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spliceosomal introns (1)^. Thus, the evolution
of GIR1 ribozymes illustrates a possible
step in the evolution of spliceosomal introns.
The homing endonuclease is expressed from
I-DirI mRNA formed by precursor ribosomal
RNA (rRNA) processing (10). The formation
of the 5 end of the I-DirI mRNAbycleavage
of the spliced out intron precludes the normal
cotranscriptional addition of a cap nucleotide.
The cap nucleotide in typical messengers serves
several functions, including protection from
degradation by 5Y3 exonucleases, and recruitment
of protein factors in initiation of
translation (15). The reaction at IPS in GIR1
results in protection of the 5 end of the I-DirI
mRNA against the activity of 5Y3 exonucleases
by formation of a lariat cap. This
potentially substitutes for one of the features
of the missing cap nucleotide. GIR1 processed
RNAs are stable when expressed in Escherichia
coli and yeast (11, 16), which supports this idea
of 5 stabilization by lariat formation.
The present observations extend the diversity
of natural ribozymes (3) and connect the group I
and group II introns, two major structural
classes of self-splicing RNAs. Despite its
overall structural similarity with other natural
group I introns, the GIR1 ribozyme is distinct
from the group I splicing ribozymes not only in
key structural features but also in the type of
reaction catalyzed and in the derived function.
References and Notes
1. C. B. Burge, T. Tuschl, P. A. Sharp, in The RNA
World, Second Edition, R.F.Gesteland,T.R.Cech,
J. F. Atkins, Eds. [Cold Spring Harbor Laboratory
(CSHL) Press, Cold Spring Harbor, New York,
1999], pp. 525–560.
2. C. R. Trotta, J. Abelson, in The RNA World, Second
Edition, R. F. Gesteland, T. R. Cech, J. F. Atkins, Eds.
(CSHL Press, Cold Spring Harbor, New York, 1999),
pp. 561–584.
Fig. 2. (A) Characterization of the 5 end of the 22-nt 3 fragment. [ 32 P]pCplabeled
3 fragment was isolated from 157.22 and 166.22 and subjected to
treatment with alkaline phosphatase (AP), AP followed by rephosphorylation
with T4 polynucleotide kinase (APxPNK), or treatment with PNK alone (PNK).
The sample denoted (157)22 166 was preincubated at reaction conditions
for 30 min before the analysis. OH and T1: Alkaline ladder and T1 digest of
[ 32 P]pCp–labeled precursor 157.22. (B) Diagram of the 22 nt lariat used for
experiments in (C) and (D). The RNA was body-labeled at the phosphates in
bold by incorporation of 32 P. Arrows indicate potential cleavage sites for mung
bean nuclease (MB) and snake venom phosphodiesterase (SV). Cleavage of the
22-nt fragment at sites labeled 1 with SV results in a protected lariat circle
(LC). Cleavage at sites labeled 1 and 2 with MB results in a protected branched
nucleotide (BR). Subsequent cleavage of BR with SV at sites labeled 3 releases
the nucleotides involved in the branch. (C) Characterization of the lariat circle
by gel purification and subsequent digestion with MB (LCxMB). The 22-nt
fragment and digests with MB or SV serves as markers. (D) Characterization of
the branched nucleotide by purification of its phosphorylated and dephosphorylated
form, and subsequent TLC analysis of nucleotides liberated by
digestion with SV. The first two runs show digests of the 22-nt fragment. The
followingshowtheisolatedbranch(BR),anddephosphorylatedbranch(BRAP),
respectively. Finally, the last two runs show the subsequent digests of these
with SV (BRxSV and BRAPxSV).
Fig. 3. (A) Outline of the reaction catalyzed by GIR1. The 2OH of the
internal residue U232 makes a nucleophilic attack at the IPS. Bond lengths
are not drawn to scale. (B) Cleavage experiment using 157.-7 ribozyme
combined with four different deoxy-substituted substrates each containing
7 nucleotides upstream and 22 nucleotides downstream of IPS.
Numbering of nucleotides is according to their position in the intron. (C)
Diagram showing the structure of the fully processed I-Dir I mRNA that encodes the homing endonuclease.
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2 SEPTEMBER 2005 VOL 309 SCIENCE www.sciencemag.org