNA with the translation apparatus(translation factors and ribosome) as a templatefor protein synthesis. Parallels have previouslybeen drawn between the processes of eukary-otic transcription and translation, likening thenear-ubiquitous mRNA 50-cap structure and 30-poly(A) tail to constitutive promoter elements,whereas cis-regulatory motifs in the UTRs wereviewed as mRNA-specific elements that act tocontrol translation in a combinatorial and con-text-specific manner akin to transcriptional reg-ulatory elements such as silencers and enhanc-ers (Sachs and Buratowski 1997; Gebauer andHentze 2004). This useful concept may be ex-tended by an analogy between DNA in chroma-tin as the “real” template for transcription andthe mRNA interacting with the translation ma-chinery packaged into complex mRNP parti-cles, which may even adopt additional states of“compaction,” for instance, in the form of re-pressive cytoplasmic foci such as stress granulesand processing bodies (Anderson and Kedersha2009; Balagopal and Parker 2009). Adoptionof such an mRNP-centric view could providesimilar conceptual breakthroughs for transla-tion research as the chromatin model did forthe transcription field.

At present, we are only scratching the surfacein terms of understanding the composition ofmRNPs, and the analogy with transcription maybreak down at the point of histones and nu-cleosomes. Even the most common RBPs (e.g.,the hnRNPs) do not appear to organize mRNPsin a regular fashion as histones do with thechromatin template. There is a pressing needfor identifying all cellular proteins interactingwith mRNA within native mRNPs (“mRNA in-teractomes”), and then to study their organiza-tion and dynamic interplay. At least the deter-mination of the first mRNA interactomes hasnow become feasible (see below).

NEW TECHNOLOGIES TO STUDYCIS-ACTING ELEMENTS ANDTRANS-ACTING FACTORS

The complete description of an mRNA inter-actome in a given cellular condition requirescataloging the proteins that bind to each ex-

pressed mRNA and a high-resolution mappingof all respective binding site(s) on the mRNA.Conventional affinity copurification and low-throughput identification of binding partnershave long been useful tools for characterizingindividual examples and have started the jour-ney along this road. However, such small-scalemethods cannot cope with the magnitude of thenew tasks at hand. Valiant attempts have beenmade to draw the power of bioinformatics intothe challenge and to predict the occurrence ofknown cis-acting motifs across larger spectra ofmRNAs. However, these approaches are limitedby the degeneracy of the primary RNA sequencemotifs involved (e.g., miRNA target sites) (Saitoand Saetrom 2010) and/or by the importantrole that RNA secondary and tertiary structureplays in defining functional regulatory motifs(Parker et al. 2011; Zhao et al. 2011). The com-putational prediction of RBPs has helped sig-nificantly in the identification of new candi-dates, but it is by definition limited to proteinsbearing known RNA-binding domains.

Identification of Cis-Acting Elementsby Cross-Linking

A long existing but recently refined and popu-larized approach to study RBPs as well as themRNAs and sites they bind to is to covalentlycross-link the two to each other in vivo. Cova-lent cross-links can be induced chemically (Va-lasek et al. 2007) or by ultraviolet (UV) light.UV light of 254 nm cross-links the naturallyphotoreactive nucleotide bases, especially py-rimidines, and specific amino acids (Phe, Trp,Tyr, Cys, and Lys) (Hockensmith et al. 1986;Brimacombe et al. 1988). UV-cross-linking re-quires direct contact (zero distance) betweenprotein and RNA and does not promote pro-tein–protein cross-linking (Greenberg 1979).A recent version of in vivo UV cross-linkingis called PAR-CLIP (photoactivatable-ribonu-cleoside-enhanced cross-linking and immuno-precipitation) (Fig. 2A) (Hafner et al. 2010).The photoactivatable nucleotide 4-thiouridine(4SU) is taken up by cultured cells and in-corporated into nascent RNAs, and efficientcross-linking is induced by 365-nm UV light

Cis-Regulatory Elements

Cite this article as Cold Spring Harb Perspect Biol 2012;4:a012245 3



irradiation, followed by affinity capture of theRBP under study, using specific antibodiesagainst the native protein itself or a tagged ver-sion of the RBP expressed in the cells. The iso-lated complex is then subjected to limited RN-ase digestion, radioactive end labeling of the

bound RNAs, and size selection by denaturinggel electrophoresis. Finally, RNA recovered fromthe complexes is used for reverse transcriptionand next-generation sequencing. The RNasetrimming step ensures that transcript regionsbound by the bait protein will be preferentially

X X X

X X

X X

XX

Denaturing lysis

Oligo(dT) capture,stringent washes

Release by RNAse

High-throughput sequencing

A

UV-cross-linking

PAR-CLIP

(Immuno)affinity-purification,RNase trimming

U32P OH

SDS-PAGE,autoradiography,band excision

X-linked RBP

Electroelution,proteinase digestion

U32P OH

RT-PCR-basedlibrary preparation

CG

B CGRNA chromatography Interactome capture

Mass spectrometry

UV-cross-linking

AAAAA

AAAAA

AAAAA

AAAAA

X

UV

Mass spectrometry

AAAAA

AAAAA

AAAAA

AAAAA

TTTTT

TTTTT

TTTTT

TTTTT

RNA bait boxB

λ-GST

Glutathionebeads

Cellular extract

+Bind

Wash

Elute

AAAAA

AAAAA

AAAAA

AAAAA

UU

U U AU

U

U

U AU

U U

U U

UV 365 nm

+4SU

X AX

X AX

X X

XX

X X AX

X AAX

X X

XX

AAAAA

AAAAA

AAAAA

AAAAA

X X AX

X AAX

X X

XX

Figure 2. Methods to study mRNP composition. (A) Photoactivatable-ribonucleoside-enhanced cross-linkingand immunoprecipitation (PAR-CLIP). Cells are cultured in media containing 4-thiouridine (4SU), leading toincorporation of the photoactivatable nucleoside into cellular RNA. Cross-linking with UV light of 365 nmleads to covalent attachment of RBPs to RNA targets that withstands partial RNAse digestion, immunoprecip-itation, and purification by denaturing gel electrophoresis. Isolated RNA fragments are identified by next-generation sequencing, aided by a tendency of the cross-linked site to show thymidine (T) to cytidine (C)transitions. (B) GRNA chromatography using specific interaction between a 21-amino-acid peptide from the lphage N anti-terminator protein and the boxB hairpin. A fusion of lN peptide with glutathione S-transferase(GST), and incorporation of the boxB hairpin into bait RNA converts glutathione Sepharose into an RNAaffinity matrix (GRNA resin), which is incubated with cellular extracts. Proteins specifically bound to the matrixare eluted and identified by mass spectrometry. (C) Interactome capture. The procedure begins with RNP cross-linking in living cells by conventional UV 254-nm cross-linking or as in the PAR-CLIP approach. Following lysis,the complete cellular complement of (m)RNPs is purified by binding to an oligo(dT) resin and stringentwashing under conditions that dissociate noncovalent RNA–protein interactions. Specifically bound proteinsare released by RNase digestion and identified by mass spectrometry. (Diagrams are based on data fromCzaplinski et al. 2005, Hafner et al. 2010, and Castello et al. 2012, respectively.)

F. Gebauer et al.




sequenced, whereas the propensity of the PAR-cross-linking chemistry to induce thymidine(T) to cytidine (C) transitions during reversetranscription helps with precise binding siteidentification. Even if PAR-CLIP is currentlyvery popular, combinations of UV-cross-linking,RNP immunoprecipitation, isolation of cross-linked RNA segments, and cDNA sequencinghave been developed before (e.g., CLIP) (Ule2003), and related methods using microarraysor high-throughput sequencing exist to profileRNAs associated with immunopurified RNA-binding proteins (RIP-Chip, RIP-Seq) (Tenen-baum et al. 2000).

Identification of Trans-Acting Factors by RNAAffinity Chromatography

The converse approach to CLIP experiments,using a given RNA under study as bait to purifyand identify interacting proteins, is also com-monly used. Here the RNA is typically engi-neered to contain a small sequence or structuraltag that will facilitate specific capture of themRNP complex. GRNA affinity chromatogra-phy is an example of such an approach (Fig. 2B)(Czaplinski et al. 2005; Duncan 2006). It usesthe specific binding of an RNA hairpin to ashort peptide from the N anti-terminator pro-tein of the l phage (termed BoxB hairpin andlN peptide, respectively, originally developed asa tool for tethered function assays) (De Grego-rio et al. 1999). For GRNA chromatography, na-tive mRNP complexes are assembled on boxB-tagged RNAs in vitro, which are then purifiedwith the help of l-GST fusion proteins boundto a solid support. Proteins that copurify withthe tagged RNA are then identified by massspectrometry. Besides the boxB/l tether, severalother combinations of RNA aptamers (e.g., thestreptomycin or tobramycin tags) or tetheringproteins (e.g., MS2 coat protein) have been usedsuccessfully (Beach and Keene 2008).

Although these approaches have been quitesuccessful in vitro, only a few success storieshave been reported from in vivo settings (Hoggand Collins 2007). Recently, the MS2 coat pro-tein fused to a tag consisting of two His6 clustersseparated by a cleavage site for the TEV protease

and an in vivo biotinylation site has been used tocapture IRES-binding proteins from 293 cells(Tsai et al. 2011). Furthermore, the developmentof “designer RNA-binding domains” that canbe engineered to bind any desired target RNAsequence (Filipovska et al. 2011; Mackay et al.2011) holds further promise for the purificationof specific native mRNPs from living cells.

Discovery of RBPs by Interactome Capture

Given the high sensitivity of contemporary massspectrometric approaches to determine thou-sands of proteins in complex mixtures, twogroups recentlyachieved the first step in the questfor complete mRNA interactomes (see above),the identification of “all” mRNA-binding pro-teins of a mammalian cell (Castello et al. 2012).This work first used efficient in vivo cross-link-ing (conventional UV254 cross-linking, or PAR-CL) to preserve physiologically relevant RNA–protein interactions, followed by capture of thepolyadenylated (m)RNAs with theircross-linkedRBPs on an oligo(dT) matrix, stringent washing,subsequent release of bound proteins by RNasedigestion, and finally identification by massspectrometry (Fig. 2C). This approach extendsvery early work on hnRNP proteins in vivo(Dreyfuss et al. 1984) and recent studies thatused hybridization of labeled mRNA prepara-tions to protein arrays to identify cellular RNA-binding proteins in vitro (Scherrer et al. 2010;Tsvetanova et al. 2010). It also has the advantageof sensitively and selectively detecting the near-complete array of native protein–mRNA inter-actions as they occur in living cells. This interac-tome capture approach can now be used oradapted to study the mRNA interactomes of oth-er cells and to investigate changes in interactomecomposition as a function of biological condi-tions, such as differences in cell growth or cellcycle phase, or forms of stress (hypoxia, oxidativestress, nutrient deprivation, etc.).

FROM COMPLEX RNPs TO DEFINEDCIS/TRANS INTERACTIONS

Proteins rarely act alone to regulate transla-tion. Rather, multi-subunit complexes assembleon (the UTRs of ) the transcript, directed by





interactions between the RBP components ofthe complex and regulatory cis-acting sequencesor structures of the mRNA. These complexesmay also contain regulatory RNAs (e.g., mi-RISC), using nucleic acid hybridization as aprinciple of site-specific binding. It has nowbecome clear that components of regulatorycomplexes and regulatory factors (or the transla-tional machinery) need to interact dynamicallyto achieve accurate translational control. In thefollowing, we focus on three illustrative examplesof coordinated translational regulation. Two ofthem involve the cooperation between multiplecis-elements and trans-acting factors to ensuretight and timely control of translation. In thethird example, temporal control of translationis achieved by the stepwise assembly and activa-tion of a regulatory complex that coordinates theexpression of a posttranscriptional operon.

TIGHT TRANSLATIONAL REPRESSION,A MULTIFUNCTIONAL RBP ANDCOMBINATORIAL REGULATION:Msl2 mRNA

MSL2 is the limiting component of the Droso-phila dosage compensation complex, a chroma-tin assembly that equalizes the expression of X-linked genes between males (XY) and females(XX) by promoting hypertranscription of thesingle male X chromosome (for review, see Gel-bart and Kuroda 2009). Dosage compensation

must be repressed in females for viability, andthis is primarily achieved by preventing MSL2expression. Two posttranscriptional controlmechanisms cooperate to inhibit msl2, andboth are exerted by the female-specific RBPSex-lethal (SXL). In the nucleus, SXL binds tooligo-uridine stretches adjacent to the splicesites of a small facultative intron in the msl2 50

UTR to inhibit its splicing; this splicing inhibi-tion retains the SXL-binding sites in the maturemRNA. In the cytoplasm, SXL inhibits msl2translation by binding to specific sites in boththe retained intron and the 30 UTR (for review,see Graindorge et al. 2011). Binding of SXLto both UTRs is necessary for tight transla-tional repression, but partial inhibition can beachieved by each UTR alone, allowing the step-wise dissection of the mechanism (Bashaw andBaker 1997; Kelley et al. 1997; Gebauer et al.1999). Extensive mutational and functional an-alyses have revealed that SXL regulates transla-tion by a dual mechanism: SXL bound to the 30

UTR inhibits the recruitment of the 43S ribo-somal complex to the mRNA, whereas SXLbound to the 50 UTR blocks the scanning ofthose complexes that have presumably escapedthe 30-UTR-mediated control (Fig. 3) (Gebaueret al. 2003; Beckmann et al. 2005). A uORFlocated just upstream of the SXL-binding sitehas been shown recently to be important for50-UTR-mediated repression (Medenbach et al.2011); SXL promotes the recognition of the

5′

3′

SXLmsl2 ORF

UNR SXL

2

1

AAAAAAAAA

43S

Y4E

43SuAUG

4G

PABPX

Figure 3. Mechanism of translational repression of msl2 mRNA. SXL binds to both the 50 and 30 UTRs of msl2 toachieve strong repression. SXL bound to the 30 UTR recruits UNR to bind to the RNA in close proximity. In turn,UNR interacts with poly(A) tail-bound PABP to inhibit 43S ribosomal complex recruitment at a step downstreamfrom closed-loop formation (1). SXL bound to the 50 UTR inhibits ribosomal scanning by promoting recognitionof an upstream AUG (uAUG), thus preventing 43S complexes from reaching the main msl2 ORF (2). Additionalunidentified factors (X, Y) are likely involved. (Adapted in modified form from Graindorge et al. 2011.)

F. Gebauer et al.




upstream initiator AUG by scanning 43S com-plexes, thus preventing them from reaching themain ORF. SXL does not simply act by sterichindrance, because PTB bound in the sameposition does not promote uAUG recognition.These data suggest that SXL establishes spe-cific contacts with additional factors and/orthe translational machinery for repression viathe 50 UTR; they also provide a first exampleof RBP regulation of a uORF.

Translational repression via the 30 UTR cer-tainly requires additional factors. Binding ofSXL alone is insufficient to repress the 43S re-cruitment step, and the highly conserved SXLhomolog from Musca domestica cannot inhib-it translation despite binding to the same siteswith similar apparent affinities, suggesting thatspecific contacts are made between SXL, themsl2 30 UTR, and other factors necessary forrepression (Gebauer et al. 2003; Grskovic et al.2003). One of the critical factors was identifiedas the protein Upstream of N-ras (UNR), a con-served regulator also known for its role in IRES-mediated translation and mRNA stability con-trol in mammals (Fig. 3) (for review, see Mihai-lovich et al. 2010). UNR is required for transla-tional repression of msl2 reporters in vitro, forrepression of endogenous msl2 in cell culture,and for inhibition of dosage compensation infemale flies (Abaza et al. 2006; Duncan 2006;Patalano et al. 2009). Binding of UNR to the30 UTR of msl2 depends on SXL, and therefore,even though UNR is present in males, it doesnot bind to msl2 and repress its translation be-cause of the absence of SXL. Thus, SXL confers asex-specific function to UNR.

How does the SXL–UNR complex functionto repress translation? Although a poly(A) tail isnot strictly required for regulation, translationalrepression via the 30 UTR is stimulated by thepoly(A) tail (Duncan et al. 2009). Interactionsbetween UNR and poly(A) tail-bound PABP arethought to underlie this stimulation by mecha-nisms that are yet unknown. PABP binds to thepoly(A) tail and contacts the cap-binding com-plex, yielding a closed-loop conformation of themRNA that is thought to be optimal for efficientribosome recruitment (Tarun and Sachs 1996;Wells et al. 1998). Intriguingly, closed-loop for-

mation of the msl2 mRNA is not affected byUNR, indicating that the SXL–UNR complexinhibits ribosome recruitment by targeting astep in translation initiation that is downstreamfrom eIF4F binding (Fig. 3) (Duncan et al.2009). Close examination of the msl2 30 UTRindicates the presence of sequences requiredfor translational repression but dispensable forSXL–UNR binding, suggesting that the full30-UTR repressor complex contains additionalfactors (C Militti, E Szostak, and F Gebauer,unpubl.). Understanding the composition ofthis complex and the interactions of its com-ponents with the translational machinery mayyield novel clues as to how the SXL/UNR-orga-nized complex on the 30 UTR of the msl2 mRNAcontrols the recruitment of ribosomes.

In summary, tight translational repressionof msl2 mRNA is achieved through an elaboratecombinatorial mechanism that involves target-ing different steps of translation initiation fromboth ends of the mRNA, involving multipleRBPs and a uORF as an additional cis-regulato-ry element. How all of these elements act togeth-er to ensure efficient and coordinated repres-sion warrants further investigation.

MORE COMPLEXITY FOR TEMPORALAND SPATIAL CONTROL OFTRANSLATION: nanos mRNA

Nanos (Nos) is a posterior determinant re-quired for the formation of abdominal seg-ments during early Drosophila development.Synthesis of Nos exclusively at the posteriorof the embryo is achieved by localization andtranslational activation of the nos transcriptat this region, combined with translational re-pression elsewhere (Gavis and Lehmann 1994).nos mRNA is transcribed and actively translatedin nurse cells, and subsequently transferred tothe adjacent growing oocyte through ring ca-nals. In the bulk cytoplasm of late oocytes andearly embryos, nos mRNA is translationally re-pressed via sequences contained in a discreteregion of the 30 UTR proximal to the stop co-don, the translational control element (TCE)(Dahanukar and Wharton 1996; Gavis et al.1996; Smibert et al. 1996). The TCE consists





of three stem–loops that are necessary for re-pression (Fig. 4). The AU-rich stem of one ofthese structures (stem IIIA, following the no-menclature of Crucs et al. 2000) is necessaryfor translational repression in oocytes and isthought to bind the hnRNP F/H protein Glor-und (Crucs et al. 2000; Forrest et al. 2004; Kalifaet al. 2006). The other two stems carry loopsconsisting of CUGGC, which are recognizedby the repressor Smaug and are therefore re-ferred to as Smaug recognition elements(SREs) (Smibert et al. 1996, 1999; Dahanukaret al. 1999). Smaug is expressed exclusively inthe early embryo and acts as a major regulator ofmaternal mRNA destabilization upon egg acti-vation (Tadros et al. 2007). Point mutations inthe SREs affect Smaug binding and lead to nosderepression in the embryo without effects onlocalization of the mRNA (Smibert et al. 1996).Smaug binds to the SRE via its SAM (sterile a

motif ) domain (Aviv et al. 2003; Green et al.2003). A central guanine in the SRE appropri-ately oriented by the stem–loop structure iscritical for SAM domain recognition (Johnsonand Donaldson 2006; Oberstrass et al. 2006).

Smaug promotes deadenylation of nosmRNA by recruitment of the CAF–CCR4–

NOT complex (Jeske et al. 2006; Zaessingeret al. 2006). Deadenylation is necessary for nosrepression, but deadenylated nos reporters canstill be strongly repressed in vitro in a mannerthat partially depends on the SREs (Jeske et al.2006). These data suggest that Smaug-mediatedrepression has two components: one that isindependent of the poly(A) tail, and one thatdepends on deadenylation. Indeed, translation-al repression is often coupled to deadenylation,which contributes to maintain the repressedstate (for review, see Miller and Olivas 2011).Smaug interacts with Cup, a protein that bindseIF4E and blocks the recruitment of eIF4G tothe cap complex (Fig. 4) (Nelson et al. 2004; forreview, see Richter and Sonenberg 2005). Mu-tation of the eIF4E-binding motifs of Cup par-tially relieves repression of nos transgenes, andthe binding of Cup and eIF4G to nos mRNPs ismutually exclusive, implying a function of Cupin mediating a translation initiation block bySmaug (Nelson et al. 2004; Jeske et al. 2011).Contradictory results have been reported con-cerning the requirement of the cap structure forSmaug-mediated repression, however (Andrewset al. 2011; Jeske et al. 2011). Furthermore, asignificant fraction of nos mRNA was found in

??

4G4G

4E4E

Cup

SmgCAFCCR4NOT

IIIA

TCE

Late oocyte Embryo

SRE SRE

(A)n(A)n

uc cg

Gloggccu g uu

c cc

c ggg

g

nosnos

Smg

Aub piRNAs

Figure 4. Mechanism of translational repression of nanos mRNA. nanos mRNA switches from a translationallyactive state in nurse cells to a silenced state in late oocytes and early embryos. Repression in late oocytes is drivenby Glorund (Glo) binding to stem IIIA of the TCE and seems to be effected primarily at the elongation step. Inembryos, Smaug (Smg) is synthesized and takes over repression by binding to the SREs within the TCE; therelative contribution of each SRE is uncertain, although SRE1 seems to contribute more to Smaug binding thanSRE2. Smg recruits the eIF4E-binding protein Cup and the CAF–CCR4–NOT complex to block translationinitiation and promote deadenylation and degradation of nos mRNA. The piwi pathway has been recentlyreported to participate in deadenylation. Additional steps of translation could be affected both in late oocytesand early embryos (see text for details).

F. Gebauer et al.




association with polysomes in early embryoseven under conditions in which the mRNAis completely unlocalized and Nos protein isundetectable, suggesting that a postinitiationstep is affected (Clark et al. 2000). Consistently,translation mediated by the Cricket paralysisvirus (CrPV) IRES, which mediates translationinitiation without requirement for any of thecellular translation initiation factors, can be in-hibited by the SREs (Jeske et al. 2011). Therefore,repression by Smaug might involve initiationand postinitiation events. Interestingly, recentdata suggest that Cup can elicit translational re-pression independent of its eIF4E-binding mo-tifs, and that Cup also promotes deadenylationdirectly via association with the CAF–CCR4–NOT complex (Igreja and Izaurralde 2011). Thisraises the possibility that Cup mediates repressorfunctions of Smaug beyond translation initia-tion. It will be interesting to analyze to what ex-tent Smaug function is preserved in Cup-deplet-ed extracts.

Recently, late ovary extracts that recapitulaterepression mediated by the IIIA stem (the Glor-und-binding site) have been developed (An-drews et al. 2011). Repression seems to be capindependent in these extracts. In addition, nosmRNA is found associated with polysomes, andGlorund is present in polysomes in associationwith the repressed mRNA, suggesting that Glor-und inhibits translation at a postinitiation step.On the other hand, repression in late oocytes ispoly(A) dependent, which may reflect an effectof Glorund on initiation as well. A comparisonof the polysomal association of nos mRNA intotal ovary extracts, which are enriched for ear-ly-stage oocytes, with that in late ovary and em-bryo extracts indicates a gradual shift to lighterfractions, consistent with the temporal acquisi-tion of distinct mechanisms of translational re-pression (Andrews et al. 2011).

Altogether, currently available data can beintegrated into the following model explainingthe temporal switch in nos mRNA expression,from activation in nurse cells to repression laterin development. In late oocytes, Glorund im-poses a block on elongation/termination thatresults in quick repression; in early embryos,Smaug consolidates nos inhibition at the initi-

ation and possibly postinitiation steps and pro-motes deadenylation and degradation of thetranscript by recruiting the CAF–CCR4–NOTcomplex (Fig. 4). Intriguingly, the piRNA path-way has recently been implicated in this mech-anism. Rouget et al. (2010) found that CCR4-mediated deadenylation of nos depends onpiRNAs complementary to a distal region ofnos 30 UTR and that Aubergine, an Argonauteprotein, interacts with Smaug and CCR4. Fur-ther experiments are required to decipher howthe piRNA pathwaycooperates with the Smaug–CCR4 complex.

SIGNAL-DEPENDENT TEMPORALCONTROL OF AN (ANTI-)INFLAMMATORYRNA OPERON: THE GAIT COMPLEX

During inflammation, the synthesis of cerulo-plasmin (Cp) is transiently induced by interfer-on (IFN)-g in myeloid cells and ceases at �24 hof IFN-g treatment by the action of a translationrepressor complex termed GAIT (IFN-g-acti-vated inhibitor of translation) (for review, seeMukhopadhyay et al. 2009). The GAIT complexrecognizes a bipartite stem–loop structure inthe 30 UTR of Cp mRNA, the GAIT element(Fig. 5) (Sampath et al. 2003). The GAIT com-plex is composed of four proteins: ribosomalprotein L13a, glutamyl-prolyl tRNA synthetase(EPRS), NS1-associated protein 1 (NSAP1),and glyceraldehyde 3-phosphate dehydrogenase(GAPDH) (Mazumder et al. 2003; Sampathet al. 2004). EPRS is the subunit that binds di-rectly to the RNA, whereas phosphorylated L13ais responsible for interactions with the transla-tional machinery (Sampath et al. 2004; Kapasiet al. 2007; Arif et al. 2009). Similar to 30-UTR-bound SXL, the GAIT complex inhibits 43S re-cruitment without affecting closed-loop forma-tion (Mazumder et al. 2001; Kapasi et al. 2007).Furthermore, because repression requires PABPand the poly(A) tail, it was proposed that mRNAcircularization actually favors the correct posi-tioning of GAIT close to the 50 UTR (Mazumderet al. 2001). L13a binds eIF4G at its eIF3-bindingsite and blocks the eIF4G–eIF3 interaction, astep required for 43S complex recruitment (Ka-pasi et al. 2007; Arif et al. 2009).





Several lessons can be learned from this in-structive example of translational control. First,temporal control of translation is achieved bythe regulated, ordered assembly of the GAITcomplex. Under control conditions, EPRS re-sides in the cytosolic tRNA multisynthetasecomplex (MSC) and acts as an enzyme catalyz-ing the addition of amino acids to tRNA. EPRScontains two catalytic domains (ERS and PRS)linked by a region that is not required for itsenzymatic activity. This region contains threehelix–turn–helix structures termed WHEP do-mains (named after the three tRNA synthetases

that contain them) that serve RNA binding (Jiaet al. 2008). Phosphorylation of EPRS by Cdk5during the early phase of the IFN-g responseinduces its release from the MSC (Sampathet al. 2004; Arif et al. 2009, 2011). Free, phos-phorylated EPRS interacts with NSAP1 throughone of the WHEP domains, resulting in a com-plex with no RNA-binding activity (Fig. 5).Later on, L13a—which resides in the large ribo-somal subunit—is phosphorylated and releasedfrom the ribosome (Mazumder et al. 2003).Phosphorylated L13a and GAPDH then interactwith the phosphorylated EPRS–NSAP1 dimer,

4E

GAIT

GAIT complex

Gapdh

60S

Nsap1

Cdk5ZIPK

DAPK

L13a

MSC

EPRS

IFN-γ

(A)n

4G43S

Cp

elF3

Figure 5. Translational repression by the GAIT complex. During inflammation, EPRS and L13a are phosphor-ylated and released from the multisynthetase complex (MSC) and the 60S ribosomal subunit, respectively. Theseproteins bind to NSAP1 and GAPDH to form the heterotetrameric GAIT complex, which binds to a split stemstructure present in the 30 UTR of target mRNAs. L13a then inhibits the recruitment of the 43S ribosomalcomplex by blocking the interaction between 43S-associated eIF3 and eIF4G.

F. Gebauer et al.




resulting in a heterotetrameric complex that ex-poses the WHEP domains for interactions withthe GAITelement (Jia et al. 2008).

The second lesson concerns the functionalversatility of RBPs. As mentioned above, EPRSand GAPDH are polypeptides that can func-tion as enzymes or contribute to translationalcontrol depending on cellular conditions. Thefounding example of an enzyme that also func-tions as an RBP is IRP1. In iron-replete cells,IRP1 bears an iron–sulfur cluster necessary forcytosolic aconitase activity. In iron-starved cells,the iron–sulfur cluster is lost, and a pocket isuncovered that binds to mRNAs encoding fac-tors involved in iron metabolism, resultingin stabilization or translational repression ofits target mRNAs (for review, see Pantopoulos2004). Interconnectivity between cellular me-tabolism and RNA regulation might be anemerging theme in gene expression (Hentzeand Preiss 2010).

The third lesson pertains to the observationthat L13a phosphorylation leads to L13a-de-pleted ribosomes that are functionally normal.Practically the entire cellular complement ofL13a is released upon phosphorylation, yieldingno defects in general translation (Mazumderet al. 2003). This result suggests that the ribo-some also serves as a repository of regulatorymolecules for release from its surface when theirfunctions are required. The concept of largemacromolecular complexes as depots for regu-latory proteins has been discussed elsewhere(Ray et al. 2007).

Finally, the fourth lesson relates to the exis-tence of an RNA regulon for GAIT-mediatedtranslational control during the inflammatoryresponse. L13a phosphorylation is the culminat-ing event of a kinase cascade in which IFN-gactivates DAPK, which, in turn, activates ZIPK(Fig. 5). Both the DAPK and ZIPK mRNAscontain GAITelements in their 30 UTRs and arerepressed by the GAIT complex, establishing anegative-feedback loop that contributes to thetemporal limitation of the inflammatory re-sponse (Mukhopadhyay et al. 2008). Further-more, in silico searches and genome-wide poly-some profiling have provided a list of candidatetargets containing GAITelements, manyof which

encode proteins involved in inflammation (Rayand Fox 2007; Vyas et al. 2009). These resultsimply that the GAIT complex appears to coordi-nate a posttranscriptional operon involved in theresolution of the inflammatory response.

CONCLUSIONS AND PERSPECTIVES

Starting with the mapping of cis-acting ele-ments and the identification of trans-actingfactors mostly by biochemical and genetic ap-proaches during the past decades, the investiga-tion of mRNA translation has now entered intoa phase of transcriptome-wide, highly parallelanalyses. These approaches have begun to helpdefine RBP-binding sites across the transcrip-tome and paint a picture of dense mRNP as-semblies involving a multitude of trans-actingfactors. Several concepts have emerged along theway: (1) The binding sites of RBPs, and RBPsthemselves, influence each other on the mRNAs,giving rise to combinatorial outcomes. (2) Es-sentially all RBPs have numerous target mRNAs,further driving combinatorial modes of trans-lational control. (3) The resulting mRNPs arehighly dynamic structures that undergo rear-rangements in response to biological signaling.(4) DExH/D RNA helicases not only remodelRNA structure by unwinding RNA–RNA du-plex structures, but can directly remodel RNPsin different biological settings (Jankowsky andBowers 2006). Reductionist biochemical workon model systems revealed that the same RBPscan influence translation by more than onemechanism, often influenced by other RBPs ina combinatorial way. We expect that reduction-ist mechanistic investigations by biochemicalapproaches and transcriptome-wide, time-re-solved in vivo analyses including ribosome pro-filing (Ingolia et al. 2011) will converge to yieldunprecedented insights into translation andtranslational control, both at the level of indi-vidual mRNAs and whole transcriptomes.

ACKNOWLEDGMENTS

Work in F.G.’s laboratory is supported by grantsBFU2009-08243 and Consolider CSD2009-00080 from MICINN; T.P. is supported by





grants from the National Health and MedicalResearch Council of Australia and the Austra-lian Research Council; M.W.H. acknowledgescontinuous support from the Deutsche For-schungsgemeinschaft. We are grateful to lab-oratory members and collaborators for theirmany critical contributions to cited work fromour laboratories, and apologize to those col-leagues whose work we could not cite for reasonsof focus.

REFERENCES

Abaza I, Coll O, Patalano S, Gebauer F. 2006. DrosophilaUNR is required for translational repression of male-spe-cific lethal 2 mRNA during regulation of X-chromosomedosage compensation. Genes Dev 20: 380–389.

Anderson P, Kedersha N. 2009. RNA granules: Post-tran-scriptional and epigenetic modulators of gene expres-sion. Nat Rev Mol Cell Biol 10: 430–436.

Andrews S, Snowflack DR, Clark IE, Gavis ER. 2011. Mul-tiple mechanisms collaborate to repress nanos translationin the Drosophila ovary and embryo. RNA 17: 967–977.

Arif A, Jia J, Mukhopadhyay R, Willard B, Kinter M, Fox PL.2009. Two-site phosphorylation of EPRS coordinatesmultimodal regulation of noncanonical translationalcontrol activity. Mol Cell 35: 164–180.

Arif A, Jia J, Moodt RA, DiCorleto PE, Fox PL. 2011. Phos-phorylation of glutamyl-prolyl tRNA synthetase bycyclin-dependent kinase 5 dictates transcript-selectivetranslational control. Proc Natl Acad Sci 108: 1415–1420.

Aviv T, Lin Z, Lau S, Rendl LM, Sicheri F, Smibert CA. 2003.The RNA-binding SAM domain of Smaug defines a newfamily of post-transcriptional regulators. Nat Struct Biol10: 614–621.

Balagopal V, Parker R. 2009. Polysomes, P bodies and stressgranules: States and fates of eukaryotic mRNAs. CurrOpin Cell Biol 21: 403–408.

Bashaw GJ, Baker BS. 1997. The regulation of the Drosophilamsl-2 gene reveals a function for Sex-lethal in translation-al control. Cell 89: 789–798.

Beach DL, Keene JD. 2008. Ribotrap: Targeted purificationof RNA-specific RNPs from cell lysates through immu-noaffinity precipitation to identify regulatory proteinsand RNAs. Methods Mol Biol 419: 69–91.

Beckmann K, Grskovic M, Gebauer F, Hentze MW. 2005. Adual inhibitory mechanism restricts msl-2 mRNA trans-lation for dosage compensation in Drosophila. Cell 122:529–540.

Brimacombe R, Stiege W, Kyriatsoulis A, Maly P. 1988. In-tra-RNA and RNA–protein cross-linking techniques inEscherichia coli ribosomes. Methods Enzymol 164: 287–309.

Castello A, Fischer B, Schuschke K, Horos R, BeckmannBM, Strein C, Davey NE, Humphreys DT, Preiss T, Stein-metz LM, et al. 2012. Insights into RNA biology froman atlas of mammalian mRNA-binding proteins. Cell (inpress).

Clark IE, Wyckoff D, Gavis ER. 2000. Synthesis of the pos-terior determinant Nanos is spatially restricted by a novelcotranslational regulatory mechanism. Curr Biol 10:1311–1314.

Crucs S, Chatterjee S, Gavis ER. 2000. Overlapping but dis-tinct RNA elements control repression and activation ofnanos translation. Mol Cell 5: 457–467.

Czaplinski K, Kocher T, Schelder M, Segref A, Wilm M,Mattaj IW. 2005. Identification of 40LoVe, a XenopushnRNP D family protein involved in localizing a TGF-b-related mRNA during oogenesis. Dev Cell 8: 505–515.

Dahanukar A, Wharton RP. 1996. The Nanos gradient inDrosophila embryos is generated by translational regula-tion. Genes Dev 10: 2610–2620.

Dahanukar A, Walker JA, Wharton RP. 1999. Smaug, a novelRNA-binding protein that operates a translational switchin Drosophila. Mol Cell 4: 209–218.

De Gregorio E, Preiss T, Hentze MW. 1999. Translation driv-en by an eIF4G core domain in vivo. EMBO J 18: 4865–4874.

Dreyfuss G, Choi YD, Adam SA. 1984. Characterization ofheterogeneous nuclear RNA–protein complexes in vivowith monoclonal antibodies. Mol Cell Biol 4: 1104–1114.

Duncan K. 2006. Sex-lethal imparts a sex-specific functionto UNR by recruiting it to the msl-2 mRNA 30 UTR:Translational repression for dosage compensation. GenesDev 20: 368–379.

Duncan KE, Strein C, Hentze MW. 2009. The SXL–UNRcorepressor complex uses a PABP-mediated mechanismto inhibit ribosome recruitment to msl-2 mRNA. MolCell 36: 571–582.

Filipovska A, Razif MFM, Nygard KKA, Rackham O. 2011.A universal code for RNA recognition by PUF proteins.Nat Chem Biol 7: 425–427.

Forrest KM, Clark IE, Jain RA, Gavis ER. 2004. Temporalcomplexity within a translational control element in thenanos mRNA. Development 131: 5849–5857.

Gavis ER, Lehmann R. 1994. Translational regulation ofnanos by RNA localization. Nature 369: 315–318.

Gavis ER, Lunsford L, Bergsten SE, Lehmann R. 1996. Aconserved 90 nucleotide element mediates translationalrepression of nanos RNA. Development 122: 2791–2800.

Gebauer F, Hentze M. 2004. Molecular mechanisms oftranslational control. Nat Rev Mol Cell Biol 5: 827–835.

Gebauer F, Corona DF, Preiss T, Becker PB, Hentze MW.1999. Translational control of dosage compensation inDrosophila by Sex-lethal: Cooperative silencing via the50 and 30 UTRs of msl-2 mRNA is independent of thepoly(A) tail. EMBO J 18: 6146–6154.

Gebauer F, Grskovic M, Hentze MW. 2003. Drosophila Sex-lethal inhibits the stable association of the 40S ribosomalsubunit with msl-2 mRNA. Mol Cell 11: 1397–1404.

Gelbart ME, Kuroda MI. 2009. Drosophila dosage compen-sation: A complex voyage to the X chromosome. Devel-opment 136: 1399–1410.

Graindorge A, Militti C, Gebauer F. 2011. Posttranscription-al control of X-chromosome dosage compensation. WileyInterdiscip Rev RNA 2: 534–545.

Green JB, Gardner CD, Wharton RP, Aggarwal AK. 2003.RNA recognition via the SAM domain of Smaug. Mol Cell11: 1537–1548.

F. Gebauer et al.




Greenberg JR. 1979. Ultraviolet light-induced crosslinkingof mRNA to proteins. Nucleic Acids Res 6: 715–732.

Grskovic M, Hentze MW, Gebauer F. 2003. A co-repressorassembly nucleated by Sex-lethal in the 30UTR mediatestranslational control of Drosophila msl-2 mRNA. EMBO J22: 5571–5581.

Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J,Berninger P, Rothballer A, Ascano M, Jungkamp A-C,Munschauer M, et al. 2010. Transcriptome-wide identi-fication of RNA-binding protein and microRNA targetsites by PAR-CLIP. Cell 141: 129–141.

Hentze MW, Preiss T. 2010. The REM phase of gene regu-lation. Trends Biochem Sci 35: 423–426.

Hentze MW, Caughman SW, Rouault TA, Barriocanal JG,Dancis A, Harford JB, Klausner RD. 1987. Identificationof the iron-responsive element for the translational reg-ulation of human ferritin mRNA. Science 238: 1570–1573.

Hentze MW, Muckenthaler MU, Galy B, Camaschella C.2010. Two to tango: Regulation of mammalian iron me-tabolism. Cell 142: 24–38.

Hinnebusch AG. 2005. Translational regulation of GCN4and the general amino acid control of yeast. Ann RevMicrobiol 59: 407–450.

Hockensmith JW, Kubasek WL, Vorachek WR, von HippelPH. 1986. Laser cross-linking of nucleic acids to proteins.Methodology and first applications to the phage T4 DNAreplication system. J Biol Chem 261: 3512–3518.

Hogg JR, Collins K. 2007. RNA-based affinity purificationreveals 7SK RNPs with distinct composition and regula-tion. RNA 13: 868–880.

Holcik M, Sonenberg N, Korneluk RG. 2000. Internal ribo-some initiation of translation and the control of celldeath. Trends Genet 16: 469–473.

Igreja C, Izaurralde E. 2011. CUP promotes deadenylationand inhibits decapping of mRNA targets. Genes Dev 25:1955–1967.

Ingolia NT, Lareau LF, Weissman JS. 2011. Ribosome pro-filing of mouse embryonic stem cells reveals the complex-ity and dynamics of mammalian proteomes. Cell 147:789–802.

Jankowsky E, Bowers H. 2006. Remodeling of ribonucleo-protein complexes with DExH/D RNA helicases. NucleicAcids Res 34: 4181–4188.

Jeske M, Meyer S, Temme C, Freudenreich D, Wahle E. 2006.Rapid ATP-dependent deadenylation of nanos mRNA ina cell-free system from Drosophila embryos. J Biol Chem281: 25124–25133.

Jeske M, Moritz B, Anders A, Wahle E. 2011. Smaug assem-bles an ATP-dependent stable complex repressing nanosmRNA translation at multiple levels. EMBO J 30: 90–103.

Jia J, Arif A, Ray PS, Fox PL. 2008. WHEP domains directnoncanonical function of glutamyl-Prolyl tRNA synthe-tase in translational control of gene expression. Mol Cell29: 679–690.

Johnson PE, Donaldson LW. 2006. RNA recognition by theVts1p SAM domain. Nat Struct Mol Biol 13: 177–178.

Kalifa Y, Huang T, Rosen LN, Chatterjee S, Gavis ER. 2006.Glorund, a Drosophila hnRNP F/H homolog, is an ovar-ian repressor of nanos translation. Dev Cell 10: 291–301.

Kapasi P, Chaudhuri S, Vyas K, Baus D, Komar AA, Fox PL,Merrick WC, Mazumder B. 2007. L13a blocks 48S assem-bly: Role of a general initiation factor in mRNA-specifictranslational control. Mol Cell 25: 113–126.

Keene JD. 2007. RNA regulons: Coordination of post-tran-scriptional events. Nat Rev Genet 8: 533–543.

Kelley RL, Wang J, Bell L, Kuroda MI. 1997. Sex lethal con-trols dosage compensation in Drosophila by a non-splic-ing mechanism. Nature 387: 195–199.

Kozak M. 1986. Influences of mRNA secondary structure oninitiation by eukaryotic ribosomes. Proc Natl Acad Sci 83:2850–2854.

Leibold EA, Munro HN. 1987. Characterization and evolu-tion of the expressed rat ferritin light subunit gene and itspseudogene family. Conservation of sequences withinnoncoding regions of ferritin genes. J Biol Chem 262:7335–7341.

Li JB, Levanon EY, Yoon JK, Aach J, Xie B, Leproust E, ZhangK, Gao Y, Church GM. 2009. Genome-wide identificationof human RNA editing sites by parallel DNA capturingand sequencing. Science 324: 1210–1213.

Mackay JP, Font J, Segal DJ. 2011. The prospects for designersingle-stranded RNA-binding proteins. Nat Struct MolBiol 18: 256–261.

Martin KC, Ephrussi A. 2009. mRNA localization: Geneexpression in the spatial dimension. Cell 136: 719–730.

Mazumder B, Seshadri V, Imataka H, Sonenberg N, Fox PL.2001. Translational silencing of ceruloplasmin requiresthe essential elements of mRNA circularization: Poly(A)tail, poly(A)-binding protein, and eukaryotic translationinitiation factor 4G. Mol Cell Biol 21: 6440–6449.

Mazumder B, Sampath P, Seshadri V, Maitra RK, DiCorletoPE, Fox PL. 2003. Regulated release of L13a from the 60Sribosomal subunit as a mechanism of transcript-specifictranslational control. Cell 115: 187–198.

Medenbach J, Seiler M, Hentze MW. 2011. Translationalcontrol via protein-regulated upstream open readingframes. Cell 145: 902–913.

Mihailovich M, Militti C, Gabaldon T, Gebauer F. 2010.Eukaryotic cold shock domain proteins: Highly versatileregulators of gene expression. BioEssays 32: 109–118.

Miller MA, Olivas WM. 2011. Roles of Puf proteins inmRNA degradation and translation. Wiley InterdiscipRev RNA 2: 471–492.

Moore MJ, Proudfoot NJ. 2009. Pre-mRNA processingreaches back to transcription and ahead to translation.Cell 136: 688–700.

Mueller PP, Hinnebusch AG. 1986. Multiple upstream AUGcodons mediate translational control of GCN4. Cell 45:201–207.

Mukhopadhyay R, Ray PS, Arif A, Brady AK, Kinter M, FoxPL. 2008. DAPK–ZIPK–L13a axis constitutes a negative-feedback module regulating inflammatory gene expres-sion. Mol Cell 32: 371–382.

Mukhopadhyay R, Jia J, Arif A, Ray PS, Fox PL. 2009. TheGAIT system: A gatekeeper of inflammatory gene expres-sion. Trends Biochem Sci 34: 324–331.

Namy O, Rousset JP, Napthine S, Brierley I. 2004. Repro-grammed genetic decoding in cellular gene expression.Mol Cell 13: 157–168.





Nelson MR, Leidal AM, Smibert CA. 2004. Drosophila Cupis an eIF4E-binding protein that functions in Smaug-mediated translational repression. EMBO J 23: 150–159.

Oberstrass FC, Lee A, Stefl R, Janis M, Chanfreau G, AllainFH. 2006. Shape-specific recognition in the structure ofthe Vts1p SAM domain with RNA. Nat Struct Mol Biol13: 160–167.

Pantopoulos K. 2004. Iron metabolism and the IRE/IRPregulatory system: An update. Ann NY Acad Sci 1012:1–13.

Parker BJ, Moltke I, Roth A, Washietl S, Wen J, Kellis M,Breaker R, Pedersen JS. 2011. New families of humanregulatory RNA structures identified by comparativeanalysis of vertebrate genomes. Genome Res 21: 1929–1943.

Patalano S, Mihailovich M, Belacortu Y, Paricio N, GebauerF. 2009. Dual sex-specific functions of Drosophila up-stream of N-ras in the control of X chromosome dosagecompensation. Development 136: 689–698.

Pelletier J, Sonenberg N. 1985. Insertion mutagenesis toincrease secondary structure within the 50 noncoding re-gion of a eukaryotic mRNA reduces translational efficien-cy. Cell 40: 515–526.

Ray PS, Fox PL. 2007. A post-transcriptional pathway re-presses monocyte VEGF-A expression and angiogenicactivity. EMBO J 26: 3360–3372.

Ray PS, Arif A, Fox PL. 2007. Macromolecular complexes asdepots for releasable regulatory proteins. Trends BiochemSci 32: 158–164.

Richter JD, Sonenberg N. 2005. Regulation of cap-depen-dent translation by eIF4E inhibitory proteins. Nature433: 477–480.

Rouget C, Papin C, Boureux A, Meunier AC, Franco B,Robine N, Lai EC, Pelisson A, Simonelig M. 2010. Ma-ternal mRNA deadenylation and decay by the piRNApathway in the early Drosophila embryo. Nature 467:1128–1132.

Sachs AB, Buratowski S. 1997. Common themes in transla-tional and transcriptional regulation. Trends Biochem Sci22: 189–192.

Saito T, Saetrom P. 2010. MicroRNAs—Targeting and targetprediction. N Biotechnol 27: 243–249.

Sampath P, Mazumder B, Seshadri V, Fox PL. 2003. Tran-script-selective translational silencing by g interferon isdirected by a novel structural element in the ceruloplas-min mRNA 30 untranslated region. Mol Cell Biol 23:1509–1519.

Sampath P, Mazumder B, Seshadri V, Gerber CA, Chavatte L,Kinter M, Ting SM, Dignam JD, Kim S, Driscoll DM, etal. 2004. Noncanonical function of glutamyl-prolyl-tRNA synthetase: Gene-specific silencing of translation.Cell 119: 195–208.

Scherrer T, Mittal N, Janga SC, Gerber AP. 2010. A screen forRNA-binding proteins in yeast indicates dual functionsfor many enzymes. PLoS ONE 5: e15499.

Schwanhausser B, Busse D, Li N, Dittmar G, Schuchhardt J,Wolf J, Chen W, Selbach M. 2011. Global quantificationof mammalian gene expression control. Nature 473:337–342.

Smibert CA, Wilson JE, Kerr K, Macdonald PM. 1996.Smaug protein represses translation of unlocalized nanos

mRNA in the Drosophila embryo. Genes Dev 10: 2600–2609.

Smibert CA, Lie YS, Shillinglaw W, Henzel WJ, MacdonaldPM. 1999. Smaug, a novel and conserved protein, con-tributes to repression of nanos mRNA translation in vitro.RNA 5: 1535–1547.

Sonenberg N, Hinnebusch AG. 2009. Regulation of transla-tion initiation in eukaryotes: Mechanisms and biologicaltargets. Cell 136: 731–745.

Squires JE, Preiss T. 2010. Function and detection of 5-meth-ylcytosine in eukaryotic RNA. Epigenomics 2: 709–715.

Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT,Parker BJ, Suter CM, Preiss T. 2012. Widespread occur-rence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res doi: 10.1093/nar/gks144.

Tadros W, Goldman AL, Babak T, Menzies F, Vardy L, Orr-Weaver T, Hughes TR, Westwood JT, Smibert CA, LipshitzHD. 2007. SMAUG is a major regulator of maternalmRNA destabilization in Drosophila and its translationis activated by the PAN GU kinase. Dev Cell 12: 143–155.

Tarun SZ Jr, Sachs AB. 1996. Association of the yeast poly(A)tail binding protein with translation initiation factoreIF-4G. EMBO J 15: 7168–7177.

Tenenbaum S, Carson C, Lager P, Keene J. 2000. IdentifyingmRNA subsets in messenger ribonucleoprotein com-plexes by using cDNA arrays. Proc Natl Acad Sci 97:14085–14090.

Tsai BP, Wang X, Huang L, Waterman ML. 2011. Quantita-tive profiling of in vivo–assembled RNA–protein com-plexes using a novel integrated proteomic approach. MolCell Proteomics 10: M110.007385.

Tsvetanova NG, Klass DM, Salzman J, Brown PO. 2010.Proteome-wide search reveals unexpected RNA-bindingproteins in Saccharomyces cerevisiae. PLoS ONE 5:e12671.

Ule J. 2003. CLIP identifies Nova-regulated RNA networksin the brain. Science 302: 1212–1215.

Valasek L, Szamecz B, Hinnebusch AG, Nielsen KH. 2007. Invivo stabilization of preinitiation complexes by formal-dehyde cross-linking. Methods Enzymol 429: 163–183.

Vyas K, Chaudhuri S, Leaman DW, Komar AA, MusiyenkoA, Barik S, Mazumder B. 2009. Genome-wide polysomeprofiling reveals an inflammation-responsive posttran-scriptional operon in g interferon-activated monocytes.Mol Cell Biol 29: 458–470.

Wells SE, Hillner PE, Vale RD, Sachs AB. 1998. Circulariza-tion of mRNA by eukaryotic translation initiation fac-tors. Mol Cell Biol 2: 135–140.

Zaessinger S, Busseau I, Simonelig M. 2006. Oskar allowsnanos mRNA translation in Drosophila embryos by pre-venting its deadenylation by Smaug/CCR4. Development133: 4573–4583.

Zhang X, Liu Z, Yi J, Tang H, Xing J, Yu M, Tong T, Shang Y,Gorospe M, Wang W. 2012. The tRNA methyltransferaseNSun2 stabilizes p16(INK4) mRNA by methylating the3-untranslated region of p16. Nat Commun 3: 712.

Zhao H, Yang Y, Zhou Y. 2011. Structure-based prediction ofRNA-binding domains and RNA-binding sites and ap-plication to structural genomics targets. Nucleic Acids Res39: 3017–3025.

F. Gebauer et al.




2012; doi: 10.1101/cshperspect.a012245Cold Spring Harb Perspect Biol Fátima Gebauer, Thomas Preiss and Matthias W. Hentze

-Regulatory Elements to Complex RNPs and BackCisFrom

Subject Collection Protein Synthesis and Translational Control

Virus-Infected CellsTinkering with Translation: Protein Synthesis in

Derek Walsh, Michael B. Mathews and Ian MohrTranslational ControlToward a Genome-Wide Landscape of

Ola Larsson, Bin Tian and Nahum SonenbergTranslational Control in Cancer Etiology

Davide Ruggero IRESsThe Current Status of Vertebrate Cellular mRNA

Richard J. Jackson

Mechanism of Gene Silencing by MicroRNAsDeadenylases Provides New Insight into the A Molecular Link between miRISCs and

IzaurraldeJoerg E. Braun, Eric Huntzinger and Elisa

Principles of Translational Control: An Overview

Michael B. MathewsJohn W.B. Hershey, Nahum Sonenberg and

Fluorescent MicroscopyImaging Translation in Single Cells Using

SingerJeffrey A. Chao, Young J. Yoon and Robert H.

PathwaysRegulation of mRNA Translation by Signaling

Philippe P. Roux and Ivan Topisirovic

OogenesisDrosophilamRNA Localization and Translational Control in

Paul LaskoInitiation: New Insights and ChallengesThe Mechanism of Eukaryotic Translation

Alan G. Hinnebusch and Jon R. Lorsch

Degradationthe Control of Translation and mRNA P-Bodies and Stress Granules: Possible Roles in

Carolyn J. Decker and Roy ParkerDynamicsSingle-Molecule Analysis of Translational

Alexey Petrov, Jin Chen, Seán O'Leary, et al.Protein Secretion and the Endoplasmic Reticulum

Adam M. Benham Control of Complex Brain FunctionCytoplasmic RNA-Binding Proteins and the

Jennifer C. Darnell and Joel D. Richter

and Back-Regulatory Elements to Complex RNPsCisFrom

HentzeFátima Gebauer, Thomas Preiss and Matthias W.

Phases of Translation in EukaryotesThe Elongation, Termination, and Recycling

Thomas E. Dever and Rachel Green

http://cshperspectives.cshlp.org/cgi/collection/ For additional articles in this collection, see

Copyright © 2012 Cold Spring Harbor Laboratory Press; all rights reserved


http://cshperspectives.cshlp.org/cgi/collection/

http://cshperspectives.cshlp.org/cgi/collection/


from cis-regulatory elements to complex rnps and...

Documents