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IMPORTANCE OF SYSTEMATIC IDENTIFICATION OF RNA-BINDING PROTEINS IN
A HYPERTHERMOPHILIC ARCHAEON
1 Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan Tel: +81-235-29-0524; Fax: +81-235-29-0525; E-ma 2 Department of Environmental Information, Keio University, Fujisawa, Kanagawa 252-8520, Japan (Received October 26, 2006 Accepted October 30, 2006) Abstract
Recent findings of huge numbers of non-coding RNAs and accumulating reports of gene regulation at the RNA level support the concept of “the RNA world” at the beginning of life on Earth. So the study of RNAs and their enzymes in a hyperthermophilic archaeon, Transcriptome
Pyrococcus furiosus, which is believed to be a very Non-coding RNAs
ancient organism, may open a new door in the life sciences. We have developed an expression cloning Proteome
method to classify and identify factors involved in the regulation of RNA metabolism in P. furiosus. Here I Metabolome
Metabolite
propose the value of the systematic analysis of regulatory RNAs and their binding proteins. Figure 1. Revised view of the central dogma: non-coding RNAs maintain potential genetic networks beyond the Keywords: Archaea, Pyrococcus furiosus, Expression
cloning, DNA/RNA-binding protein, RNA world, Non-coding RNA Functional classification of archaeal proteome by
expression cloning

Introduction
Recent progress in genome projects has revealed the Approximately 50 years has passed since the complete genomic DNA sequences in many species, establishment of the central dogma of the genetic from Bacteria to Archaea to Eukarya. However, only code—the concept of information flow from DNA to half of all proteins deduced from these sequences RNA to protein. During this period, this flow has been could be assigned putative cellular roles. The rest are manipulated thanks to the discovery of reverse considered to be conserved hypothetical proteins or transcriptase. RNA is mostly regarded as the simply hypothetical proteins. This is mainly because information transmitter, and it is widely considered annotations of proteins are made by searching that it has always had this role. However, genome homologies against a limited set of databases of mapping, and especially the recent elucidation of functionally known proteins. Therefore, a systematic non-coding RNA (untranslated RNA), has seemingly way of annotating or analyzing the functions of entrenched the concept of RNA as more active proteins from the genome level would be valuable for functional molecules. Our group has found and characterizing proteins in the post-genome era. In this characterized non-coding RNAs in a variety of respect, the use of an expression cloning method in the organisms, including mouse (Mus musculus) [1], fruit test tube [8, 9] or a biochemical genomics approach in fly (Drosophila melanogaster) [2], nematode yeast [10] may turn out to be very useful. We have (Caenorhabditis elegans) [3], and a bacterium developed an efficient, highly sensitive method for (Escherichia coli) [4]. It is now believed that cells house huge numbers of non-coding RNAs, which may hyperthermophilic archaeon Pyrococcus furiosus at function beyond the central dogma (Figure 1), the genome level. This system has several advantages: although the functions of most remain unknown. In • P. furiosus has only about 2000 genes, and the addition, the “RNA world” hypothesis, which assumes complete genomic nucleotide sequence has been that genetic information was originally controlled by determined (http://www.genome.utah.edu/). It is RNA molecules, apparently renders RNA research less than half the size of the E. coli genome. more important. Since hyperthermophilic archaeons, • The encoded proteins are mostly heat stable and especially Pyrococcus species, which grow in the deep sea at around 100 °C, are believed to be very ancient • Most of the genes involved in nucleic acid organisms, analyzing RNA metabolism in them could metabolism in the Archaea are similar to those bring new insights into the fundamental regulation of found in the Eukarya, but the regulation no significant homology with any protein whose functions are well known at either the nucleotide or Our strategy for the systematic identification of the amino acid level, we found weak homology with DNA/RNA-binding proteins is described in Figure 2. the RNase E/RNase G protein family at the We made a genomic DNA expression library of P. N-terminus (25%–30% identity). No ribonuclease furiosus [5]. Briefly, after the P. furiosus genomic activity was found, however, in the purified FAU-1 DNA was prepared, partially digested DNA fragments protein fractions, suggesting that FAU-1 is a novel (about 7 kb average size) were ligated into a pRSET A RNA-binding protein. To determine the most suitable plasmid vector. Although the vector contained the T7 RNA sequence for recognition by FAU-1, we RNA polymerase promoter sequence, P. furiosus performed in vitro selection experiments (SELEX genes were expressed in E. coli without induction by analysis) with RNA ligands and found that FAU-1 T7 RNA polymerase. Next, we prepared protein pools binds specifically to an AU-rich sequence in a loop (one pool consisting of 30 independent colonies of E. coli in the library). These protein pools were In recent years, it has been well accepted that heat-treated to kill endogenous proteins from E. coli, RNA secondary structures, including stem-loop reducing the background noise and revealing the structures, are involved in many stages of gene DNA/RNA-binding activities of proteins derived from regulation, such as transcription, splicing, translation, P. furiosus. Because the genome of P. furiosus is and degradation. In particular, a stem-loop RNA about 2 megabases long, screening of 1200 clones (40 structure located near the translation start AUG codon pools of 30 clones) should cover the whole genome. appears to be the key regulator for translation. In the Thus, half a day is enough to screen a genome of this next round of screening, we therefore used an oligoribonucleotide probe with a specific RNA secondary structure (a stem-loop RNA oligo containing an AUG sequence in the loop region), and Genom ic DN A prep aratio n
isolated the gene encoding thymidylate synthase from Pyr ococcus fu rios us
(Pf-Thy1) as an RNA-binding protein [7]. Pf-Thy1 Partia l dig estio n
also bound to the stem-loop structure located near the wi th res tri cti on enzy me
translational start codon AUG in its own mRNA. In Sau3AI
vitro translation tests using E. coli lysate indicated that Purification of abou t 7 k b D NA
fragm en ts and su bcloning in to

the stem-loop structure of Pf-Thy1 mRNA might work p la smid vector
as a translational repressor (Figure 3). Also, Pf-Thy1 inhibits the in vitro translation system. This evidence Tran sforma tio n o f E . coli
(Plasm id lib ra ry)
N um berin g
I nd uction of recomb inan t proteins
in E. coli an d h ea t treatmen t
Pf-Thy1 mRNA
(Prot ein library)
Translation
Fun ctional screen in g in vitro
Pf-Thy1
Gen e id en tification
Figure 2. Expression cloning of P. furiosus genes to Thymidine synthesis
identify protein function at the proteome level. Figure 3. A model for autoregulation of Pf-Thy1 mRNA translation. RNA secondary structures and their binding
A stem-loop RNA secondary structure is located proteins
around the translation start AUG codon of Pf-Thy1 mRNA and acts as an inhibitory regulator of After making the P. furiosus genomic expression translation through a Shine–Dalgarno (SD)-like library, we screened the library to isolate novel genes sequence in the stem region. Addition of Pf-Thy1 for DNA/RNA-binding proteins by using a series of into the in vitro translation system also inhibits translation. These results suggest that thymidylate synthases of this class regulate their own translation. Flavin mononucleotide (FMN) is required for the DNA/RNA-binding activities, and isolated and thymidine synthetase activity of the enzyme. characterized one gene product, named FAU-1 (P. furiosus AU-binding protein-1), which is able to bind the r(A-U)10 sequence [5]. Although FAU-1 showed strongly suggests that Pf-Thy1 controls its own mRNA as an RNA-binding protein. This finding is biology, biochemistry, bioinformatics, and structural consistent with the fact that another thymidylate biology at the whole genome level is very useful for synthase, ThyA, acts as an RNA-binding protein creating a new biology in the post-genome era. This is against its own mRNA [11, 12], although it belongs to an ideal strategy of “systems biology”. a different class of thymidylate synthases. Now we are advancing our expression cloning method using a variety of oligoribonucleotide probes to pick up novel Acknowledgments
Analyzing specific RNA secondary structures in I would like to thank my many collaborators in the the untranslated region (UTR) of mRNAs may help us understand a new RNA-protein network system possibly involved in gene regulation at the RNA level. References
One of these basic secondary structures is the Numata, K., Kanai, A., Saito, R., Kondo, S., Adachi, J., stem-loop. Some stem-loop RNA structures in Wilming, L. G., Hume, D. A., Hayashizaki, Y., and Tomita, mRNAs, or “RNA domains”, are functional M. Identification of putative noncoding RNAs among the RIKEN mouse full-length cDNA collection, Genome Res 13, cis-elements such as riboswitches, internal ribosome entry sites (IRES), or selenocysteine-insertion Inagaki, S., Numata, K., Kondo, T., Tomita, M., Yasuda, K., sequences (SECIS). In the post-genome era, it is Kanai, A., and Kageyama, Y. Identification and expression extremely important to summarize these RNA analysis of putative mRNA-like non-coding RNA in Drosophila, Genes Cells 10, 1163-1173 (2005). domains at the genome level, through prediction of Watanabe, Y., Yachie, N., Numata, K., Saito, R., Kanai, A., RNA secondary structures and mapping of the and Tomita, M. Computational analysis of microRNA targets positions of these structures in transcripts. Based on in Caenorhabditis elegans, Gene 365, 2-10 (2006). these analyses and our strategy for gene identification, Yachie, N., Numata, K., Saito, R., Kanai, A., and Tomita, M. the systematic identification of RNA-binding proteins Prediction of non-coding and antisense RNA genes in Escherichia coli with gapped Markov model, Gene 372, that bind to specific RNA secondary structures will contribute to the mapping of RNA–protein interaction Kanai, A., Oida, H., Matsuura, N., and Doi, H. Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus, Biochem J 372, 253-261 (2003). It is also true that classical non-coding RNAs Sato, A., Kanai, A., Itaya, M., and Tomita, M. Cooperative such as rRNAs and tRNAs work with various types of regulation for Okazaki fragment processing by RNase HII RNA-binding proteins. So far, experimental and and FEN-1 purified from a hyperthermophilic archaeon, bioinformatics approaches have predicted and Pyrococcus furiosus, Biochem Biophys Res Commun 309, identified a huge amount of non-coding RNAs. Kanai, A., Sato, A., Imoto, J., and Tomita, M. Archaeal However, there are few reports of the functional Pyrococcus furiosus thymidylate synthase 1 is an analysis of these non-coding RNAs. As described RNA-binding protein, Biochem J 393, 373-379 (2006). above, some RNA-binding proteins specifically Stukenberg, P. T., Lustig, K. D., McGarry, T. J., King, R. W., regulate their target RNA domains in the Kuang, J., and Kirschner, M. W. Systematic identification of mitotic phosphoproteins, Curr Biol 7, 338-348 (1997). protein-coding transcripts. It is possible that King, R. W., Lustig, K. D., Stukenberg, P. T., McGarry, T. J., secondary structures found in non-coding RNAs and Kirschner, M. W. Expression cloning in the test tube, interact with certain RNA-binding proteins. These could be identified by searching for common Martzen, M. R., McCraith, S. M., Spinelli, S. L., Torres, F. secondary structures among non-coding RNAs. For M., Fields, S., Grayhack, E. J., and Phizicky, E. M. A biochemical genomics approach for identifying genes by the example, mapping of conserved RNA secondary activity of their products, Science 286, 1153-1155 (1999). structures predicts thousands of functional non-coding Chu, E., Koeller, D. M., Casey, J. L., Drake, J. C., Chabner, B. A., Elwood, P. C., Zinn, S., and Allegra, C. J. Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase, Proc Natl Acad Sci U S A 88, 8977-8981 (1991). Conclusions
Chu, E., Voeller, D., Koeller, D. M., Drake, J. C., Takimoto, C. H., Maley, G. F., Maley, F., and Allegra, C. J. This review introduces a systematic methodology for Identification of an RNA binding site for human thymidylate characterizing RNA-binding proteins. The synthase, Proc Natl Acad Sci U S A 90, 517-521 (1993). methodology can identify new functional proteins Washietl, S., Hofacker, I. L., Lukasser, M., Huttenhofer, A. because the cloning strategy is based on biological, and Stadler, P. F. Mapping of conserved RNA secondary structures predicts thousands of functional noncoding RNAs chemical, or physical characteristics, not on the in the human genome, Nat Biotechnol 23, 1383-1390 (2005). primary sequence of amino acids or on homology. It Sugahara, J., Yachie, N., Sekine, Y., Soma, A., Matsui, M., further makes it possible to add new functional Tomita, M., and Kanai, A. SPLITS: a new program for properties (such as DNA/RNA-binding, kinase, predicting split and intron-containing tRNA genes at the genome level, In Silico Biology 6, 0039 (2006). protease, or other enzymatic capabilities) to already Fujishima, K., Imoto, J., Kanai, A., and Tomita, M. A new annotated proteins. In addition, functional method for characterizing functionally-unknown proteins classification using a bioinformatics approach is using specific amino acid frequency and periodicity at the becoming important for revealing RNA molecules proteome level, Genome Informatics 14, 526-527 (2003). [14] and unknown proteins [15]. Collaborative 16. Okada, K., Takahashi, M., Sakamoto, T., Kawai, G., Nakamura, K., and Kanai, A. Solution structure of a GAAG structural analysis of RNA and RNA-binding proteins tetraloop in helix 6 of SRP RNA from Pyrococcus furiosus, is valuable [16, 17]. The combination of molecular Nucleosides Nucleotides Nucleic Acids 25, 383-395 (2006). Okada, K., Matsuda, T., Sakamoto, T., Muto, Y., Yokoyama, publication
S., Kanai, A., and Kawai, G. (1)H, (13)C and (15)N resonance assignments of the 2′-5′ RNA ligase-like protein from Pyrococcus furiosus, J Biomol NMR online

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