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AU-Rich Elements Target Small Nuclear Rnas As Well As Mrnas for Rapid Degradation

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AU-Rich Elements Target Small Nuclear Rnas As Well As Mrnas for Rapid Degradation Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation Xinhao Cynthia Fan, Vic E. Myer,1 and Joan A. Steitz2 Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut 06520 USA AU-rich elements (AREs, usually containing repeated copies of AUUUA), when present in the 3*-untranslated regions (UTRs) of many mammalian mRNAs, confer instability on their host RNA molecules. The viral small nuclear RNA (snRNA) Herpesvirus saimiri U RNA 1 (HSUR 1) also contains an AUUUA-rich sequence. Here, we report that this ARE induces rapid degradation of HSUR 1 itself and of other snRNAs including HSUR 2 and cellular U1. Mutational analyses of the viral ARE establish that sequence requirements for mRNA and snRNA decay are the same, suggesting a similar mechanism. Moreover, the in vivo degradation activity of mutant AREs correlates with their in vitro binding to the HuR protein, implicated previously as a component of the mRNA degradation machinery. Our results suggest that ARE-mediated instability can be uncoupled from both ongoing translation and deadenylation of the target RNA. [Key Words: ARE; HSUR 1; RNA degradation; HuR; Herpesvirus saimiri] Received June 5, 1997; revised version accepted August 4, 1997. Targeted mRNA instability represents an important interleukin-3 (IL-3) mRNAs have also been reported to mode of post-transcriptional gene regulation in eukary- be selectively stabilized in a human T cell line upon otic cells. Much attention has focused on AU-rich ele- anti-CD28 antibody treatment (Lindsten et al. 1989). ments (AREs) that are present in the 38-untranslated re- The AREs in ERG mRNAs contain multiple copies of gions (38 UTRs) of the mRNAs from many early response the sequence AUUUA (Caput et al. 1986; Shaw and Ka- genes (ERGs), including cytokines, lymphokines, and men 1988; for review, see Belasco and Brawerman 1993; proto-oncogenes (Caput et al. 1986; Shaw and Kamen Chen and Shyu 1995). Using a serum-inducible c-fos pro- 1988; for review, see Belasco and Brawerman 1993; Chen moter and a reporter gene system in transiently trans- and Shyu 1995). The expression of ERGs is both sensitive fected NIH-3T3 cells, two groups have performed exten- to extracellular stimuli and transient, requiring rapid sive mutagenesis to identify the minimum sequence mRNA removal through destabilization following the that directs mRNA degradation. Both found that an iso- cessation of transcription. The ARE is a major determi- lated single AUUUA is not active, whereas an AUUUA nant for the rapid degradation of these mRNAs. Insertion flanked by two Us (UUAUUUAUU) has weak destabili- of a 51-nucleotide AU-rich sequence derived from the zation activity and two separate or overlapping copies of GM-CSF (granulocyte–macrophage colony-stimulating UUAUUUAUU are highly destabilizing (Lagnado et al. factor) into the 38 UTR of b-globin mRNA greatly short- 1994; Zubiaga et al. 1995). Using the same serum-induc- ens its normally long half-life (Shaw and Kamen 1988). ible promoter system, Shyu and coworkers provided evi- Conversely, removal of the ARE stabilizes the labile dence that ARE-mediated mRNA decay proceeds in a proto-oncogene c-fos mRNA and confers a transforming biphasic manner, with shortening of the poly(A) tail fol- phenotype (Miller et al. 1984; Meijlink et al. 1985; W. lowed by a first order degradation of the remaining RNA Lee et al. 1988). Depending on cell type, there appear to (Shyu et al. 1991; Chen et al. 1995). Whether the latter be different pathways regulating ARE-mediated mRNA step involves exo- or endonucleases is not yet estab- decay. For instance, in a monocyte tumor cell line, c-fos lished. Also unknown is whether ARE-mediated mRNA mRNA is degraded constitutively, whereas the GM-CSF decay is obligatorily coupled to ongoing translation, be- mRNA is stable (Schuler and Cole 1988). GM-CSF and cause evidence both in favor of and against this linkage has been reported (Koeller et al. 1991; Savant-Bhonsale 1Present address: Whitehead Institute for Biomedical Research, 9 Cam- and Cleveland 1992; Aharon and Schneider 1993; Chen bridge Center, Cambridge, Massachusetts 02142 USA. 2Corresponding author. et al. 1995; Curatola et al. 1995; Winstall et al. 1995; see E-MAIL [email protected]; FAX (203) 624-8213. below). GENES & DEVELOPMENT 11:2557–2568 © 1997 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/97 $5.00 2557 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Fan et al. Although many proteins have been charaterized as fac- AGGUA, an alteration that is known both to stabilize a tors recognizing AREs (Malter 1989; Bohjanen et al. b-globin reporter mRNA in NIH-3T3 cells and to abolish 1991, 1992; Brewer 1991; Malter and Hong 1991; Vakalo- binding to HuR (Myer et al. 1997). The CUU sequence poulou et al. 1991; Hamilton et al. 1993; Zhang et al. that follows the ARE was also mutated (to CGG) in this 1993; Katz et al. 1994; Nakagawa et al. 1995), we have construct (Fig. 1A). HSUR promoters are similar to the concentrated on an apparent 32-kD protein, first identi- well-characterized U1 snRNA promoter (S. Lee et al. fied by cross-linking to the c-fos ARE in HeLa cell ex- 1988), whose transcription by RNA polymerase II is not tracts (Vakalopoulou et al. 1991). This protein also binds affected by internal coding sequences (Dahlberg and to several of the seven small nuclear RNAs (snRNAs) Lund 1988). To achieve comparable transcription rates, that are highly expressed in marmoset T cells trans- we cloned wild-type and mutant HSUR 1 (Mut), as well formed by Herpesvirus saimiri (Myer et al. 1992). These as HSUR 3 (which does not have an ARE and serves as a H. saimiri U RNAs (HSURs 1–7) are similar to cellular U transfection control; Fig. 1A), between the U1 promoter RNAs in that they are transcribed by RNA polymerase II, and 38 termination box in a pUC plasmid (Fig. 1A; Yuo possess 58 trimethylated guanosine caps at their 58 ends, and Weiner 1989). and assemble into small nuclear ribonucleoprotein par- HSURs were shown previously to be efficiently ex- ticles (snRNPs) of the Sm class (Murthy et al. 1986; S. pressed and assembled into Sm-precipitable snRNPs Lee et al. 1988; Wassarman et al. 1989; Lee and Steitz even in transiently transfected cells that do not serve as 1990; Albrecht and Fleckenstein 1992). At their 58 ends, hosts for H. saimiri (Lee and Steitz 1990). Murine L929 HSUR 1, HSUR 2, and HSUR 5 contain four, two, and cells were chosen for transient transfection in our study one copies of AUUUA, respectively. Characterization of because they have been used previously to study the purified 32-kD protein demonstrated that it is iden- AUUUA-mediated mRNA destabilization (Savant-Bhon- tical to HuR (Myer et al. 1997), a ubiquitously expressed sale and Cleveland 1992). Either wild-type or the mu- member of the ELAV family (Embryonic Lethal, Abnor- tant pUC–U1–HSUR 1 was cotransfected with pUC–U1– mal Vision; Ma et al. 1996). Like other ELAV proteins, HSUR 3 into L929 cells with DEAE–dextran. To monitor HuR contains three RNA recognition motifs (RRMs) and the transfection efficiency, a plasmid encoding b-galac- binds in vitro to ARE sequences (Levine et al. 1993; Gao tosidase was transfected in parallel. X-gal staining et al. 1994; Ma et al. 1996; Myer et al. 1997). Detailed showed that transfection efficiencies ranged between analyses revealed a direct correlation between the in 25% and 30% (data not shown). Total RNA was har- vitro affinity of an ARE sequence for HuR and its ability vested 45–50 hr following transfection and was analyzed to direct in vivo degradation of a reporter mRNA, sug- by T1 RNase protection. gesting the involvement of HuR in the destabilization (or We were surprised to observe that the wild-type HSUR stabilization) of ARE-containing mRNAs (Myer et al. 1 level (Fig. 1B, lane 2) was significantly lower (one- 1997). eighth) than that of the mutant (lane 3) when normalized We set out to test a model whereby the HSURs func- to the cotransfected control HSUR 3. Comparable results tion to stabilize mRNAs normally targeted for rapid deg- were obtained in other cell lines of human, monkey, or radation by competitively binding cellular components mouse origin; HeLa cells, HEK-293 cells, Jurkat cells, of the mRNA degradation machinery (Myer et al. 1992). COS cells, and NIH-3T3 cells all produced wild-type By transiently expressing HSUR 1, which has a strong HSUR 1 at one-sixth to one-tenth the level of the mutant affinity for HuR (Myer et al. 1992), in mouse L929 cells, (data not shown). we expected to observe elevated levels of ARE-contain- To determine whether the lower level of wild-type ing mRNAs. Instead, we discovered that wild-type compared with mutant HSUR 1 was attributable to HSUR 1 is expressed at a much lower level than a mu- greater instability or to some unexpected effect of the tant HSUR 1 with its AUUUA repeats converted to internal HSUR 1 sequence on promoter activity, we AGGUA, a mutation already known to stabilize mRNAs performed run-on transcription assays. Whole cell rath- (Shaw and Kamen 1988; Vakalopoulou et al. 1991; Myer er than the standard nuclear assay was used because et al. 1997). After demonstrating that this is a post-tran- of the low [a-32P]UTP incorporation into snRNAs in scriptional phenomenon, we have gone on to show that the nuclear system (Sheldon et al.
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