HSUR

Abstract: Targeted mRNA instability represents an important mode of post-transcriptional gene regulation in eukaryotic cells. Much attention has focused on AU-rich elements (AREs) that are present in the 3′-untranslated regions (3′ UTRs) of the mRNAs from many early response genes (ERGs), including cytokines, lymphokines, and proto-oncogenes (Caput et al. 1986; Shaw and Kamen 1988; for review, see Belasco and Brawerman 1993; Chen and Shyu 1995). The expression of ERGs is both sensitive to extracellular stimuli and transient, requiring rapid mRNA removal through destabilization following the cessation of transcription. The ARE is a major determinant for the rapid degradation of these mRNAs. Insertion of a 51-nucleotide AU-rich sequence derived from the GM-CSF (granulocyte–macrophage colony-stimulating factor) into the 3′ UTR of β-globin mRNA greatly shortens its normally long half-life (Shaw and Kamen 1988). Conversely, removal of the ARE stabilizes the labile proto-oncogene c-fos mRNA and confers a transforming phenotype (Miller et al. 1984; Meijlink et al. 1985; W. Lee et al. 1988). Depending on cell type, there appear to be different pathways regulating ARE-mediated mRNA decay. For instance, in a monocyte tumor cell line, c-fos mRNA is degraded constitutively, whereas the GM-CSF mRNA is stable (Schuler and Cole 1988). GM-CSF and interleukin-3 (IL-3) mRNAs have also been reported to be selectively stabilized in a human T cell line upon anti-CD28 antibody treatment (Lindsten et al. 1989). The AREs in ERG mRNAs contain multiple copies of the sequence AUUUA (Caput et al. 1986; Shaw and Kamen 1988; for review, see Belasco and Brawerman 1993; Chen and Shyu 1995). Using a serum-inducible c-fos promoter and a reporter gene system in transiently transfected NIH-3T3 cells, two groups have performed extensive mutagenesis to identify the minimum sequence that directs mRNA degradation. Both found that an isolated single AUUUA is not active, whereas an AUUUA flanked by two Us (UUAUUUAUU) has weak destabilization activity and two separate or overlapping copies of UUAUUUAUU are highly destabilizing (Lagnado et al. 1994; Zubiaga et al. 1995). Using the same serum-inducible promoter system, Shyu and coworkers provided evidence that ARE-mediated mRNA decay proceeds in a biphasic manner, with shortening of the poly(A) tail followed by a first order degradation of the remaining RNA (Shyu et al. 1991; Chen et al. 1995). Whether the latter step involves exo- or endonucleases is not yet established. Also unknown is whether ARE-mediated mRNA decay is obligatorily coupled to ongoing translation, because evidence both in favor of and against this linkage has been reported (Koeller et al. 1991; Savant-Bhonsale and Cleveland 1992; Aharon and Schneider 1993; Chen et al. 1995; Curatola et al. 1995; Winstall et al. 1995; see below). Although many proteins have been charaterized as factors recognizing AREs (Malter 1989; Bohjanen et al. 1991, 1992; Brewer 1991; Malter and Hong 1991; Vakalopoulou et al. 1991; Hamilton et al. 1993; Zhang et al. 1993; Katz et al. 1994; Nakagawa et al. 1995), we have concentrated on an apparent 32-kD protein, first identified by cross-linking to the c-fos ARE in HeLa cell extracts (Vakalopoulou et al. 1991). This protein also binds to several of the seven small nuclear RNAs (snRNAs) that are highly expressed in marmoset T cells transformed by Herpesvirus saimiri (Myer et al. 1992). These H. saimiri U RNAs (HSURs 1–7) are similar to cellular U RNAs in that they are transcribed by RNA polymerase II, possess 5′ trimethylated guanosine caps at their 5′ ends, and assemble into small nuclear ribonucleoprotein particles (snRNPs) of the Sm class (Murthy et al. 1986; S. Lee et al. 1988; Wassarman et al. 1989; Lee and Steitz 1990; Albrecht and Fleckenstein 1992). At their 5′ ends, HSUR 1, HSUR 2, and HSUR 5 contain four, two, and one copies of AUUUA, respectively. Characterization of the purified 32-kD protein demonstrated that it is identical to HuR (Myer et al. 1997), a ubiquitously expressed member of the ELAV family (Embryonic Lethal, Abnormal Vision; Ma et al. 1996). Like other ELAV proteins, HuR contains three RNA recognition motifs (RRMs) and binds in vitro to ARE sequences (Levine et al. 1993; Gao et al. 1994; Ma et al. 1996; Myer et al. 1997). Detailed analyses revealed a direct correlation between the in vitro affinity of an ARE sequence for HuR and its ability to direct in vivo degradation of a reporter mRNA, suggesting the involvement of HuR in the destabilization (or stabilization) of ARE-containing mRNAs (Myer et al. 1997). We set out to test a model whereby the HSURs function to stabilize mRNAs normally targeted for rapid degradation by competitively binding cellular components of the mRNA degradation machinery (Myer et al. 1992). By transiently expressing HSUR 1, which has a strong affinity for HuR (Myer et al. 1992), in mouse L929 cells, we expected to observe elevated levels of ARE-containing mRNAs. Instead, we discovered that wild-type HSUR 1 is expressed at a much lower level than a mutant HSUR 1 with its AUUUA repeats converted to AGGUA, a mutation already known to stabilize mRNAs (Shaw and Kamen 1988; Vakalopoulou et al. 1991; Myer et al. 1997). After demonstrating that this is a post-transcriptional phenomenon, we have gone on to show that the HSUR 1 ARE can target the in vivo degradation of other snRNAs, as well as a reporter mRNA. Mutational analyses of the viral ARE reveal a correlation between binding of the HuR protein and in vivo degradation. We discuss the relevance of our findings to current understanding of the mechanism of ARE-mediated mRNA degradation and of the biological functioning of HSUR 1.

Abstract: EBER 1, a small noncoding viral RNA abundantly expressed in all cells transformed by Epstein-Barr virus (EBV), has been shown to associate with the human ribosomal protein L22. Here we present in vitro binding studies using purified RNAs and recombinant proteins. Electrophoretic mobility-shift assays (EMSAs) show that recombinant L22 (rL22) and maltose-binding protein (MBP)-tagged L22 protein bind EBER 1 in vitro, both forming three specific protein-dependent mobility shifts. Use of a mixture of rL22 and MBP-L22 indicates that these three shifts contain one, two, or three L22 proteins per EBER 1 molecule. EMSAs performed with EBER 1 deletion constructs and EBER 1 stem-loops inserted into a nonbinding RNA, HSUR 3, identify stem-loops I, III, and IV as L22 binding sites. The existence of multiple L22 binding sites on EBER 1 inside cells is demonstrated by in vivo UV cross-linking. Our results are discussed with respect to the function of EBER 1 in EBV-infected human B cells.