INTRON AND RNA SPLICING IN ARCHAEA
Yoh-ichi Watanabe1* and Shigeo Yoshinari1,2
1
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
2
Present address: Department of Biological Sciences, State University of New York at Buffalo,Buffalo, NY 14260, USA.
*Corresponding author: Department of Biomedical Chemistry, Graduate School of Medicine, The
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan, Fax: +81-3-5841-3444,
e-mail: ywatanab@m.u-tokyo.ac.jp

Abstract
In Archaea, almost all introns in pre-tRNA,pre-rRNA, and pre-mRNA are spliced through two
common steps by protein enzymes: cleavage of theprecursor with splicing endonuclease, and ligation of
the exons with RNA ligase. We found the firstexamples of archaeal pre-mRNA splicing andcleavage of the pre-mRNA with a novel subclass ofarchaeal splicing endonuclease. We further solved thenovel tertiary structure of the splicing endonuclease,and revealed that the lineage-specific insertion ofamino acid residues in the endonuclease expands therecognition of the substrate precursor RNA. We alsodiscuss the possible involvement of tRNA splicing
and its machinery in the origin of the tRNA molecule.

with a 2’,3’-cyclic phosphate residue and the 5’ endof the 3’ exon without a phosphate residue, and uses
the phosphate residue at the 3’ end of the 5’ exon as
the linkage between the exons [14].

(Keywords)
intron, RNA splicing, Archaea, splicing endonuclease,
RNA ligase, bulge-helix-bulge motif

Discovery of archaeal pre-mRNA splicing, andidentification of structural elements in archaeal
splicing endonuclease for expansion of substrate
RNA recognition
In 2002, we identified the first examples ofintrons in archaeal protein coding genes [15]. Furtheranalysis revealed that pre-mRNA fragments werecleaved at the exon-intron boundaries withheteromeric crencarcheal splicing endonuclease [5].
Notably, some of the exon-intron boundaries inorthologous genes in related species may not form
strict BHB motifs, including mismatches in thehelices and/or bulge loops with fewer or more than 3bases (Fig. 1B) [5,15,16]. Conventional euryarchaeal

Introduction
In bacteria and eukaryotes, some types of RNA
splicing occur by precursor RNAs themselves, orwith the aid of trans-factors including RNA-protein
complexes [1]. However, in Archaea, with a few
exceptions of group II introns found in Euryarchaeota[2], almost all of the introns in pre-tRNA [3],pre-rRNA [4], and pre-mRNA [5] are thought to be
spliced through two steps catalyzed by protein-based
enzymes: cleavage at exon-intron boundaries inprecursor RNA with splicing endonuclease [6], andligation of the exon fragments with RNA ligase [7].
The splicing endonuclease recognizes a specificstructure in the substrate precursor RNA
bulge-helix-bulge (BHB) motif, composed of a4-basepair central helix flanked by two 3-base bulge
loops at the 3’ sides together with basepairs, andcleaves the specific sites in the “bulge” region [8, Fig.
1A]. The recognition of the substrate with theenzyme is independent of the other part of thesubstrate, including the majority of the exon region[9]. Archaeal splicing endonucleases are classified
into three subclasses based on subunit structures:homo-tetrameric enzymes, homo-dimeric enzymes,
and hetero-tetrameric (alpha2beta2) enzymes [6].
Archaeal splicing endonuclease shares a commonancestor with eukaryotic tRNA splicing endonuclease[10].
The recently identified archaeal RNA ligase[11] shares a common ancestor with a bacterial RNA
repair enzyme [12] and a human tRNA ligase [13].
Archaeal RNA ligase joins the 3’ end of the 5’ exonFig 1. BHB motifs predicted in archaeal cbf5 pre-mRNAs[15]. A, a conventional BHB motif in Aeropyrum pernix
cbf5 pre-mRNA. B, a relaxed BHB motif in Sulfolobus
tokodaii cbf5 pre-mRNA. Rectangles in blue represent thecentral 4-bp helices. Triangles in green represent the 3-basebulge loops. Note that in S. tokodaii pre-mRNA, the Cresidue at the 3’ side of the 3’ bulge (indicated in red)cannot form a Watson-Crick basepair with the A residue(also in red) at the 5’ side of the central helix. Arrows
represent cleavage sites with the splicing endonucleaseconfirmed in vitro [5,18]. Upper case, exon regions; lower
case, intron regions.

Viva Origino 41 (2013) 12 - 13
© 2012 by SSOEL Japan12Viva Origino 41 (2013) 12 - 13

homo-tetramericandhomo-dimericsplicing
endonucleases cannot cleave such relaxed forms of
the BHB motif [6]. To reveal the structural elementsthat expand the substrate recognition in the
crenarchaeal heteromeric enzyme, we determined the
crystal structures of two crenarchaeal enzymes, andfound that they take a hetero-tetrameric (alpha2beta2)
subunit structure [17,18]. Moreover, we showed thatthe specific insertion of amino acid sequences in the
crenarchaeal enzymes, rather than the subunit
structure of the crenarchaeal enzymes, is importantfor substrate recognition [17,18]. Transplantation of
the insertion sequences into a euryarchaeal
homo-dimericenzyme
expanded
substrate
recognition [19,20, Yoshinari, S. and Watanabe, Y.
unpublished results]. A specific lysine residue in theinserted sequence is important for the substrate
recognition/catalysis of the enzyme [18,19,20].
Although the nanoarchaeal enzyme with the
expanded substrate recognition does not have theinsertion at the corresponding position [21], a novel
insertion at the alternative position may have asimilar role [18].

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16. Yokobori, S., Itoh, T., Yoshinari, S., Nomura, N., Sako, Y.,
Yamagishi, A., Oshima, T., Kita, K., and Watanabe, Y. Gain
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17. Yoshinari, S., Shiba, T., Inaoka, D.K., Itoh, T., Kurisu, G.,Harada, S., Kita, K., and Watanabe, Y. Functional importanceof crenarchaea-specific extra-loop revealed by an X-ray
structure of a heterotetrameric crenarchaeal splicing
endonuclease. Nucleic Acids Res. 37, 4787-4798 (2009)
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conserved lysine residue in the crenarchaea-specific loop is
important for the crenarchaeal splicing endonuclease activity.
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tRNA splicing and the origin of tRNA
It has been proposed that tRNA was born by afusion of two half RNA molecules which included asingle stem-loop flanked by single strand regions atboth the 5’ and 3’ sides [22]. Interestingly, some ofthe eukaryotic pre-tRNAs including introns in theanticodon loops have autocatalytic hydrolysis at ornear the position of exon-intron boundaries [23],suggesting that a pre-tRNA molecule with an intron
may be related to the prototypical tRNA. It would beinteresting to investigate the autocatalytic hydrolysisof archaeal pre-tRNAs. Furthermore, many bacteria
have homologs of archaeal and eukaryotic (t)RNA
ligase, although bacteria do not have tRNA splicing
driven by splicing endonucleases and RNA ligases[7], suggesting that this type of RNA ligase may exist
in the last common ancestor of the three domains oflife, and may have played a role in forming a
prototype tRNA from two tRNA half molecules.
Acknowledgments
Our studies were partly supported by themetabolic protein group of the National Project on
Protein Structural and Functional Analyses, andGrants-in-Aid of the Ministry of Science, Sports andCulture, Japan to YW and SY.
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