Multiple domains of EBER 1, an Epstein-Barr virus noncoding RNA, recruit human ribosomal protein L22

Abstract

EBER 1, asmall 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 1i nv itro, both forming three specific protein-dependent mobility shifts. Use of am ixture of rL22 and MBP-L22 indicates that these three shifts contain one, two, or three L22 proteins per EBER 1m olecule. EMSAs performed with EBER 1d eletion constructs and EBER 1s tem-loops inserted into an onbinding RNA, HSUR 3, identify stem-loops I, III, and IV as L22 binding sites. The existence of multiple L22 binding sites on EBER 1inside cells is demonstrated by in vivo UV cross-linking. Our results are discussed with respect to the function of EBER 1i nE BV-infected human Bc ells.

AUF1/hnRNP D is a novel protein partner of the EBER1 noncoding RNA of Epstein-Barr virus

“…Surprisingly, although EBER1 could be selected by an antisense oligonucleotide, La and L22, the best-characterized EBER1 interacting partners, were not coselected. The region of EBER1 we identified as a target for hybridization and RNase H cleavage overlaps with stemloop III, previously characterized as the major binding site for L22 (Toczyski and Steitz 1993;Fok et al 2006b). Whereas in prior immunoprecipitation experiments >95% of total EBER1 was precipitated by anti-L22 antibodies (Toczyski and Steitz 1993), oligonucleotide selection captured only 5% (Fig.…”

“…This behavior is reminiscent of RNP complex formation between EBER1 and L22, in which multiple stem-loops within EBER1 contribute to the RNA-protein interaction (Fok et al 2006b). AUF1 and L22, however, form distinctly different RNP complexes with EBER1, since single stem-loop deletions did not result in a decrease in the overall number of AUF1-EBER1 RNP complexes (data not shown), in contrast to L22 (Fok et al 2006b). EBER1 and the TNFa-ARE formed RNP complexes at comparable AUF1 concentrations (Fig.…”

“…ARE, EBER1, and VAI RNAs were in vitro transcribed by T7 polymerase and gel-purified from polyacrylamide gels according to the method previously described (Fok et al 2006b). Purified RNAs were alkaline-phosphatase-treated to remove the 59-phosphate, followed by end-labeling with [g-32 P]ATP using T4 polynucleotide kinase.…”

Expression and sequence analysis of candidates for the 1p36.31 tumor suppressor gene deleted in neuroblastomas

“…There is no published evidence to support a role for most genes in the region (Table 1) as TSGs in cancer generally, or neuroblastomas in particular. RPL22 recruits the EpsteinBarr early RNA and is linked to lymphoma associated with Epstein-Barr virus (Elia et al, 2004;Fok et al, 2006). ICMT is an isoprenylcysteine methyltransferase that has been implicated in carcinogenesis and as a target for anticancer therapy (Bergo et al, 2004;WinterVann et al, 2005).…”

The Challenge of Viral snRNPs

“…Previous in vitro binding studies suggested that EBER1 stem-loops III and IV serve as binding sites for L22 (Toczyski and Steitz 1993;Dobbelstein and Shenk 1995). Recently, we carried out experiments using recombinant L22 protein in electrophoretic mobility-shift assays and found that the EBER1 molecule harbors three L22-binding sites (Fok et al 2006b). These are the above- Some gammaherpesviruses encode nuclear noncoding RNAs (ncRNAs) that assemble with host proteins.…”