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Mini review
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.
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Corden International (Magyarország) Kft. Központi Vizsgálati Árjegyzéke A vizsgálat neve A vizsgálat rövid neve, más használatban levő elnevezése Listaár Inzulin-rezisztencia index, IR% (inzulinból és vércukorból számított index). Az ár csak az index kiszámítását fedezi, a vizsgálatok pluszban fizetendők. R atio of O varian MA lignancy. HE4 és CA 125 II ér
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