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Transfer RNA Modification Status Influences Retroviral Ribosomal Frameshifting

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Transfer RNA Modification Status Influences Retroviral Ribosomal Frameshifting Virology 255, 2–8 (1999) Article ID viro.1998.9569, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector RAPID COMMUNICATION Transfer RNA Modification Status Influences Retroviral Ribosomal Frameshifting Bradley A. Carlson,* So Yeon Kwon,† Mario Chamorro,‡ Stephen Oroszlan,§ Dolph L. Hatfield,* and Byeong Jae Lee†,1 *Section on the Molecular Biology of Selenium, Laboratory of Basic Research and ‡The Varmus Laboratory, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; †Laboratory of Molecular Genetics, Institute for Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Korea; and §ABL-Basic Research Program, NCI-FCRDC, Frederick, Maryland 21702 Received August 26, 1998; returned to author for revision November 4, 1998; accepted December 11, 1998 The possibility of whether tRNAs with and without a highly modified base in their anticodon loop may influence the level of retroviral ribosomal frameshifting was examined. Rabbit reticulocyte lysates were programmed with mRNA encoding UUU or AAC at the frameshift site and the corresponding Phe tRNA with or without the highly modified wyebutoxine (Y) base on the 39 side of its anticodon or Asn tRNA with or without the highly modified queuine (Q) base in the wobble position of its anticodon added. Phe and Asn tRNAs without the Y or Q base, respectively, stimulated the level of frameshifting, suggesting that the frameshift event is influenced by tRNA modification status. In addition, when AAU occurred immediately upstream of UUU as the penultimate frameshift site codon, addition of tRNAAsn without the Q base reduced the stimulatory effect of tRNAPhe without the Y base, whereas addition of tRNAAsn with the Q base did not alter the stimulatory effect. The addition of tRNAAsn without the Q base and tRNAPhe with the Y base inhibited frameshifting. The latter studies suggest an interplay between the tRNAs decoded at the penulimate frameshift and frameshift site codons that is also influenced by tRNA modification status. These data may be intrepreted as indicating that a hypomodified isoacceptor modulates frameshifting in an upward manner when utilized at the frameshift site codon, but modulates frameshifting in a downward manner when utilized at the penultimate frameshift site codon. © 1999 Academic Press In many eukaryotic retroviruses, the reading frame is anticodon, and tRNAAsn contains queuine (Q) base in extended beyond gag and into pro-pol or pol by ribo- position 34, which is the wobble base within its antico- somal frameshifting in the 21 direction (4, 11). The don (25). Since the presence or absence of these bases elements in retroviral mRNA that dictate the shift in the has a dramatic effect on the decoding properties of the reading frame consist of a heptanucleotide sequence corresponding tRNA (2, 3, 13, 22, 24, 27), it is tempting to upon which the shift occurs and a secondary or tertiary speculate that the modification status of these tRNAs structure that occurs just downstream of the frameshift may affect their shiftiness in ribosomal frameshifting site. The heptanucleotide sequence X XXY YYZ, desig- (see below). nated the frameshift signal, contains only certain codons, Several years ago, we observed that a correlation e.g., UUU, UUA, or AAC, at the YYZ or frameshift site exists between the occurrence of hypomodified Phe and codon. The observation that only certain codons exist at Asn tRNAs in retrovirus-infected cells and their utilization this site and the finding that generating mutations within in translating codons within their respective frameshift these codons inhibited frameshifting led Jacks et al. (19) signals (12 ). This observation led us to propose that the to propose that ribosomal frameshifting is promoted by lack of a highly modified base within the tRNA anticodon specialized “shifty” tRNAs. loop would create more space in and around the frame- Transfer RNAs that decode UUU (phenylalanine) and shift site, which in turn would facilitate frameshifting by AAC (asparagine) are characterized by the fact that they allowing greater flexibility of movement of the anticodon normally contain highly modified bases within their anti- (13, 17 ). We also suggested that the shifty tRNAs pro- codon loops. Transfer RNAPhe contains wyebutoxine (Y) posed by Varmus and collaborators (19 ) to mediate base in position 37, which is the base on the 39 side of its frameshifting are likely to be devoid of a highly modified base in their anticodon loop. Other investigators, work- ing in prokaryotic systems, proposed a different model in 1 To whom correspondence and reprint requests should be ad- which frameshifting is carried out by normal, highly mod- dressed. Fax: 82-2-872-9019. E-mail: [email protected]. ified tRNAs (6). However, it is unlikely that ribosomal 0042-6822/99 $30.00 Copyright © 1999 by Academic Press 2 All rights of reproduction in any form reserved. RAPID COMMUNICATION 3 FIG. 1. Constructs encoding mutant and wild-type frameshift signals harboring an upstream GST gene. The 214-bp MMTV gag-pro region (see upper portion of the figure) was prepared by amplifying this fragment, which begins 39 bases upstream of the frameshift site, and cloning it into BamHI–SmaI within the multiple cloning site in-frame with the GST gene. Constructs were amplified by transforming DH5a competent cells (Gibco) with the appropriate plasmid, growing transformed cells in 1 L batches, and extracting and purifying plasmids with a Qiafilter Kit (Qiagen). frameshifting in prokaryotes is comparable to that in Our approach in examining whether tRNA modification eukaryotes (6, 13). status may affect ribosomal frameshifting was to supple- In the present study, we asked whether the modifica- ment rabbit reticulocyte lysates programmed with mRNA tion status of tRNA (i.e., whether the presence or ab- generated from constructs encoding glutathione trans- sence of a highly modified base in the anticodon loop of ferase (GST) attached upstream of the 39 terminus of the tRNA decoded within the ribosomal frameshift signal) mouse mammary tumor virus (MMTV) gag-pro frameshift can influence or modulate the frameshift event. We uti- region (see Fig. 1) or encoding the MMTV gag-pro-pol Phe Phe lized tRNA without Y base (tRNA2Y ) from rabbit re- region (7) with the above described tRNAs. The GST ticulocytes and have taken advantage of the fact that constructs contain A AAA AAC (designated GST-A6C), A Asn yeast tRNA does not contain Q base to determine AAU UUU (GST-A3U4), or A AAU UUC (GST-A3U3C) as the whether these isoacceptors will enhance frameshifting frameshift signal (Fig. 1), while the MMTV constructs when the corresponding tRNA is required to decode the contain either A AAA AAC (designated MMTV-A6C) or A frameshift site. Furthermore, we examined the effect of AAA AAU (MMTV-A6U) as the frameshift signal (7). The Asn Asn tRNA with the Q base (tRNA1Q) or without the Q base role of Phe and Asn tRNAs with and without Y or Q base, Asn (tRNA2Q) on ribosomal frameshifting when Asn was de- respectively, could be readily determined with constructs coded within the frameshift signal, but immediately up- encoding UUU/C (Phe) or AAU/C (Asn) at the frameshift stream of the frameshift site. site. Since UUU and UUC code for the same Phe tRNA 4 RAPID COMMUNICATION FIG. 2. Influence of Y base decoded at the frameshift site on frameshifting. Rabbit reticulocyte lysates (nuclease treated; Promega) contained in m 35 m a 25- l volume [ S]methionine (sp act 1200 Ci/mmol; NEN), 19 unlabeled amino acids, and 5 g of mRNA generated from (A) GST-A3T4 and (B) Phe Phe GST-A3T3C and no tRNA (lane 1), tRNA1Y (lane 2), or tRNA2Y (lane 3). GST constructs were cut with EcoRI and mRNA was generated using a Ribomax Phe m Phe m transcription kit (Promega). Five pmoles of tRNA1Y (1.5 g of tRNA) or tRNA2Y (5.4 g of tRNA) was added to reactions where indicated. At the end of the incubation period, 5 ml of each reaction was added to 0.5 ml of 0.1 M KOH for 1–2 min to deacylate tRNA, and the sample was precipitated with 5% trichloroacetic acid, collected, and counted as described (16 ) to determine the amount of [35S]methionine incorporated into protein. One microliter of each sample was diluted with an equal volume of H2O followed by an equal volume of loading buffer and electrophoresed on 12% polyacrylamide gels and the level of frameshifting was determined by analyzing developed gels using a Molecular Dynamics PhosphorImager. The number of methionine residues in the 0 frame and the 21 frame were compensated for in determining the level of frameshifting. % FS in the figure designates percentage frameshifting. and AAU and AAC code for the same Asn tRNA, codon acylating tRNAs in the presence of 14C-labeled amino variations at this site permitted us to monitor the effi- acid ([14C]phenylalanine, sp act 505.3 mCi/mmol, from ciency of frameshifting with synonymous codons that are NEN; and [14C]asparagine, sp act 195 mCi/mmol, from normally read at similar efficiencies in protein synthesis Moravek Biochemicals) and rabbit reticulocyte synthe- (2, 24). Furthermore, the construct GST-A AAT TTT (GST- tases under limiting levels of tRNA (14 ). Phe A3T4) afforded us an opportunity to explore the effect of To determine the effect of tRNA2Y decoded at the Asn Asn Phe tRNA1Q or tRNA2Q on the incoming tRNA decoded at frameshift site codon on ribosomal frameshifting, reticu- Asn the frameshift site following transfer of tRNA1Q or locyte lysates were programmed with mRNA generated Asn tRNA2Q to the ribosomal P site. The advantage of using from the construct encoding the GST-A3T4 frameshift 2 the MMTV-A6C and A6U constructs is that the level of signal.
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