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tion E(157)22 RNA^ with AP (alkaline phosphatase)
or AP and PNK (polynucleotide kinase),
or PNK alone all shifted the mobility of the
fragment one position upward in the gel, which
was consistent with the removal of the 3-
phosphate of the pCp label. In contrast, a 3
fragment that resulted from cleavage at IPS1
without the IPS2 modification E(166)22 RNA^
was shifted two positions upward with AP, one
position when phosphorylated with PNK after
AP treatment, and one position with PNK
alone. This is consistent with removal of the
3-phosphate (from the pCp) as well as an additional
phosphate at the 5 endleftbyIPS1
cleavage. Thus, the phosphate at the 5 end of
the 22-nt 3 fragment is accessible to phosphatase
in the absence of the IPS2 modification
but inaccessible when the IPS2 modification is
present. This feature of the IPS2 modification
could be removed by incubation of (157)22
RNA with 166 RNA before the analysis, as
shown in the last panel in Fig. 2A. Thus, both
the primer extension stop at IPS2 and blocking
of the 5 end are reversible. An explanation
for these observations is that GIR1
cleavage occurs by a transesterification reaction
in which cleavage at IPS1 is coupled to
formation of a 2,5-phosphodiester bond between
C230 and U232. This explains the
primer extension stop at IPS2, the blocking of
the 5 end, the conservation of internal energy
after cleavage, and the reversibility of the reaction.
Consistent with the engagement of the
2OH of U232, this nucleotide is resistant to
alkaline hydrolysis in (157)22 RNA, in contrast
to (166)22 RNA (fig. S3).
Branches in RNA are resistant to digestion
with various RNases including mung bean
nuclease (13). A resistant fragment was found
in mung bean nuclease digests of bodylabeled
(157)22 RNA but not (166)22 RNA
(fig. S4 and SOM text). Digestion of (157)22
RNA with the exonuclease snake venom
phosphodiesterase resulted in a resistant fragment
corresponding to the 4-nt lariat circle
(Fig. 2B) that could subsequently be cleaved
by the endonuclease mung bean nuclease to
release the branched nucleotide and pA (Fig.
2C). These analyses are consistent with the
presence of the proposed 2,5-phosphodiester
bond between C230 and U232. The sequence
of the branch was verified by thin-layer
chromatography (TLC) analysis of the nucleotides
liberated by snake venom phosphodiesterase
cleavage of purified branch nucleotide
(Fig. 2D).
Formation of the branched nucleotide implies
a reaction mechanism in which the
2OH of U232 makes a nucleophilic attack
at the phosphodiester bond at IPS (Fig. 3A).
To test this mechanism, we made cleavage
analyses combining a ribozyme truncated in L9
(157.-7) and site-specifically deoxy-substituted
substrates that complemented the truncated
ribozyme (7.22). Only the dU232 substrate did
not support cleavage (Fig. 3B). Weak cleavage
with the dA231 substrate is ascribed to a
critical structural role of this nucleotide. The
cleavage in the all-RNA, dC230, dA231, and
dC233 substrates was by transesterification as
shown by primer extension analysis (fig. S5).
We have shown here that GIR1 cleaves by
transesterification, not by hydrolysis as proposed
previously (5). The reaction leaves a 5 fragment
containing a fully active ribozyme with a
3OH, and a 3 fragment in which the first and
the third nucleotides are linked by a 2,5-
phosphodiester bond. A 4-nt lariat was found
by nuclear magnetic resonance (NMR) imaging
to have an unusual structure with the sugars in
the lariat ring locked in a rigid South-type
conformation (14). We refer to the similarly
sized lariat in Didymium as the lariat cap
because it is found to cap the cellular I-Dir I
mRNA (Fig. 3C). Other studies have shown
that IPS1-cleaved RNA cannot be reactivated
for modification at IPS2, which suggests a
mechanistic coupling of the two reactions.
Hydrolytic IPS1 cleavage thus appears as a
failure to link the 5-phosphate of C230 to the
A
B
U
A
P4
P6
GIR1
G
A
U
C
C
G C
A C
A
U
C
C
G
U
C
C
U
A
C
A
G
G
G
G
G
U
G
G
C
A
G
G
C
A
P2.1
G
C
A
A
A
P15
HEG
A
A
U
C
G
G
G
U
U
G
A
A
C
A
C
U
U
A
A
U
U
A
U
G
G
C
C
U
A
A
G
U
A
A
C
U
U
G
U
G
IPS1
P5 . IPS2
P10
.
.
GIR2
U
P2
157 166
A
A
G
G
A
IPS1 A
G
C
A
G
C
C
U
U
G
C
U
G
A
C
A
A
U
U
G
G
G
U
G
G
A
U
.
.
U
SSU rDNA
Dir.S956-1
166.22 RNA
157.22 RNA
(166)22 RNA
(157)22 RNA
166 RNA
157 RNA
157.-7 RNA
7.22 RNA
U
U
A G GGU
U
GG
- 5’
A C
G G U A C U A U G A
GU UGGUU
U
A . .
C C A U G A U G C U C C CA
A CA AUC
A
G
A A
U
A
U
C A
- ORF- 3’
22
P9
C
A
G
A
C
U
G
C
A
C
G
G
C
C
C
U
G
C
C
U
C
P7
P3
P8
C
D
E
F
2OH of U232 and is considered an in vitro
phenomenon. We propose that IPS1 is denoted
IPS, and that IPS2 is replaced by BP (branch
point) in line with the nomenclature used for
group II and spliceosomal introns.
Our results show that a ribozyme with a
group I intron–like architecture can carry out an
RNA branching reaction similar to the first step
of splicing in group II introns and spliceosomal
introns. The GIR1 ribozyme has clear structural
distinctions from group I self-splicing ribozymes
including the lack of a P1 helix carrying the
5 splice site (5, 6). The two known GIR1
ribozymes from Didymium and Naegleria
show striking similarity to individual members
of group I eubacterial transfer RNA (tRNA)
introns to which the similarity in the core
structure is in the 60 to 80% range (6). Therefore,
GIR1 ribozymes may have arisen from
splicing ribozymes several times during evolution.
The rearrangements that led to the evolution
of GIR1 transformed a conventional 3
splice site (G,) into a 5 splice site AG, [incidentally
the consensus sequence of the exon
part of the 5 splice site of the major class of
G A T C
G A T C
166.22 157.22
166.22
157.22
(157)22 (157)22 (157)22
+ 157 + 166
M1 M2
32
P-22 nt
+ unlab. 157
32
P-22 nt
+ unlab. 166
IPS1
IPS2
22
IPS1
IPS2
22 nt
166.22
Fig. 1. (A) Schematic drawing of the structure of the Dir.S956-1 intron and the GIR1 RNAs described in
the text. (166)22 RNA refers to a 22-nt fragment isolated from cleavage of a 166.22 RNA precursor. (B)
Structure diagram of Didymium GIR1. (C) Primer extension analysis of RNA from an experiment
parallel to that shown in fig. S1. A sequencing ladder is shown to the left. (D) Cleavage analysis
performed as in fig. S1A, but by using precursor RNA that was labeled at its 3 end with [ 32 P]pCp
instead of body-labeling with [a- 32 P]UTP. (E) Primer extension analysis of gel-isolated and
reincubated (157)22 RNA alone, with 157 RNA, and with 166 RNA. The time points are 0, 1, 4,
and 8 hours. (F) Ligationofa22-nt3 fragment to a 166-nt 5 fragment. The 3 fragment was labeled
at its 3 end with [ 32 P]pCp. The 5 fragment was unlabeled. The time points are 0 and 20 min, and 1,
2, 3, and 4 hours. M1 and M2: 166.22 and 157.22, respectively, cleaved and labeled with [ 32 P]pCp.
www.sciencemag.org SCIENCE VOL 309 2 SEPTEMBER 2005 1585