IMPORTANCE OF SYSTEMATIC IDENTIFICATION OF RNA-BINDING PROTEINS INA HYPERTHERMOPHILIC ARCHAEON
Akio Kanai 1,2
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan
Tel: +81-235-29-0524; Fax: +81-235-29-0525; E-mail: akio@sfc.keio.ac.jp
2
Department of Environmental Information, Keio University, Fujisawa, Kanagawa 252-8520, Japan
1(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 theRNA level support the concept of “the RNA world” atthe beginning of life on Earth. So the study of RNAs
and their enzymes in a hyperthermophilic archaeon,
Pyrococcus furiosus, which is believed to be a veryancient organism, may open a new door in the life
sciences. We have developed an expression cloning
method to classify and identify factors involved in theregulation of RNA metabolism in P. furiosus. Here I
propose the value of the systematic analysis ofregulatory RNAs and their binding proteins.
Keywords: Archaea, Pyrococcus furiosus, Expression
cloning, DNA/RNA-binding protein, RNA world,
Non-coding RNA
Introduction
Approximately 50 years has passed since theestablishment of the central dogma of the genetic
code—the concept of information flow from DNA toRNA to protein. During this period, this flow has been
manipulated thanks to the discovery of reverse
transcriptase. RNA is mostly regarded as theinformation transmitter, and it is widely consideredthat it has always had this role. However, genome
mapping, and especially the recent elucidation of
non-coding RNA (untranslated RNA), has seemingly
entrenched the concept of RNA as more activefunctional molecules. Our group has found andcharacterized non-coding RNAs in a variety oforganisms, including mouse (Mus musculus) [1], fruit
fly (Drosophila melanogaster) [2], nematode(Caenorhabditis elegans) [3], and a bacterium(Escherichia coli) [4]. It is now believed that cells
house huge numbers of non-coding RNAs, which may
function beyond the central dogma (Figure 1),
although the functions of most remain unknown. In
addition, the “RNA world” hypothesis, which assumes
that genetic information was originally controlled byRNA molecules, apparently renders RNA researchmore important. Since hyperthermophilic archaeons,especially Pyrococcus species, which grow in the deepsea at around 100 °C, are believed to be very ancientorganisms, analyzing RNA metabolism in them could
bring new insights into the fundamental regulation ofgenes [5–7].

GenomeTranscriptome

DNAmRNA
Non-coding RNAs

Proteome

Protein

Metabolome

Metabolite

Figure 1. Revised view of the central dogma: non-coding
RNAs maintain potential genetic networks beyond thecentral dogma.

Functional classification of archaeal proteome byexpression cloning
Recent progress in genome projects has revealed thecomplete genomic DNA sequences in many species,
from Bacteria to Archaea to Eukarya. However, only
half of all proteins deduced from these sequences
could be assigned putative cellular roles. The rest areconsidered to be conserved hypothetical proteins or
simply hypothetical proteins. This is mainly because
annotations of proteins are made by searchinghomologies against a limited set of databases offunctionally known proteins. Therefore, a systematicway of annotating or analyzing the functions ofproteins from the genome level would be valuable forcharacterizing proteins in the post-genome era. In thisrespect, the use of an expression cloning method in thetest tube [8, 9] or a biochemical genomics approach inyeast [10] may turn out to be very useful. We havedeveloped an efficient, highly sensitive method for
identifying
newgenespresentinthehyperthermophilic archaeon Pyrococcus furiosus at
the genome level. This system has several advantages:• P. furiosus has only about 2000 genes, and thecomplete genomic nucleotide sequence has been
determined (http://www.genome.utah.edu/). It isless than half the size of the E. coli genome.
• The encoded proteins are mostly heat stable andare easy to handle biochemically.
• Most of the genes involved in nucleic acidmetabolism in the Archaea are similar to thosefound in the Eukarya, but the regulation

mechanisms in the Archaea are simpler.

no significant homology with any protein whosefunctions are well known at either the nucleotide or
the amino acid level, we found weak homology with
the RNase E/RNase G protein family at theN-terminus (25%–30% identity). No ribonucleaseactivity was found, however, in the purified FAU-1
protein fractions, suggesting that FAU-1 is a novel
RNA-binding protein. To determine the most suitable
RNA sequence for recognition by FAU-1, weperformed in vitro selection experiments (SELEXanalysis) with RNA ligands and found that FAU-1
binds specifically to an AU-rich sequence in a loop
region of a possible RNA ligand [5].
In recent years, it has been well accepted thatRNA secondary structures, including stem-loop
structures, are involved in many stages of gene
regulation, such as transcription, splicing, translation,and degradation. In particular, a stem-loop RNAstructure located near the translation start AUG codon
appears to be the key regulator for translation. In thenext round of screening, we therefore used anoligoribonucleotide probe with a specific RNA
secondary structure (a stem-loop RNA oligo
containing an AUG sequence in the loop region), andisolated the gene encoding thymidylate synthase(Pf-Thy1) as an RNA-binding protein [7]. Pf-Thy1
also bound to the stem-loop structure located near thetranslational start codon AUG in its own mRNA. Invitro translation tests using E. coli lysate indicated thatthe stem-loop structure of Pf-Thy1 mRNA might work
as a translational repressor (Figure 3). Also, Pf-Thy1
inhibits the in vitro translation system. This evidence

Our strategy for the systematic identification of
DNA/RNA-binding proteins is described in Figure 2.
We made a genomic DNA expression library of P.
furiosus [5]. Briefly, after the P. furiosus genomic
DNA was prepared, partially digested DNA fragments(about 7 kb average size) were ligated into a pRSET A
plasmid vector. Although the vector contained the T7
RNA polymerase promoter sequence, P. furiosus
genes were expressed in E. coli without induction by
T7 RNA polymerase. Next, we prepared protein pools(one pool consisting of 30 independent colonies of E.
coli in the library). These protein pools were
heat-treated to kill endogenous proteins from E. coli,
reducing the background noise and revealing theDNA/RNA-binding activities of proteins derived fromP. furiosus. Because the genome of P. furiosus isabout 2 megabases long, screening of 1200 clones (40pools of 30 clones) should cover the whole genome.
Thus, half a day is enough to screen a genome of thissize.
Step 1

Genom ic DN A prep aratio nfrom Pyr ococcus fu rios us

Partial digestion
wi th res tri cti on enzy me
Sau3AI

Step 2

Purification of abou t 7 k b D NA
fragm en ts and su bcloning in top la smid vector+
Step 3

Tran sforma tio n o f E . coli
(Plasm id lib ra ry)
＃1

＃2

＃3

＃4

AU G

＃N

N um berin g

Step 4I nd uction of recomb inan t proteinsin E. coli an d h ea t treatmen t(Prot ein library)＃1＃1

Step 5

＃2

＃3

SD

＃4

Pf-Thy1 mRNATranslation＃N

Fun ctional screen in g in vitro

Pf-Thy1
Step 6

Gen e id en tification

＃X

＃X

FMN

Figure 2. Expression cloning of P. furiosus genes toidentify protein function at the proteome level.

Thymidine synthesis
Figure 3. A model for autoregulation of Pf-Thy1 mRNAtranslation.
A stem-loop RNA secondary structure is located

RNA secondary structures and their binding
proteinsaround the translation start AUG codon of Pf-Thy1
mRNA and acts as an inhibitory regulator oftranslation through a Shine–Dalgarno (SD)-likesequence in the stem region. Addition of Pf-Thy1
into the in vitro translation system also inhibitstranslation. These results suggest that thymidylate
synthases of this class regulate their own translation.
Flavin mononucleotide (FMN) is required for thethymidine synthetase activity of the enzyme.

After making the P. furiosus genomic expression
library, we screened the library to isolate novel genes
for DNA/RNA-binding proteins by using a series ofartificial probes such as r(A-U)10, r(G-C)10, d(A-T)10,We
detectedabout50
andd(G-C)10.
DNA/RNA-binding activities, and isolated andcharacterized one gene product, named FAU-1 (P.
furiosus AU-binding protein-1), which is able to bindthe r(A-U)10 sequence [5]. Although FAU-1 showedstrongly suggests that Pf-Thy1 controls its own

2mRNA as an RNA-binding protein. This finding is
consistent with the fact that another thymidylate
synthase, ThyA, acts as an RNA-binding protein
against its own mRNA [11, 12], although it belongs toa different class of thymidylate synthases. Now we areadvancing our expression cloning method using avariety of oligoribonucleotide probes to pick up novel
RNA-binding proteins in the Archaea.
Analyzing specific RNA secondary structures in
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.
One of these basic secondary structures is thestem-loop. Some stem-loop RNA structures inmRNAs, or “RNA domains”, are functional
cis-elements such as riboswitches, internal ribosome
entry sites (IRES), or selenocysteine-insertion
sequences (SECIS). In the post-genome era, it isextremely important to summarize these RNA
domains at the genome level, through prediction of
RNA secondary structures and mapping of thepositions of these structures in transcripts. Based onthese analyses and our strategy for gene identification,
the systematic identification of RNA-binding proteinsthat bind to specific RNA secondary structures willcontribute to the mapping of RNA–protein interaction
networks at the genome level

biology, biochemistry, bioinformatics, and structural
biology at the whole genome level is very useful for
creating a new biology in the post-genome era. This is
an ideal strategy of “systems biology”.
Acknowledgments
I would like to thank my many collaborators in theresearch.
References
1.

2.

3.
4.

5.

It is also true that classical non-coding RNAs
such as rRNAs and tRNAs work with various types ofRNA-binding proteins. So far, experimental andbioinformatics approaches have predicted andidentified a huge amount of non-coding RNAs.
However, there are few reports of the functional
analysis of these non-coding RNAs. As describedabove, some RNA-binding proteins specificallyregulate their target RNA domains in theprotein-coding transcripts. It is possible that
secondary structures found in non-coding RNAsinteract with certain RNA-binding proteins. These
could be identified by searching for common
secondary structures among non-coding RNAs. For
example, mapping of conserved RNA secondarystructures predicts thousands of functional non-coding
RNAs in the human genome [13].

6.

7.
8.
9.
10.

11.

Conclusions

12.

This review introduces a systematic methodology forcharacterizingRNA-bindingproteins.
Themethodology can identify new functional proteinsbecause the cloning strategy is based on biological,chemical, or physical characteristics, not on theprimary sequence of amino acids or on homology. Itfurther makes it possible to add new functional
properties (such as DNA/RNA-binding, kinase,protease, or other enzymatic capabilities) to alreadyannotated proteins. In addition, functionalclassification using a bioinformatics approach is
becoming important for revealing RNA molecules[14] and unknown proteins [15]. Collaborative
structural analysis of RNA and RNA-binding proteinsis valuable [16, 17]. The combination of molecular

13.

14.

15.

16.

3

Numata, K., Kanai, A., Saito, R., Kondo, S., Adachi, J.,Wilming, L. G., Hume, D. A., Hayashizaki, Y., and Tomita,
M. Identification of putative noncoding RNAs among theRIKEN mouse full-length cDNA collection, Genome Res 13,
1301-1306 (2003).
Inagaki, S., Numata, K., Kondo, T., Tomita, M., Yasuda, K.,
Kanai, A., and Kageyama, Y. Identification and expression
analysis of putative mRNA-like non-coding RNA inDrosophila, Genes Cells 10, 1163-1173 (2005).
Watanabe, Y., Yachie, N., Numata, K., Saito, R., Kanai, A.,and Tomita, M. Computational analysis of microRNA targets
in Caenorhabditis elegans, Gene 365, 2-10 (2006).
Yachie, N., Numata, K., Saito, R., Kanai, A., and Tomita, M.
Prediction of non-coding and antisense RNA genes in
Escherichia coli with gapped Markov model, Gene 372,
171-181 (2006).
Kanai, A., Oida, H., Matsuura, N., and Doi, H. Expression
cloning and characterization of a novel gene that encodes theRNA-binding protein FAU-1 from Pyrococcus furiosus,
Biochem J 372, 253-261 (2003).
Sato, A., Kanai, A., Itaya, M., and Tomita, M. Cooperative
regulation for Okazaki fragment processing by RNase HII
and FEN-1 purified from a hyperthermophilic archaeon,Pyrococcus furiosus, Biochem Biophys Res Commun 309,247-252 (2003).
Kanai, A., Sato, A., Imoto, J., and Tomita, M. Archaeal
Pyrococcus furiosus thymidylate synthase 1 is an
RNA-binding protein, Biochem J 393, 373-379 (2006).
Stukenberg, P. T., Lustig, K. D., McGarry, T. J., King, R. W.,
Kuang, J., and Kirschner, M. W. Systematic identification ofmitotic phosphoproteins, Curr Biol 7, 338-348 (1997).
King, R. W., Lustig, K. D., Stukenberg, P. T., McGarry, T. J.,
and Kirschner, M. W. Expression cloning in the test tube,Science 277, 973-974 (1997).
Martzen, M. R., McCraith, S. M., Spinelli, S. L., Torres, F.
M., Fields, S., Grayhack, E. J., and Phizicky, E. M. A
biochemical genomics approach for identifying genes by theactivity of their products, Science 286, 1153-1155 (1999).
Chu, E., Koeller, D. M., Casey, J. L., Drake, J. C., Chabner, B.
A., Elwood, P. C., Zinn, S., and Allegra, C. J. Autoregulationof human thymidylate synthase messenger RNA translationby thymidylate synthase, Proc Natl Acad Sci U S A 88,8977-8981 (1991).
Chu, E., Voeller, D., Koeller, D. M., Drake, J. C., Takimoto,C. H., Maley, G. F., Maley, F., and Allegra, C. J.
Identification of an RNA binding site for human thymidylatesynthase, Proc Natl Acad Sci U S A 90, 517-521 (1993).
Washietl, S., Hofacker, I. L., Lukasser, M., Huttenhofer, A.
and Stadler, P. F. Mapping of conserved RNA secondarystructures predicts thousands of functional noncoding RNAsin the human genome, Nat Biotechnol 23, 1383-1390 (2005).
Sugahara, J., Yachie, N., Sekine, Y., Soma, A., Matsui, M.,
Tomita, M., and Kanai, A. SPLITS: a new program forpredicting split and intron-containing tRNA genes at thegenome level, In Silico Biology 6, 0039 (2006).
Fujishima, K., Imoto, J., Kanai, A., and Tomita, M. A newmethod for characterizing functionally-unknown proteinsusing specific amino acid frequency and periodicity at theproteome level, Genome Informatics 14, 526-527 (2003).
Okada, K., Takahashi, M., Sakamoto, T., Kawai, G.,Nakamura, K., and Kanai, A. Solution structure of a GAAG
tetraloop in helix 6 of SRP RNA from Pyrococcus furiosus,
Nucleosides Nucleotides Nucleic Acids 25, 383-395 (2006).

17.

publication
02 February 2006
(doi:10.1007/s10858-005-5581-8) (2006).

Okada, K., Matsuda, T., Sakamoto, T., Muto, Y., Yokoyama,S., Kanai, A., and Kawai, G. (1)H, (13)C and (15)N
resonance assignments of the 2′-5′ RNA ligase-like proteinfrom Pyrococcus furiosus, J Biomol NMR online

4