Translation initiation factor a/eIF2(-␥) counteracts 5؅ to 3؅ mRNA decay in the archaeon Sulfolobus solfataricus David Haseno¨ hrl*, Tania Lombo*, Vladimir Kaberdin*, Paola Londei†, and Udo Bla¨ si*‡ *Department of Microbiology and Immunobiology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria; and †Department of Cellular Biotechnology and Hematology, University of Rome La Sapienza, Viale Regina Elena 324, 00161 Rome, Italy Edited by Carl R. Woese, University of Illinois at Urbana–Champaign, Urbana, IL, and approved December 18, 2007 (received for review September 19, 2007) The trimeric translation initiation factor a/eIF2 of the crenarchaeon and ␥-subunits are required for tRNAi binding (19, 20), whereas Sulfolobus solfataricus is pivotal for binding of initiator tRNA to the in eIF2, the ␤- and ␥-subunits (18) are responsible for this task. ribosome. Here, we present in vitro and in vivo evidence that the Some proteins of the translation machinery have additional a/eIF2 ␥-subunit exhibits an additional function with resemblance functions. For instance, E. coli ribosomal protein L4 has a second to the eukaryotic cap-complex. It binds to the 5؅-triphosphate end function in transcription termination, whereas S1 is part of the of mRNA and protects the 5؅ part from degradation. This unprec- replicase of the E. coli phage Q␤ (21, 22). The ribosomal proteins edented capacity of the archaeal initiation factor further indicates S16 of E. coli and L32–2 of Schizosaccharomyces pombe display that 5؅ 3 3؅ directional mRNA decay is a pathway common to all endonuclease activity (23) and act as a transcription activator domains of life. (24), respectively. Here, we show that the a/eIF2 ␥-subunit (a/eIF2-␥)ofS. solfataricus has a dual function. Beside its n Escherichia coli, the decay of most RNA transcripts appears requirement for tRNAi binding to the ribosome, the initiation Ito be initiated by 5Ј pyrophosphate removal (1), followed by factor binds to the triphosphorylated 5Ј end of mRNA and endonucleolytic cleavages that are generated by RNase E (2, 3). counteracts 5Ј 3 3Ј directional decay. The intermediate cleavage products are further degraded by the 3Ј 3 5Ј exonucleases polynucleotide phosphorylase (PNPase), Results RNase II, and oligoribonuclease, converting the decay interme- a/eIF2(-␥) Binds to the 5؅ End of mRNA. During our efforts to diates into poly- and mononucleotides (4). At variance with E. establish an in vitro translation initiation assay for S. solfataricus, coli, Bacillus subtilis possesses a 5Ј 3 3Ј exonuclease activity, we observed that translation initiation factor a/eIF2 binds to the which could explain why RNAs in this organism are stabilized for 5Ј end of RNA. Primer extension experiments in the presence of great distances downstream of stable secondary structures or a/eIF2 and S. solfataricus 2508sh mRNA revealed an a/eIF2- bound ribosomes (5). In eukaryotes, mRNA decay is mainly specific stop signal positioned at nucleotide ϩ4 with respect to catalyzed by exonucleases (6). Eukaryotic mRNAs generally the 5Ј end of the RNA (Fig. 1A, lane 5). The position of the stop have a 7-methylguanosine cap at their 5Ј end and a poly(A) tail signal was independent of the RNA substrate because a ϩ4 stop at their 3Ј end. Removal of these terminal modifications is signal was also observed with heterologous and other homolo- considered rate-limiting for mRNA decay (7). Translation ini- gous mRNAs including E. coli ompA and ␭ cI mRNAs as well as tiation factor eIF4E binds to the 7-methylguanosine cap and S. solfataricus 104 and tfb-1 mRNAs (data not shown). We then thereby protects the cap structure from the decapping enzyme asked which individual subunit of a/eIF2 is required for 5Ј end and consequently the mRNA from 5Ј 3 3Ј exonucleolytic decay binding. As shown in Fig. 1A, lane 4, a/eIF2-␥ alone was able to (7). Different RNases with either endo- or exonuclease activity bind to the 5Ј end, i.e., block the reverse transcriptase at position (8–11) have been inferred or described in Archaea. In S. ϩ4, whereas the ␣- and ␤-subunits failed to do so (Fig. 1A, lanes solfataricus a3Ј 3 5Ј directional decay by a multisubunit 2 and 3). Because a/eIF2 and a/eIF2-␥ binding was independent exosome complex has been demonstrated in vitro (10). In of the nature of the mRNAs, the only constant recognition motif addition, the Sulfolobus solfataricus exosome is able to polyad- for the protein appeared to be the triphosphate group at the 5Ј enylate the 3Ј end of RNAs in the presence of ADP (12). With end. Hence, we tested whether a/eIF2 or a/eIF2-␥ bind to the exception of the exosome, no other endo- or exonuclease 5Ј-dephosphorylated 2508sh mRNA. As shown in Fig. 1A (lanes activities have been described in S. solfataricus. At variance with 7–10), the a/eIF2-dependent stop signal of the reverse transcrip- a previous study wherein the longevity of selected S. solfataricus tase was not obtained with 5Ј-dephosphorylated RNA, indicat- mRNAs was found to be rather high (13), a recent microarray- ing that the triphosphate group is required for 5Ј-end binding of based analysis (14) indicated a rather short mRNA half-life, a/eIF2-␥. comparable with that of bacterial mRNAs. Next, the binding affinity of a/eIF2 and a/eIF2-␥ for 2508sh Two different mechanisms for translational initiation seem to mRNA was determined by using filter-binding assays. The exist in Sulfolobus (15). One is based on a canonical SD/anti-SD apparent Kd of a/eIF2 and of cross-linked a/eIF2 for 2508sh interaction and operates on internal cistrons of polycistronic mRNA was calculated with Ϸ10 nM (Fig. 1B). The binding mRNAs. In contrast, monocistronic mRNAs and proximal genes affinity of a/eIF2-␥ was indistinguishable from that of a/eIF2 or of polycistronic mRNAs are frequently devoid of a 5Ј untrans- lated region. Decoding of these leaderless mRNAs requires, analogously to Bacteria (16), pairing of the start codon with Author contributions: D.H. and U.B. designed research; D.H. and T.L. performed research; initiator-tRNA (15). The complexity of archaeal translational D.H., T.L., V.K., P.L., and U.B. analyzed data; and D.H. and U.B. wrote the paper. initiation seems to be underscored by the presence of a larger- The authors declare no conflict of interest. than-bacterial set of factors because Archaea encode Ϸ10 or- This article is a PNAS Direct Submission. thologs of eukaryal and bacterial initiation factors (17). Like its ‡To whom correspondence should be addressed. E-mail: [email protected]. eukaryotic counterpart eIF2 (18), the archaeal factor a/eIF2 This article contains supporting information online at www.pnas.org/cgi/content/full/ consists of three subunits, ␣, ␤, and ␥, and recruits initiator 0708894105/DC1. tRNA (tRNAi) to the 30S ribosomal subunit. In a/eIF2, the ␣- © 2008 by The National Academy of Sciences of the USA 2146–2150 ͉ PNAS ͉ February 12, 2008 ͉ vol. 105 ͉ no. 6 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708894105 Downloaded by guest on September 27, 2021 Fig. 1. The a/eIF2 ␥-subunit binds to the 5Ј end of triphosphorylated 2508sh mRNA. (A) Lanes 1–6, primer extension reactions with 2508sh mRNA (0.05 nM) without addition of factors (lane 1), in the presence of 0.1 ␮M the a/eIF2 ␣-subunit (lane 2), the a/eIF2 ␤-subunit (lane 3), the a/eIF2 ␥-subunit (lane 4), reconstituted a/eIF2 (lane 5), or cross-linked a/eIF2 (lane 6). Lanes 7–10, primer extension reactions with 5Ј-dephosphorylated 2508sh mRNA (0.05 nM) with- out addition of factors (lane 7), in the presence of 0.1 ␮M concentrations of the a/eIF2 ␥-subunit (lane 8), reconstituted a/eIF2 (lane 9), or cross-linked a/eIF2 (lane 10). G, A, and T represent sequencing reactions obtained with 2508sh mRNA containing a 21-nt extension at the 5Ј end. The sequence of the 5Ј-proximal part of 2508sh mRNA is shown below the autoradiograph. The MICROBIOLOGY start codon is in bold. The arrow indicates the position of the a/eIF2(-␥)- dependent stop signal. (B) Radiolabeled 2508sh mRNA (1 ␮M) was incubated with increasing amounts of the a/eIF2 ␣-subunit (E), the a/eIF2 ␤-subunit (ᮀ), the a/eIF2 ␥-subunit (■), with reconstituted a/eIF2 (Œ), and with cross-linked a/eIF2 (⌬) at 70°C. Dephosphorylated radiolabeled 2508sh mRNA (1 ␮M) was incubated with increasing amounts of a/eIF2 (F) at 70°C. Samples were with- drawn after 5 min, and the amount of RNA retained on the filter was determined. The experiment was performed in triplicate. The error bars represent standard deviations. cross-linked a/eIF2 (Fig. 1B). As expected from the results shown in Fig. 1A, neither the a/eIF2 ␣- nor the ␤-subunit displayed any Fig. 2. The a/eIF2 ␥-subunit protects the 5Ј end of triphosphorylated 2508 binding activity, and a/eIF2 did not bind to dephosphorylated mRNA from degradation in vitro. The 2508 full-length mRNA (0.25 ␮M) was 2508sh mRNA (Fig. 1B) or to monophosphorylated 2508sh preincubated in the absence (white bars) or presence (gray bars) of 0.5 ␮M mRNA generated by tobacco acid pyrophospatase (data not a/eIF2-␥ for 5 min at 70°C. S. solfataricus S100 extract was added, and samples shown). The a/eIF2 ␥-subunit contains the guanine nucleotide- were withdrawn after 0, 10, 30, 60, and 90 min. The remaining RNA was deter- mined with DNA probes complementary to the 5Ј end (A), the central part (B), or binding pocket of a/eIF2 (20), and binding of a/eIF2 to tRNAi the 3Ј end (C)of2508 full-length mRNA. An autoradiograph of a corresponding depends on GTP (19).