proceeding through 3'-to-5' exosome-dependent decapping

2002 8: 1526-1537 RNA N. D. Rodgers, Z. Wang and M. Kiledjian

proceeding through 3'-to-5' exosome-dependent decappingRegulated alpha-globin mRNA decay is a cytoplasmic event

References

http://www.rnajournal.org/cgi/content/abstract/8/12/1526#otherarticlesArticle cited in:

serviceEmail alerting

click heretop right corner of the article or Receive free email alerts when new articles cite this article - sign up in the box at the

Notes

http://www.rnajournal.org/subscriptions/ go to: RNATo subscribe to

© 2002 RNA Society

on February 14, 2006 www.rnajournal.orgDownloaded from

http://www.rnajournal.org/cgi/content/abstract/8/12/1526#otherarticles

http://www.rnajournal.org/cgi/alerts/ctalert?alertType=citedby&addAlert=cited_by&saveAlert=no&cited_by_criteria_resid=rna;8/12/1526&return_type=article&return_url=http%3A%2F%2Fwww.rnajournal.org%2Fcgi%2Freprint%2F8%2F12%2F1526.pdf

http://www.rnajournal.org/subscriptions/

http://www.rnajournal.org

Regulated a-globin mRNA decay is a cytoplasmicevent proceeding through 3 9-to-59exosome-dependent decapping

NANCY D. RODGERS, ZUOREN WANG, and MEGERDITCH KILEDJIANDepartment of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082, USA

ABSTRACT

The a-globin mRNA contains a C-rich stability element (CRE) in its 3 9 untranslated region (3 9 UTR) which is critical forthe stability of this long-lived mRNA. A protein complex, termed the a-complex, forms on the CRE and has been shownto contribute to stabilization of the mRNA by at least two mechanisms, first by interacting with the poly(A)-bindingprotein (PABP) to prevent deadenylation, and second by protecting the mRNA from attack by an erythroid endoribo-nuclease. In this report, we demonstrate that the a-globin 3 9 UTR can confer stability on a heterologous mRNA in cells,and this stability is dependent on the a-complex. Moreover, the stability was exclusively detected with cytoplasmicmRNA, suggesting that the regulation of a-globin mRNA stability is a cytoplasmic event. An additional mechanism bywhich the a-complex can confer stability on an RNA in vitro was also identified and shown to involve inhibition of 3 9to 59 exonucleolytic degradation. Furthermore, using an in vitro mRNA decay system, we were able to follow thedemise of the a-globin RNA and demonstrate that the decay was initiated by deadenylation followed by 3 9-to-59 decaycarried out by the exosome and ultimately hydrolysis of the residual cap structure.

Keywords: DcpS; deadenylation; exosome; mRNA stability

INTRODUCTION

Our understanding of gene expression at the posttran-scriptional level is becoming increasingly clear, and agrowing number of genes regulated at the level of mRNAstability are being identified+All eukaryotic mRNAs con-tain stability elements at either termini that include the59 m7G cap and a poly(A) tail at the 39 terminus (Ross,1995; Sachs et al+, 1997)+ Essential to the stability pro-vided by these cis elements is the binding of specificprotein factors, which are the cap binding proteins andthe poly(A)-binding protein (PABP) that protect anmRNA from exonucleolytic degradation (Shatkin, 1985;Bernstein et al+, 1989; Wickens, 1990; Sachs, 1993;Ross, 1996; Ford et al+, 1997; Coller et al+, 1998;Wanget al+, 1999)+ Additional stability elements present inmRNAs are often located within the 39 untranslatedregion (39 UTR) of the transcript and may function topromote stability or instability of the transcript (Jack-son, 1993;Chen & Shyu, 1995;Decker & Parker, 1995)+

The a-globin mRNA is an example of an RNA that con-tains a stabilizing element within its 39 UTR (Weiss &Liebhaber, 1995;Russell et al+, 1997;Wang et al+, 1999)+

Globin mRNAs are among the most stable mRNAsknown,with estimated half-lives ranging from 24 to 60 h(Lodish & Small, 1976; Volloch & Housman, 1981;Ross& Sullivan, 1985)+ A specific ribonucleoprotein com-plex, termed the a-complex, whose presence corre-lates with mRNA stability, has been shown to bind to aC-rich element (CRE) in the a-globin 39 UTR (a39UTR;Weiss & Liebhaber, 1994, 1995; Wang et al+, 1995;Wang & Kiledjian, 2000a, 2000b)+ It has recently beendemonstrated that the a-globin and b-globin RNAs formsimilar protein complexes on their 39 UTRs, and mayshare a regulatory pathway (Yu & Russell, 2001)+ Theb-globin and a-globin mRNAs are equally stable, andexperiments in which the b39UTR has been replacedwith the a39UTR have shown that this chimeric mRNAis also stable (Russell & Liebhaber, 1996)+ Thus, it ap-pears that the stability determinants of a-globin residein its 39 UTR+ However, the possibility that sequenceswithin the coding region of the stable globin genes arerequired along with the 39 UTR cannot be completelyruled out since the role of the a39UTR was character-ized in the context of a stable mRNA+

Reprint requests to: Megerditch Kiledjian, Department of CellBiology and Neuroscience, Rutgers University, 604 Allison Road,Piscataway, New Jersey 08854-8082, USA; e-mail: kiledjia@biology+rutgers+edu+

RNA (2002), 8:1526–1537+ Cambridge University Press+ Printed in the USA+Copyright © 2002 RNA Society+DOI: 10+1017/S1355838202029035

1526



Several mechanisms by which the a-complex stabi-lizes RNA have been identified+ The a-complex wasoriginally identified as a multiprotein complex (Wanget al+, 1995; Kiledjian et al+, 1997) that included a familyof polycytidylate [poly(C)]-binding proteins aCP1 andaCP2 (Kiledjian et al+, 1995; Leffers et al+, 1995)+ Sub-sequently, the aCP proteins were shown to bind thea39UTR directly (Chkheidze et al+, 1999), and many ofthe stabilizing functions of the a-complex can be re-capitulated by the aCP protein in vitro (Wang & Kiled-jian, 2000b)+ The aCP proteins have been implicated inmRNA stabilization as well as in the regulation of trans-lation (Makeyev & Liebhaber, 2002)+ These proteinsare required for the stability of a-globin mRNA and forthe formation of the a-complex+ Sequestration of theseproteins with poly (C) or poly (dC) results in destabili-zation of a-globin mRNA (Wang et al+, 1999) as well asdisruption of the a-complex (Kiledjian et al+, 1995, 1999;Wang et al+, 1995)+

Studies in vivo using transgenic mice that expressthe human a-globin gene suggested that the a-complexmay play a role in deadenylation (Morales et al+, 1997)+The poly(A) tail of wild-type a-globin mRNA was longerthan that of a mutant a-globin mRNA (a-ConstantSpring) in which ribosomal read through into the a39UTRdisrupts the a-complex (Morales et al+, 1997)+ This ob-servation was confirmed and expanded through theuse of a cell-free mRNA decay system that uses acapped and polyadenylated mRNA substrate with S130cytosolic extract (Wang et al+, 1999)+ These studiesshowed that indeed, the a-complex functions, in part,by impeding the deadenylation of a-globin mRNA, andthat this is mediated through an interaction of the aCPproteins with PABP to increase the binding efficiency ofPABP to the poly(A) tail (Wang et al+, 1999; Wang &Kiledjian, 2000b)+ A second function of the a-complexin a-globin mRNA stability is to protect an endonucle-olytic site within the a39UTR that is a target site for anerythroid-enriched endoribonuclease (ErEN; Wang &Kiledjian, 2000a, 2000b)+ Clearly, the aCP proteins playa major role in a-globin mRNA stability+ The aCP pro-teins have also been shown to bind the collagen, tyro-sine hydroxylase, erythropoietin, and CD81 mRNAs,indicating that they may also stabilize these mRNA aswell (Holcik & Liebhaber, 1997; Stefanovic et al+, 1997;Czyzyk-Krzeska & Bendixen, 1999; Paulding & Czyzyk-Krzeska, 1999; Trifillis et al+, 1999)+

Much of what we know about mRNA turnover in eu-karyotes comes from studies in yeast, where the pre-dominant mechanism of decay is deadenylation followedby decapping and subsequent 59-to-39 exonucleolyticdecay (Larimer et al+, 1992; Decker & Parker, 1994;Muhlrad et al+, 1995; Beelman et al+, 1996; Caponigro& Parker, 1996; LaGrandeur & Parker, 1998)+ Alter-natively, mRNAs may be degraded in the 39-to-59direction following deadenylation by a complex of exo-nucleases called the exosome (Mitchell et al+, 1997;

Jacobs et al+, 1998)+ The residual cap is hydrolyzed bya scavenger decapping enzyme,Dcs1 (formerly yDcpS),which has recently been identified in yeast (Wang &Kiledjian, 2001; Liu et al+, 2002)+ These pathways ap-pear to have been conserved from yeast to mammalsand the elucidation of the pathways of mRNA turnoverin mammalian cells has been greatly advanced withthe use of cell-free mRNA decay systems+

The first step in degradation of many mRNAs in mam-mals appears to be deadenylation (Wilson & Treisman,1988; Shyu et al+, 1991)+ A deadenylating enzyme hasbeen identified termed poly(A) ribonuclease (PARN;Korner & Wahle, 1997; Korner et al+, 1998; Dehlin et al+,2000)+ PARN appears to be the major deadenylaseactivity in mammalian cells, as deadenylation is inhib-ited in HeLa extracts when PARN is immunodepleted(Gao et al+, 2000)+ Also, microinjection of anti-PARNantibodies inhibits deadenylation in Xenopus oocytesduring progesterone-induced maturation (Korner et al+,1998)+ The fate of the mRNA after deadenylation, how-ever, was unclear until recently+ It appears that 39-to-59decay may be the major degradation pathway of mRNAin mammalian cells (Wang & Kiledjian, 2001)+ The prom-inence of the 39-to 59 decay pathway has been shownin an in vitro mRNA decay system, with generic RNAsintroduced into cells and with an endogenous mRNA(Wang & Kiledjian, 2001)+ It also appears that ARE-containing mRNAs undergo 39-to-59 exosome-mediateddecay (Chen et al+, 2001; Mukherjee et al+, 2002)+ Themammalian scavenger decapping enzyme,DcpS,whichis responsible for the hydrolysis of the residual capstructure following 39-to-59 decay was recently identi-fied (Wang & Kiledjian, 2001; Liu et al+, 2002)+ DcpSassociates with the exosome (Wang & Kiledjian, 2001)and functions only on RNAs less than 10 nt long pro-duced from nucleolytic decay (Liu et al+, 2002)+ In ad-dition to DcpS, the human Dcp2 protein (hDcp2) hasrecently been identified as a decapping enzyme capa-ble of hydrolyzing the intact mRNA (Wang et al+, 2002)+hDcp2 activity is inhibited by the poly(A) tail (Wanget al+, 2002), suggesting that the deadenylation-dependent decapping pathway is also functional inmammals+ We now present data illustrating the mech-anistic turnover pathway of the a-globin mRNA, whichinitiates with deadenylation of the mRNA followedby exosome-dependent decay and decapping of themessage+

RESULTS

Differential stability of wild-type andmutant a39UTRs can be conferredon a heterologous mRNA

To confirm that the stability determinants within thea39UTR are able to act independently of the proteincoding region, we determined whether differential sta-

a-globin mRNA decay 1527



bility of a wild-type and mutant a39UTR could be con-ferred on a heterologous mRNA substrate in cells+Heterologous mRNAs were constructed in which thea39UTR was placed after the coding region of the fireflyluciferase gene, hereafter referred to as luc-awt+ Theluciferase gene was chosen because the half-life ofluciferase mRNA has been shown to be approximately3+5 h in cells (Wood, 1995)+ A hybrid construct in whicha mutant a39UTR (Wang et al+, 1999) was placed afterthe luciferase gene was also constructed, hereafter re-ferred to as luc-aDmt+ This mutation is a deletion inwhich a majority of the CRE has been removed+ Thesetwo constructs were separately cotransfected with acontrol plasmid encoding the b-galactosidase gene intomurine erythroleukemia (MEL) cells+ After 24 h, thecells were collected and luciferase and b-galactosidaseactivities were assayed+ The level of luciferase activitywas normalized relative to that of the b-galactosidaseactivity to control for transfection efficiency and proteinconcentrations+ The results of three independent ex-periments are shown in Figure 1+ The luciferase activityin extracts prepared from luc-awt-transfected cells wasover twofold greater than that obtained from luc-aDmt-transfected cells+ These data suggest that the luc-awt

mRNA is more stable than the luc-aDmt mRNA+

Stability of the a39UTR is regulatedin the cytoplasm

To ensure that the difference in luciferase activity wasa result of differential mRNA stability and not a result oftranslatability, mRNA levels were tested directly+ MELcells were cotransfected with either luc-awt or luc-aDmt

and a plasmid encoding the neomycin resistance gene

as a control for transfection efficiency+ Twenty-four hoursafter transfection, cells were treated with actinomycinD to inhibit transcription and collected at 4 and 8 hfollowing drug addition+ Total cellular RNA was ex-tracted and an RNase protection assay (RPA) wascarried out using luciferase and neomycin resistancegene probes+ The intensity of the protected bandswas quantitated, and the signal from the luciferasemRNA was normalized to the signal of the neomycinresistance mRNA to account for transfection effi-ciency and any differences in the amount of RNAused in the assays+ Surprisingly, no major differencein mRNA abundance was observed between the luc-awt and luc-aDmt mRNAs (data not shown)+ Becauseonly cytoplasmic mRNA would be translated, we rea-soned that perhaps the differential protein levels ob-served reflected differences in mRNA stability in thecytoplasm versus nuclear RNA+ Consistent with thispremise, much of erythroid differentiation occurs dur-ing an enucleated posttranscriptional state (Papayan-nopoulou et al+, 2000), implying that at least part ofthe regulated a-globin mRNA stability must occur inthe cytoplasm+ However, it is not known if a-globinmRNA stability is also regulated in the nucleus+ Con-sidering that the chimeric luciferase/a39UTR RNAstranscribed from a transfected plasmid do not containintrons, it was possible that a disproportionate amountof the chimeric RNA remained in the nucleus andcould account for the discrepancy+

To address the apparent contradiction between theluciferase protein activity and luciferase mRNA levels,the stability conferred by the a39UTR within the twocellular fractions was directly tested+ RNA was isolatedfrom either the cytoplasmic or nuclear compartmentfollowing actinomycin D treatment and analyzed by RPA+As shown in Figure 2A, when normalized relative to theneomycin mRNA control, the luc-awt RNA is approxi-mately twofold more abundant than the luc-aDmt RNAin the cytoplasmic fraction+ This differential stability is afunction of the mRNA stabilizing a-complex, as a sim-ilar twofold greater stability was also detected betweenluc-awt and a mutant a39UTR, luc-a19 (Fig+ 2A), con-taining a previously described 5-nt mutation within theCRE (aH19 ; Weiss & Liebhaber, 1995)+ This mutationfails to form the mRNA stability a-complex and resultsin an unstable a-globin mRNA (Wang et al+, 1995;Weiss& Liebhaber, 1995)+ Interestingly, the relative stability ofthe luciferase mRNA containing the SV40 early 39 UTRwas twofold less than the luc-awt mRNA and similar toboth mRNAs containing a CRE mutation (Fig+ 2A)+Theseresults further reinforce the significance of the CRE inthe observed stability+

Analysis of RNA from nuclear fractions revealed thatall four 39 UTR-containing luciferase mRNAs were sim-ilarly stable (Fig+ 2B)+ Although these mRNAs wereslightly more stable than the neomycin mRNA over time,differential stability relative to the neomycin mRNA was

FIGURE 1. Luciferase activity of hybrid RNAs containing the wild-type or mutant a-globin 39 UTRs+ Luciferase and b-galactosidaseactivity was assayed using protein extract prepared from cells co-transfected with a plasmid encoding the b-galactosidase gene andluc-awt (open bar) or luc-aDmt (shaded bar)+ The value obtained forluciferase activity was normalized relative to b-galatosidase activityand plotted as arbitrary units+ Standard deviations from three inde-pendent experiments are shown+

1528 N.D. Rodgers et al.



FIGURE 2. Differential stability of the a39UTR is maintained in a heterologous system within the cytoplasm+MEL cells werecotransfected with a plasmid encoding the neomycin resistance gene and luc-awt, luc-aDmt, luc-a19, or luc-SV40+ RNA wasextracted from nuclear and cytoplasmic fractions of the cells, which were treated with actinomycin D for the indicated times24 h posttransfection+ RPA was carried out on both fractions of RNA using a probe against luciferase and one against theneomycin resistance mRNA+ A:An RPA using cytoplasmic RNA is shown in the top panel+ Constructs used to transfect MELcells are labeled above the brackets and hours of actinomycin D treatment is indicated+ The protected bands correspondingto the luciferase and neomycin resistance RNA are shown+ The intensities of the detected luciferase mRNA were quantitatedand normalized relative to the neomycin resistance mRNA+ Values for the mRNAs are plotted for each time point+ B: RPAof the nuclear fraction of RNA is labeled and plotted as in A+ C: Cytoplasmic (lane 1) or nuclear (lane 2) RNA was reversetranscribed using a 32P-end-labeled oligonucleotide specific for the nucleus restricted RNA, U6+ The RT product is shownwith the arrow+ D: RT-PCR for c-myc and b-globinmaj+ Cytoplasmic RNA from A was reverse transcribed with oligo d(T) andPCR amplified with primers specific for c-myc and b-globinmaj++ PCR products are labeled and total RNA or a specific timepoint is indicated above each lane+




not detected (Fig+ 2B)+ These data indicate that theregulated differential stability observed in the cyto-plasm was not evident in the nucleus and only oc-curred in the cytoplasm+ The efficiency of cellularfractionation is shown in Figure 2C, where the fate ofthe abundant, nuclear restricted U6 RNA was followed+U6 RNA was detected by reverse transcription and ispredominately found in the nucleus as expected (Fig+ 2B,compare lane 2 to lane 1)+ To ensure that transcrip-tional shutoff was efficient, RT-PCR was carried out todetect c-myc and b-globinmaj mRNAs+ c-myc is an un-stable mRNA with a half-life of 15–40 min (Dani et al+,1984)+ As expected, no signal was detected after 4 hof actinomycin D treatment (Fig+ 2D)+ In contrast,b-globinmaj, a stable mRNA, was detected even after8 h of drug treatment (Fig+ 2D)+ Furthermore, the ob-served affects were not specific to actinomycin D, asblocking transcription with DRB, a different inhibitor,gave similar results (data not shown)+

Collectively, three conclusions can be drawn fromthe above data+ First, the differential stability of wild-type a-globin versus 39 UTR mutants can be conferredon a heterologous mRNA+ Second, the regulation ofa-globin mRNA stability mediated by the a39UTR is acytoplasmic event, as the RNA levels are equivalent inthe nucleus, and third, the CRE is essential for thisstability+ Consistent with our inability to detect differen-tial stability of the luciferase/a39UTR mRNAs using to-tal RNA, mRNA levels for the transfected genes wereapproximately twice as abundant in the nuclear fractionthan in the cytoplasmic fraction at time zero (data notshown)+The CRE-dependent regulated cytoplasmic sta-bility is also consistent with in vitro decay studies thatrecapitulate a39UTR-mediated stability using only cyto-solic extract (Wang et al+, 1999)+

The a-complex can block 3 9-to-59exonucleolytic decay

Having demonstrated that the a39UTR can stabilizea heterologous mRNA, we next asked whether thea-complex binding region was sufficient to stabilize achimeric RNA+ Binding of the a-complex to the a39UTRhas been previously demonstrated to require 20 nt withinthe a39UTR (nt 41–60; Holcik & Liebhaber, 1997)+ Toensure efficient binding of the a-complex to a chimericRNA, nt 30–70 were cloned into the pcDNA3 polylinkerand the distance of the CRE from the 39 end of theRNA was maintained so that it is the same distancefrom the 39 end as it is in the a39UTR+ This constructwill be referred to as pcP-CRE+ A mutant, pcP-mtCRE,was also constructed in which the pyrimidines withinthe CRE,which are critical for a-complex binding (Weiss& Liebhaber, 1995; Holcik & Liebhaber, 1997), werechanged to purines+ The stability of these RNAs wastested in an in vitro decay system which we have pre-viously shown recapitulates the observed in vivo dif-

ferential stability of a39UTR mutants (Wang et al+, 1999)+Cap-labeled RNA probes from these constructs wereprepared and their stabilities were compared in K562S130 extract (Fig+ 3A)+ Since the RNA is cap-labeled,only degradation occurring in the 39-to-59 direction ismonitored+ In Figure 3B the percent of RNA remainingat each time point relative to the internal control wasplotted+ The RNA containing the wild-type CRE wastwofold more stable than the mutant, which is consis-tent with our in vivo observations (Fig+ 2)+ Similar re-sults were obtained using extract derived from MELcells or using uniformly labeled RNA (data not shown)+These data demonstrate that the CRE is an auto-nomous stability element that stabilizes a chimeric RNAto the same extent as the full-length a39UTR in vitro+Because the RNA was labeled at the 59 end, these dataalso suggest that the a-complex can stabilize RNA byblocking 39-to-59 decay+

The a39UTR is degraded by the exosomein the 3 9-to-59 direction

We have recently demonstrated that a major path-way of mRNA degradation in mammalian cells pro-ceeds in the 39-to-59 direction, most likely by theexosome, following deadenylation (Wang & Kiledjian,2001)+ Because the a-complex is able to block the39-to-59 decay of an RNA, we asked whether it isblocking degradation by the exosome or whether non-exosome components were also involved in the 39-to-59 decay+ The exosome is a protein complexcomposed of 11 proteins in yeast, most of which haveexonucleolytic activity (Mitchell et al+, 1997; Jacobset al+, 1998; Bousquet-Antonelli et al+, 2000)+ Humanhomologs of all the yeast components have recentlybeen identified (Chen et al+, 2001)+ The exosome wasimmunodepleted from K562 S130 extract using a com-bination of antibodies against two components of theexosome, hRrp40 and hRrp46 (Brouwer et al+, 2001)+The stability of cap-labeled a39UTR was comparedover time in extract that was mock depleted usingbeads alone (Fig+ 4, lanes 2–4), depleted with anunrelated control antibody (Fig+ 4, lanes 5–7), or de-pleted with antibodies against exosome components(Fig+ 4, lanes 8–10)+ The degradation of the a39UTRin exosome-depleted extract was greatly decreasedcompared to the controls (Fig+ 4, compare lanes 2–7to lanes 8–10) and the extent of 39-to-59 decay wasindependent of the RNA size (data not shown)+ Theefficiency of exosome depletion was assessed by west-ern analysis in Figure 4B, which shows that the de-pletion was complete with negligible amounts ofhRrp40p detected in the depleted extract (lane 3)+The stability of the 59-end-labeled RNA in theexosome-depleted extract suggests that the degrada-tion of the a39UTR following deadenylation is mainlycarried out by the exosome+




FIGURE 3. The a-globin CRE can stabilize a chimeric RNA in vitro+ A: Cap-labeled pcP-CRE and pcP-mtCRE RNAs wereused in an in vitro decay assay with K562 S130 extract+ The RNAs are labeled above the brackets and were incubated withextract at 37 8C for the indicated times, separated on a denaturing 7 M urea-8% polyacrylamide gel and visualized byautoradiography+ The migration of the internal control is indicated+ The RNA used is depicted schematically on the bottomwhere the cap is represented by the m7GpppG and the asterisk denotes the labeled phosphate+ The presence of the CREor mtCRE is designated in the boxes+ B: The data in A was normalized to the internal control and plotted from threeindependent experiments+ Values for pcP-CRE RNA are shown by the solid line and squares and values for pcP-mtCRERNA are shown by the dotted line and circles with corresponding error bars+

FIGURE 4. Degradation the a39UTR by the exosome in the 39-to-59 direction+ A: Cap-labeled a39UTR was incubated inK562 S130 extract that was mock depleted (lanes 2–4), depleted with control antisera (lanes 5–7), or depleted with antiseraagainst two exosome components, hRrp40 and hRrp46 (lanes 8–10)+ The incubation times are indicated above each laneand migration of the internal control is shown on the right+ The RNA substrate is shown schematically on the bottom withthe cap represented by m7GpppG and the asterisk denoting the labeled phosphate+ B: Western analysis was carried outwith each of the depleted extracts to determine the extent of exosome depletion+ Protein from 50 mg of extract, as indicatedabove each lane, was resolved on SDS-PAGE and blotted to nitrocellulose+ The blot was probed with antisera againsthRrp40 and the corresponding band is labeled on the left+




Decay of the a39UTR initiates withdeadenylation followed by 3 9-to-59 decayand subsequent decapping

To directly observe the mechanistic order of decay forthe a-globin mRNA, we carried out an in vitro RNAdecay reaction with capped and polyadenylated RNA+The turnover of the a39UTR proceeds through an initialdeadenylation step (Wang et al+, 1999), which can beaccentuated by the addition of oligo(dC) competitorthat serves to sequester the aCP mRNA stability pro-teins and disrupt the aCP-PABP interaction (Wang &Kiledjian, 2000b)+ Cap-labeled a39UTR was used assubstrate to determine the relationship between de-adenylation, decay, and decapping within a single re-action+ Oligo(dC) was added to facilitate the decayprocess (Wang et al+, 1999)+At each time point, half thereaction was used to follow the deadenylation and de-cay of the RNA on a polyacrylamide gel, whereas thesecond half was used to monitor the extent of decap-ping by polyethyleneimine (PEI) cellulose thin-layer chro-matography (TLC)+ As can be seen in Figure 5A, theRNA is increasingly deadenylated over time and un-adenylated RNA accumulates by 15 min and increasesby 30 min and is then chased into smaller decay prod-ucts resulting from 39-to-59 degradation+ To determinethe relationship between deadenylation and decay ofthe RNA, the values obtained from quantifying the ad-enylated RNA were compared to the total decay of theRNA, which includes both the adenylated and unade-nylated RNA+ As shown in Figure 5C, deadenylation(represented by the solid line and open circles) pro-ceeds rapidly with 80% of the RNA deadenylated by90 min+ The decay of the RNA (represented by thedashed line and closed circles) is slower than deadenyl-ation and reaches approximately 70% by 90 min+ Twoconclusions can be made from these data+ The first isthat deadenylation precedes decay of the mRNA body+Second, there appears to be a lag between the de-adenylation and the decay of the mRNA, as unadeny-lated RNA accumulates and is detected+

We next determined at which point the RNA is de-capped+ The extent of decapping was followed andfound to be slower than deadenylation and decay+ De-capping products first become obvious at 15 min andincrease up to the 90-min time point (Fig+ 5B)+ The topspot is m7GMP released by the DcpS decapping en-zyme and the bottom spot is the subsequent hydrolysisof this product that releases the 32Pi (Wang & Kiledjian,2001)+ The graph in Figure 5C demonstrates that de-capping of the RNA occurs last+ We were unable todetect deadenylation-dependent decapping similar tothat observed in yeast with these assay conditions+Furthermore, the decay patterns observed with uni-formly labeled RNA (Fig+ 5D) were indistinguishablefrom the decay using 59-end-labeled RNA (Fig+ 5A)+This further confirms the minimal contribution of the

deadenylation-dependent decapping and 59-to-39 de-cay pathway, at least in vitro+ Thus, the order of eventsin a-globin mRNA turnover can be drawn from thesedata and suggest that the first step in the reaction isdeadenylation, which we have previously reported(Wang et al+, 1999), followed by 39-to-59 decay, andthen decapping+ This demonstrates the significance ofdeadenylation and decay in mammalian mRNA decap-ping and confirms a functional link between exosomedegradation and the decapping process in vitro+

DISCUSSION

We have used a heterologous mRNA to confirm thatthe a-globin 39 UTR can function as a stability elementindependent of the globin coding region sequences+Using chimeric luciferase constructs containing eithera wild type, a CRE deletion mutant, or a previouslydescribed a39UTR mutant, the relative abundance ofthe mutant mRNAs was reduced, and this differencecan be extrapolated into the luciferase protein activity(Figs+ 1 and 2A)+ Furthermore, the stability of the chi-meric RNAs containing the mutant a39UTRs was com-parable to a chimeric RNA containing the SV40 39UTR(Fig+ 2A)+ Therefore, the differential stability of thea39UTRs is maintained in a heterologous system, andthe a39UTR is an autonomous stability element+ Theseobservations are in agreement with Weiss and Lieb-haber (1994), who used the entire a-globin gene andnoticed the wild-type 39 UTR was more stable than themutants+ However, it is interesting that we observe dif-ferential stability only with cytoplasmic RNA (Fig+ 2),suggesting that a-globin stability is not regulated in thenucleus+ Cytoplasmic regulation of globin mRNA sta-bility correlates well with red blood cell biology+Midwaythrough erythroid terminal differentiation, the nucleuscondenses and is extruded from the cell (Papayan-nopoulou et al+, 2000); therefore, regulation that takesplace after this event would have to occur cytoplasmi-cally+ Weiss and Liebhaber (1994) were able to ob-serve differential stability of full-length a-globin with awild-type or mutant 39 UTR using total cellular RNA+This difference can be attributed to the use of genomicDNA constructs, which mimic the endogenous RNAsthat are efficiently transported to the cytoplasm;therefore, the proportion of nuclear mRNA was mostlikely negligible+ The fortuitous accumulation of nuclearluciferase/a39UTR RNA using the transfection systemallowed us to determine the stability of the RNA in bothcellular compartments+

We have also shown that the binding of the a-complexto an RNA stabilizes the RNA in vitro (Fig+ 3)+ We in-troduced the a-complex binding site, the CRE, into achimeric RNA and the RNA was stabilized twofold(Fig+ 3)+ Combining this data with previous reports fromour laboratory suggests that the a-complex is able tostabilize an RNA by multiple mechanisms+ The first is to




FIGURE 5. a-globin mRNA decay proceeds through a deadenylation-dependent decay and decapping mechanism+ A:RNA deadenylation and decay was monitored in an in vitro decay reaction using 10 mg of K562 S130 for the indicated timesusing 59 cap-labeled (m7G*pppG-) a39UTR-A60 RNA in the presence of 0+75 pmol oligo(dC) competitor to facilitate de-adenylation (Wang et al+, 1999)+ The migration of the unadenylated a39UTR is indicated on the left+ The RNA products wereresolved and visualized as describe in Figure 3+ The RNA used is shown on the bottom and is labeled as in Figure 4A+ B:An in vitro decay reaction identical to that described in A was carried out, and decapping activity was monitored by PEI-TLC+Standards are shown on the left+ C: Quantitation of deadenylation, decay, and decapping of the a39UTR in A and B isplotted+ The percentage of RNA deadenylated, which is shown by the solid line and open circles, was determined from theamount of adenylated RNA remaining at each time point relative to time zero+ The percentage of decay of the RNA,represented by the dashed line and closed circles, was determined from the amount of RNA remaining at each time pointincluding both adenylated and unadenylated a39UTR relative to the time zero+ The percentage of decapping, where the sumof both decapping products was used, is shown as a dotted line and squares+ D: An in vitro decay assay identical to thatin A is shown except that uniformly labeled a39UTR was used+ Labeling is as in A+




prevent deadenylation of the RNA through a physicalinteraction with the poly(A) binding protein,PABP (Wanget al+, 1999; Wang & Kiledjian, 2000b)+ The second isby protecting an endonucleolytic site within an RNAfrom cleavage by an endoribonuclease (Rodgers et al+,2002; Wang & Kiledjian, 2000a)+ The third is by block-ing exosome-mediated 39-to-59 decay of an unadeny-lated RNA (Fig+ 4)+ Whether the a-complex is able toinhibit the activity of the exosome through a physicalinteraction with exosome components or whether it sim-ply acts as a steric hindrance is unknown at present+Given the fact that stem loop structures are able toblock 39-to-59 degradation (Ford & Wilusz, 1999), it ispossible that any structure or protein complex stablyassociated with an RNA may be able to restrict exo-some activity to some degree+ The aCP proteins havealso been shown to bind to the 39 UTR of a number ofother RNAs+ These include the stable collagen, tyro-sine hydroxylase, and erythropoietin mRNAs (Ste-fanovic et al+, 1997; Czyzyk-Krzeska & Bendixen, 1999;Paulding & Czyzyk-Krzeska, 1999)+The b-globin 39 UTRalso forms a ribonucleoprotein complex on its 39 UTRthat appears to contain aCP proteins or proteins closelyrelated to the aCP proteins (Yu & Russell, 2001)+ TheseRNAs may also be stabilized in similar mechanisms bythe a-complex+

It is now well established that RNA decay in mamma-lian cells or extract proceeds through an initial step ofdeadenylation (Wilson & Treisman, 1988; Shyu et al+,1991) and this is also true for the a39UTR in vitro (Wanget al+, 1999)+Recently, it was shown using a generic RNAthat the subsequent events in mammalian mRNA de-cay occur through 39-to-59 decay and that the residualcap structure was hydrolyzed by DcpS (Wang & Kiled-jian, 2001)+ In those experiments, each step of turnoverwas analyzed individually+ To follow the entire turnovermechanism for the a-globin mRNA,we designed an ex-periment that would allow us to determine the pathwayof mRNA turnover in a single experiment (Fig+ 5)+ By fol-lowing the degradation of adenylated, 32P-cap-labeleda39UTR in a single reaction, we can unambiguouslydemonstrate that deadenylation precedes decay of theRNA (Fig+ 5A) and decapping of the RNA occurs last(Fig+ 5B)+Quantitation of the adenylated, unadenylated,and decapped products at each time point allowed us tofollow the turnover pathway for the a39UTR+ The use ofuniformly labeled substrate RNA resulted in an identicaldeadenylation and decay pattern, suggesting that thedeadenylation-dependent decapping and decay path-way is minor under these assay conditions+ These datademonstrate that the decay of a natural mRNA also oc-curs through a decay pathway similar to that previouslyreported for a generic RNA, where deadenylation pro-ceeds via 39-to-59 decay of the RNA by the exosome fol-lowed by DcpS decapping+

The data presented in Figure 5 also demonstrate thatthere is an exchange step between deadenylation and

decay of the RNA body as determined by the detectionof deadenylated product+ The accumulation of the de-adenylated RNA is usually not detected unless the de-adenylation step is accentuated by either the removalof PABP (addition of poly(A) competitor or poly(A) de-pletion) or sequestration of the aCP proteins with oligo-(dC), which disrupts the interaction with PABP andresults in an increase of deadenylation of the a-globinRNA (Wang et al+, 1999; Wang & Kiledjian, 2000b)+ Ittherefore seems that there is a hand-off between thetwo steps where prominent deadenylation activity ismediated by PARN (Gao et al+, 2000; our unpubl+ ob-servations), followed by the 39-to-59 decay by the exo-some+ It appears that normally, deadenylation is therate-limiting step and the subsequent 39-to-59 decayrapidly clears the RNA+ However, when the rate of de-adenylation is increased,more of the unadenylated RNAaccumulates, indicating that deadenylation and RNAdecay can be uncoupled+

We have previously reported that the a39UTR is thesubstrate of a endoribonuclease called ErEN (Wang &Kiledjian, 2000a)+ However, the contribution of ErEN toa-globin turnover appears to be minor under these as-say conditions+ Displacement of the a-complex is re-quired to observe cleavage by ErEN and even underthese conditions ErEN activity is limited+ Following ErENcleavage, 39-to-59 decay intermediates can be ob-served, suggesting that the RNA is degraded in the39-to-59 direction (Wang & Kiledjian, 2000a) and shouldbe a substrate for DcpS+ It is possible that under spe-cific cellular conditions or during differentiation of redblood cells, ErEN activity may increase and may berequired for a-globin turnover+

This report confirms that the a39UTR is an auto-nomous stability element both in vivo and in vitro, andthat the turnover of a-globin mRNA is a cytoplasmicevent that further validates our use of cytosolic extractin the in vitro decay system+We have also been able toelucidate the major mechanism of decay for the a39UTR,which proceeds through deadenylation, followed byexosome-mediated decay and decapping+ Future ef-forts will focus on the transition between deadenylationand 39-to-59 decay as well as how the a-complex influ-ences the exosome+

MATERIALS AND METHODS

Plasmid constructs

pSV2AL-awt (Luc-awt) was constructed by PCR amplifyingthe a39UTR from pSV2neoa2 (Weiss et al+, 1990) with prim-ers that introduced a BamHI restriction site at the 59 end andan EcoRI restriction site at the 39 end+ This PCR fragmentwas used to replace the SV40 39UTR (nt ;1811–3423) fromthe plasmid pSV2ALD59 at the same sites (de Wet et al+,1987)+ pSV2AL-aD9–21 (Luc-aDmt) and pSV2AL-Ha19 weresimilarly constructed using pSV2AneoaD9–21 and pSV2Aneo-




aH19+ pSV2Aneo-aH19 has been previously described (Weiss& Liebhaber, 1995)+ pSV2AneoaD9–21 contains the a39UTRwith a 33-nt deletion corresponding to codons 9 through 21according to the nomenclature of Weiss and Liebhaber (1995)+The plasmid was generated by joining the HindIII site of mu-tants aH9 and aH21 (Weiss & Liebhaber, 1995)+ The 59 por-tion of the a39UTR was excised from aH9 with BstEII andHindIII and used to replace the 59 portion of aH21 at the samesites to link codons 9 to 21+ pcDNA3-LucRP was generatedby ligating a 453-nt fragment, corresponding to the 39 end ofluciferase, obtained from an EcoRI and EcoRV digestion ofpSV2ALD59, into the pcDNA3 vector (Invitrogen)+ PlasmidpSV-b-gal expresses b-galactosidase and was obtained fromPromega+ The pSV2Aneo plasmid containing the neomycinresistance gene has previously been reported (Kadesch &Berg, 1986) as has the pGEM4-Neo283 to generate the neo-mycin resistance gene riboprobe (Kiledjian & Kadesch, 1990)+The pcP-CRE and pcP-mtCRE constructs were made by an-nealing complementary oligonucleotides corresponding to ei-ther the wild-type CRE (59-AATTCCGATGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACC and 59-TCGAGGTGCAAGGAGGGGAGGAGGGCCCGTTGGGAGGCCCATCGG)or a mutant CRE (59-AATTCCGATGGGGATGAGAACGGGGAGACGACGAGTACTTGCAGA and 59-TCGATCTGCAAGTACTCGTCGTCTCCCCGTTCTCATCCCCATCGG)+ Theoligonucleotides also contained EcoRI and XhoI overhangs+The annealed products were phosphorylated with T4 Poly-nucleotide Kinase (NEB), and were inserted into pcDNA3that was digested with EcoRI and XhoI restriction enzymes+

RT-PCR

RNA was reverse transcribed using the 1st Strand Synthesiskit according to the manufacturer (Stratagene)+ c-myc wasamplified from 1+5 mL of RT product using Taq polymerase(Promega) in 10 mM Tris:HCl, pH 8+3, 50 mM KCl, and 3 mMmagnesium chloride with 0+2 mM each of dATP, dGTP, anddTTP, 50 mM dCTP, and the following primers: 59-CTCTCCTTCCTCGGACTCGCTG-39 and 59-GTTGTTGCTGATCTGCTTCAGG-39 for 18 cycles at 92 8C for 30 s, 60 8C for 30 s,and 72 8C for 30 s, after which 7 mCi of [a-32P]dCTP wasadded for 3 additional cycles+ b-globinmaj was PCR amplifiedsimilarly with the following primers: 59-GCTGCTGTCTCTTGCCTGTG-39 and 59-CTGAAGTTCTCAGGATCCAC-39 for 18cycles at 92 8C for 30 s, 59 8C for 30 s, and 72 8C for 30 s,after which 7 mCi of [a-32P]dCTP was added for 3 additionalcycles+ A portion of the PCR sample was then resolved on a6% acrylamide-7 M urea gel and the dried gel was exposedto Kodak BioMax film+ The U6 RNA was specifically reversetranscribed by using a 32P-end-labeled oligonucleotide spe-cific for U6 (59-GCTAATATTCTCTGTATCG-39) and using0+25 mg of either cytoplasmic or nuclear RNA+

Cell culture and transfections

Murine erythroleukemia (MEL) cells were grown in Dulbec-co’s Modified Eagle Medium (D-MEM) supplemented with10% fetal bovine serum containing 100 U/mL penicillin and100 mg/mL streptomycin+ Cells were grown at 37 8C in a 5%CO2 incubator+ Transfections were carried out with a total of4 mg of DNA+ Two micrograms of each plasmid, pSV2-b-gal

and pSV2AL-aWT or pSV2AL-aD9–21 were used for proteinassays and 3 mg of pSV2ALD59, pSV2AL-aWT, pSV2A-aH19

or pSV2AL-aD9–21 and 1 mg of pSV2Aneo were used forexperiments where RNA was to be extracted from cells+ TheDNA was mixed with 12 mL of TransFast reagent (Promega)in 500 mL of serum-free D-MEM+ This mixture was incubatedfor 15 min at room temperature before addition to 1 3 106

cells that had been washed twice with serum-free D-MEMand resuspended in 500 mL serum-free D-MEM+ Cells werethen incubated at 37 8C in a 5% CO2 incubator for 3 h prior tothe addition of 5 mL of complete media+ For protein assays,the cells were collected after 24 h and treated as describedbelow+ For RNA extraction, three individual transfections werepooled after 24 h and one-third of the cells were collected asthe zero time point+ The remaining cells were treated with5 mg/mL actinomycin D for 4 and 8 h+

b-galactosidase and Luciferase assays

The b-galactosidase Enzyme Assay System was used forb-galactosidase assays as described by the manufacturer(Promega) except cells were lysed in 300 mL of 13 ReporterLysis Buffer and 40 mL of extract was used in the assays+Luciferase assays were carried out using the Luciferase As-say System according to the manufacturer’s instructions (Pro-mega) and 15 mL of extract were used in the assays+All of theassays were done in duplicate+

Cell fractionation and RNA isolation

Cells, ;2–3 3 106, were resuspended in 300 mL of RSB-100(10 mM Tris:HCl, pH 7+4, 100 mM sodium chloride, 2+5 mMmagnesium chloride, and 0+5% Triton X-100) by passing thesolution through a 1-cc insulin syringe three times+ The intactnuclei were pelleted with a quick spin and the supernatantwas transferred to a tube containing 800 mL of TRIzolT re-agent (Gibco-BRL)+ This was repeated once more, and 800 mLof TRIzol reagent was added to the nuclei pellet+ The RNAwas extracted with 200 mL of chloroform/1 mL TRIzol re-agent, precipitated with isopropanol and washed with 75%ethanol+ The RNA pellet was air-dried and resuspended inDEPC-H2O+

RNA synthesis and RNase protection assays

Template for the luciferase riboprobe was generated bylinearizing the plasmid pcDNA3-LucRP with XhoI+ The neo-mycin resistance gene riboprobe was generated from thepGEM4-Neo283 plasmid linearized with EcoRI+ Five hundrednanograms of these templates were used to produce uni-formly labeled riboprobes with T7 polymerase (Promega) ac-cording to the manufacturer’s protocol, in the presence of[a-32P]UTP+ The pcP-CRE and pcP-mtCRE RNA was tran-scribed by T7 RNA polymerase from a PCR-generatedtemplate using the T7 and SP6 promoter primers+ The a39UTRwas PCR amplified with primers that introduce a T7bacteriophage promoter at the 59 end and 60 adenylatenucleotides at the 39 end for adenylated probes and hasbeen previously described (Wang et al+, 1999)+ Uniformlylabeled transcripts were generated in the presence ofm7G(59)ppp(59)G cap analog (Pharmacia) as described pre-




viously (Wang et al+, 1999)+ Cap-labeled RNAs were gener-ated and gel purified as described by Wang and Kiledjian(2000a)+ RNase protection assays were carried out as de-scribed by Kiledjian and Kadesch (1990) using the luciferaseand neomycin resistance riboprobes described above+ Theprotected RNA was resolved on a 6% denaturing polyacryl-amide-7 M urea gel, dried, and exposed on Kodak BioMaxfilm+ Quantitation was carried out using a Molecular Dynam-ics PhosphorImager and the ImageQuant software+

In vitro mRNA decay assays

Preparation of K562 S130 extract and in vitro mRNA decayassays were performed as described by Wang et al+ (1999)+Reactions were carried out with 0+1 pmol (103 cpm) of RNAand incubated with 50 or 10 mg of K562 S130 extract or15 mg of the depleted extracts in IVDA buffer (10 mMTris:HCl, pH 7+5, 100 mM potassium acetate, 2 mM magne-sium acetate, 2 mM DTT, 10 mM creatine phosphate, 1 mMATP, 0+4 mM GTP, and 0+1 mM spermine) for the indicatedtimes at 37 8C+ For the reactions in Figure 5 containing thea39UTR, 0+75 pmol of thioated oligo (dC16) were included tosequester the aCP proteins and accelerate a-globin mRNAdeadenylation (Wang et al+, 1999)+ Reactions were stoppedwith the addition of 150 mL of ULB (7 M urea, 2% SDS,0+35 M sodium chloride, 10 mM EDTA, and 10 mM Tris:HCl,pH 7+5), containing a 32P-labeled oligonucleotide that wasused as an internal control for quantitation+ The RNA wasresolved on an 8% denaturing polyacrylamide-7 M urea gel,following phenol-chloroform extraction, ethanol precipitationwith 15 mg of glycogen carrier, and resuspension in 80%formamide dye+ The dried gel was exposed to Kodak BioMaxfilm and quantitations were carried out on a Molecular Dy-namics PhosphorImager using the ImageQuant software+

Immunoprecipitation

Immunoprecipitations were carried out with 15 mL of H77control antisera or 7+5 mL of each antisera against hRrp40and hRrp46 bound to 40 mL of protein A beads+ K562 S130extract (300 mg) was incubated in PBS/0+25% Tween-20 forthree rounds of 1-h incubations at 4 8C with the antibody-coupled beads or beads alone for the mock depletion+ Thedepleted extract was dialyzed against buffer A (10 mM Tris,pH 7+5, 1 mM potassium acetate, 1+5 mM magnesium ace-tate, 2 mM DTT) and used in decay assays and for westernanalysis+

Western analysis

Western analysis was carried out as described previously(Wang et al+, 1999)+ The hRrp40 protein was detected by arabbit anti-hRrp40 antibody, which was generated against aHis-tagged fusion protein (Brouwer et al+, 2001)+ A 1:800dilution of the primary antibody was used and visualizedby enhanced chemiluminescence using a horseradishperoxidase-conjugated goat anti-rabbit secondary antibody(1:10,000 dilution; Jackson ImmunoResearch Laboratories,Inc+)+

ACKNOWLEDGMENTS

We thank G+J+M+ Pruijin and R+ Raijmakers for the exosomeantisera and A+ Carr-Schmid for critical reading of the manu-script+ This work was supported by funds from the NationalInstitutes of Health DK51611 to M+K+

Received June 27, 2002; returned for revisionAugust 8, 2002; revised manuscript receivedSeptember 12, 2002

REFERENCES

Beelman CA, Stevens A, Caponigro G, LaGrandeur TE, Hatfield L,Fortner DM, Parker R+ 1996+ An essential component of the de-capping enzyme required for normal rates of mRNA turnover+Nature 382:642–646+

Bernstein P, Peltz SW, Ross J+ 1989+ The poly(A)-poly(A)-bindingprotein complex is a major determinant of mRNA stability in vitro+Mol Cell Biol 9:659–670+

Bousquet-Antonelli C, Presutti C, Tollervey D+ 2000+ Identification ofa regulated pathway for nuclear pre-mRNA turnover+ Cell 102:765–775+

Brouwer R, Allmang C, Raijmakers R, van Aarssen Y, Egberts WV,Petfalski E, van Venrooij WJ, Tollervey D, Pruijn GJ+ 2001+ Threenovel components of the human exosome+ J Biol Chem 276:6177–6184+

Caponigro G, Parker R+ 1996+ Mechanisms and control of mRNAturnover in Saccharomyces cerevisiae+Microbiol Rev 60:233–249+

Chen CY, Gherzi R, Ong SE, Chan EL, Raijmakers R, Pruijn GJ,Stoecklin G, Moroni C, Mann M, Karin M+ 2001+ AU binding pro-teins recruit the exosome to degrade ARE-containing mRNAs+Cell 107:451–464+

Chen CY, Shyu AB+ 1995+ AU-rich elements: Characterization andimportance in mRNA degradation+ Trends Biochem Sci 20:465–470+

Chkheidze AN, Lyakhov DL, Makeyev AV, Morales J, Kong J, Lieb-haber SA+ 1999+ Assembly of the alpha-globin mRNA stabilitycomplex reflects binary interaction between the pyrimidine-rich 39untranslated region determinant and poly(C) binding proteinalphaCP+ Mol Cell Biol 19:4572–4581+

Coller JM,Gray NK,Wickens MP+ 1998+mRNA stabilization by poly(A)binding protein is independent of poly(A) and requires translation+Genes & Dev 12:3226–3235+

Czyzyk-Krzeska MF, Bendixen AC+ 1999+ Identification of the poly(C)binding protein in the complex associated with the 39 untranslatedregion of erythropoietin messenger RNA+ Blood 93:2111–2120+

Dani C, Blanchard JM, Piechaczyk M, El Sabouty S, Marty L, Jean-teur P+ 1984+ Extreme instability of myc mRNA in normal andtransformed human cells+ Proc Natl Acad Sci USA 81:7046–7050+

Decker CJ, Parker R+ 1994+ Mechanisms of mRNA degradation ineukaryotes+ Trends Biochem Sci 19:336–340+

Decker CJ, Parker R+ 1995+ Diversity of cytoplasmic functions for the39 untranslated region of eukaryotic transcripts+ Curr Opin CellBiol 7:386–392+

Dehlin E,Wormington M, Korner CG,Wahle E+ 2000+ Cap-dependentdeadenylation of mRNA+ EMBO J 19:1079–1086+

de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S+ 1987+Firefly luciferase gene: Structure and expression in mammaliancells+ Mol Cell Biol 7:725–737+

Ford LP, Bagga PS, Wilusz J+ 1997+ The poly(A) tail inhibits theassembly of a 39-to-59 exonuclease in an in vitro RNA stabilitysystem+ Mol Cell Biol 17:398–406+

Ford LP, Wilusz J+ 1999+ 39-Terminal RNA structures and poly(U)tracts inhibit initiation by a 39 r 59 exonuclease in vitro+ NucleicAcids Res 27:1159–1167+

Gao M, Fritz DT, Ford LP, Wilusz J+ 2000+ Interaction between apoly(A)-specific ribonuclease and the 59 cap influences mRNAdeadenylation rates in vitro+ Mol Cell 5:479–488+

Holcik M, Liebhaber SA+ 1997+ Four highly stable eukaryotic mRNAsassemble 39 untranslated region RNA–protein complexes shar-




ing cis and trans components+ Proc Natl Acad Sci USA 94:2410–2414+

Jackson RJ+ 1993+ Cytoplasmic regulation of mRNA function: Theimportance of the 39 untranslated region+ Cell 74:9–14+

Jacobs JS, Anderson AR, Parker RP+ 1998+ The 39 to 59 degradationof yeast mRNAs is a general mechanism for mRNA turnover thatrequires the SKI2 DEVH box protein and 39 to 59 exonucleases ofthe exosome complex+ EMBO J 17:1497–1506+

Kadesch T, Berg P+ 1986+ Effects of the position of the simian virus 40enhancer on expression of multiple transcription units in a singleplasmid+ Mol Cell Biol 6:2593–2601+

Kiledjian M, Day N, Trifillis P+ 1999+ Purification and RNA bindingproperties of the polycytidylate-binding proteins alphaCP1 andalphaCP2+ Methods 17:84–91+

Kiledjian M, DeMaria CT, Brewer G, Novick K+ 1997+ Identification ofAUF1 (heterogeneous nuclear ribonucleoprotein D) as a compo-nent of the alpha-globin mRNA stability complex+ Mol Cell Biol17:4870–4876+

Kiledjian M, Kadesch T+ 1990+ Analysis of the human liver/bone/kidney alkaline phosphatase promoter in vivo and in vitro+ NucleicAcids Res 18:957–961+

Kiledjian M, Wang X, Liebhaber SA+ 1995+ Identification of two KHdomain proteins in the alpha-globin mRNP stability complex+EMBOJ 14:4357–4364+

Korner CG, Wahle E+ 1997+ Poly(A) tail shortening by a mammalianpoly(A)-specific 39-exoribonuclease+ J Biol Chem 272:10448–10456+

Korner CG,Wormington M, Muckenthaler M, Schneider S, Dehlin E,Wahle E+ 1998+ The deadenylating nuclease (DAN) is involved inpoly(A) tail removal during the meiotic maturation of Xenopusoocytes+ EMBO J 17:5427–5437+

LaGrandeur TE, Parker R+ 1998+ Isolation and characterization ofDcp1p, the yeast mRNA decapping enzyme+ EMBO J 17:1487–1496+

Larimer FW, Hsu CL, Maupin MK, Stevens A+ 1992+ Characterizationof the XRN1 gene encoding a 59 r 39 exoribonuclease: Se-quence data and analysis of disparate protein and mRNA levelsof gene-disrupted yeast cells+ Gene 120:51–57+

Leffers H, Dejgaard K, Celis JE+ 1995+ Characterisation of two majorcellular poly(rC)-binding human proteins, each containing threeK-homologous (KH) domains+ Eur J Biochem 230:447–453+

Liu H, Rodgers ND, Jiao X, Kiledjian M+ 2002+ The Scavenger mRNAdecapping enzyme, DcpS, is a member of the HIT family of py-rophosphatases+ EMBO J 21:4699–4708+

Lodish HF, Small B+ 1976+ Different lifetimes of reticulocyte messen-ger RNA+ Cell 7:59–65+

Makeyev AV, Liebhaber SA+ 2002+ The poly(C)-binding proteins: Amultiplicity of functions and a search for mechanisms+ RNA8:265–278+

Mitchell P, Petfalski E, Shevchenko A, Mann M, Tollervey D+ 1997+The exosome: A conserved eukaryotic RNA processing complexcontaining multiple 39r59 exoribonucleases+ Cell 91:457–466+

Morales J, Russell JE, Liebhaber SA+ 1997+ Destabilization of humanalpha-globin mRNA by translation anti-termination is controlledduring erythroid differentiation and is paralleled by phased short-ening of the poly(A) tail+ J Biol Chem 272:6607–6613+

Muhlrad D, Decker CJ, Parker R+ 1995+ Turnover mechanisms of thestable yeast PGK1 mRNA+ Mol Cell Biol 15:2145–2156+

Mukherjee D, Gao M, O’Connor JP, Raijmakers R, Pruijn G, Lutz CS,Wilusz J+ 2002+ The mammalian exosome mediates the efficientdegradation of mRNAs that contain AU-rich elements+ EMBO J21:165–174+

Papayannopoulou T,Abkowitz J, D’Andrea A+ 2000+ Biology of eryth-ropoiesis, erythroid differentiation, and maturation+ In: HoffmanR, Benz, EJ, Shattil, SJ, Furie, B, Cohen, HJ, Silberstein, LE,McGlave P, eds+ Hematology: Basic principles and practice. NewYork: Churchill Livingstone+ pp 202–219+

Paulding WR, Czyzyk-Krzeska MF+ 1999+ Regulation of tyrosinehydroxylase mRNA stability by protein-binding, pyrimidine-richsequence in the 39-untranslated region+ J Biol Chem 274:2532–2538+

Rodgers ND, Wang Z, Kiledjian M+ 2002+ Characterization and puri-

fication of a mammalian endoribonuclease specific for the alpha-globin mRNA+ J Biol Chem 277:2597–2604+

Ross J+ 1995+ mRNA stability in mammalian cells+ Microbiol Rev59:423–450+

Ross J+ 1996+ Control of messenger RNA stability in higher eukary-otes+ Trends Genet 12:171–175+

Ross J, Sullivan TD+ 1985+ Half-lives of beta and gamma globinmessenger RNAs and of protein synthetic capacity in culturedhuman reticulocytes+ Blood 66:1149–1154+

Russell JE, Liebhaber SA+ 1996+ The stability of human beta-globinmRNA is dependent on structural determinants positioned withinits 39 untranslated region+ Blood 87:5314–5323+

Russell JE, Morales J, Liebhaber SA+ 1997+ The role of mRNA sta-bility in the control of globin gene expression+ Prog Nucleic AcidRes Mol Biol 57:249–287+

Sachs AB+ 1993+ Messenger RNA degradation in eukaryotes+ Cell74:413–421+

Sachs AB, Sarnow P, Hentze MW+ 1997+ Starting at the beginning,middle, and end: Translation initiation in eukaryotes+ Cell 89:831–838+

Shatkin AJ+ 1985+ mRNA cap binding proteins: Essential factors forinitiating translation+ Cell 40:223–224+

Shyu AB, Belasco JG, Greenberg ME+ 1991+ Two distinct destabiliz-ing elements in the c-fos message trigger deadenylation as a firststep in rapid mRNA decay+ Genes & Dev 5:221–231+

Stefanovic B, Hellerbrand C, Holcik M, Briendl M, Liebhaber SA,Brenner DA+ 1997+ Posttranscriptional regulation of collagenalpha1(I) mRNA in hepatic stellate cells+ Mol Cell Biol 17:5201–5209+

Trifillis P, Day N, Kiledjian M+ 1999+ Finding the right RNA: Identifi-cation of cellular mRNA substrates for RNA-binding proteins+RNA5:1071–1082+

Volloch V, Housman D+ 1981+ Stability of globin mRNA in terminallydifferentiating murine erythroleukemia cells+ Cell 23:509–514+

Wang X, Kiledjian M, Weiss IM, Liebhaber SA+ 1995+ Detection andcharacterization of a 39 untranslated region ribonucleoprotein com-plex associated with human alpha-globin mRNA stability+Mol CellBiol 15:1769–1777+

Wang Z, Day N, Trifillis P, Kiledjian M+ 1999+ An mRNA stability com-plex functions with poly(A)-binding protein to stabilize mRNA invitro+ Mol Cell Biol 19:4552–4560+

Wang Z, Jiao X, Carr-Schmid A, Kiledjian M+ 2002+ The hDcp2 pro-tein is a mammalian mRNA decapping enzyme+ Proc Natl AcadSci USA 99:12663–12668+

Wang Z, Kiledjian M+ 2000a+ Identification of an erythroid-enrichedendoribonuclease activity involved in specific mRNA cleavage+EMBO J 19:295–305+

Wang Z, Kiledjian M+ 2000b+ The poly(A)-binding protein and anmRNA stability protein jointly regulate an endoribonuclease ac-tivity+ Mol Cell Biol 20:6334–6341+

Wang Z, Kiledjian M+ 2001+ Functional link between the mammalianexosome and mRNA decapping+ Cell 107:751–762+

Weiss I, Cash FE, Coleman MB, Pressley A, Adams JG, San-guansermsri T, Liebhaber SA, Steinberg MH+ 1990+ Molecularbasis for alpha-thalassemia associated with the structural mutanthemoglobin Suan-Dok (alpha 2109leu–arg)+ Blood 76:2630–2636+

Weiss IM, Liebhaber SA+ 1994+ Erythroid cell-specific determinantsof alpha-globin mRNA stability+ Mol Cell Biol 14:8123–8132+

Weiss IM, Liebhaber SA+ 1995+ Erythroid cell-specific mRNA stabilityelements in the alpha 2-globin 39 nontranslated region+ Mol CellBiol 15:2457–2465+

Wickens M+ 1990+ In the beginning is the end: Regulation of poly(A)addition and removal during early development+ Trends BiochemSci 15:320–324+

Wilson T, Treisman R+ 1988+ Removal of poly(A) and consequentdegradation of c-fos mRNA facilitated by 39 AU-rich sequences+Nature 336:396–399+

Wood KV+ 1995+ Marker proteins for gene expression+ Curr OpinBiotechnol 6:50–58+

Yu J,Russell JE+ 2001+ Structural and functional analysis of an mRNPcomplex that mediates the high stability of human beta-globinmRNA+ Mol Cell Biol 21:5879–5888+




Documents

proceeding through 3'-to-5' exosome-dependent decapping