Group

I and group
ROLAND

SALDANHA,

II introns
GEORG MOHR

MARLENE

BELFORT

AND ALAN M. LAMBOWITZ,i

Departments
of Molecular Genetics and Biochemistry,
and the Biotechnology
Columbus, Ohio 43210, USA; and tMolar
Genetics Program,
Wadsworth
New York State Department
of Health, Albany, New York 12201-0509, USA

ABSTRACT
Group
I and
group
II introns
are two
types
of RNA enzymes,
ribozymes,
that catalyze
their
own splicing
by different
mechanisms.
In this review, we
summarize
current
information
about
the structures
of
group I and group II introns,
their RNA-catalyzed
reactions,
the facilitation
of RNA-catalyzed
splicing
by protein factors,
and the ability
of the introns
to function
as
mobile
elements.
The RNA-based
enzymatic
reactions
and intron
mobility
provide
a framework
for considering
the role of primordial
catalytic
RNAs in evolution
and the
origin
of introns
in higher
organisms.Saldanha,
R.,
Mohr, G., Belfort,
M., and Lambowitz,
A. M. Group I
and group
II introns.
FASEBJ. 7: 15-24; 1993.
Ky

Words: catalytic RNA

mobile int,vn
reverse transcriptase
evolution

GROUP

site-specific endonucleose

I INTRONS

Group
I introns
are present
in rRNA,
tRNA,
and proteincoding genes. They are particularly
abundant
in fungal and
plant mitochondrial
DNAs (mtDNAs),2
but have also been
found in nuclear
rRNA
genes of Tetrahymena and other lower
eukaryotes,
in chloroplast
DNAs
(ctDNAs),
in bacteriophage,
and recently
in several tRNA
genes in eubacteria
(see refs 1-3). Most group I introns
have a highly variable
distribution,
even in related organisms,
apparently
reflecting
their dispersal
as mobile elements.
On the other hand,
the
same group
I intron
is present
in the tRNA
genes of
ctDNAs
and five different
cyanobacterial
species, suggesting
that it existed before the evolution
of plastids
and could be
1 to 3.5 billion years old. The finding that some group I introns are ancient
is consistent
with the idea that they are
remnants
of a primordial
RNA world. Indeed,
the catalytic
activities
of group I introns
have begun
to provide
insight
into how primitive
RNAs
could have catalyzed
their own
replication
and contributed
to the evolution
of protein
synthesis.
Splicing

mechanism

and

structure

As first shown for the Tetrahymena large rRNA

intron,
group
I introns splice by a mechanism
involving
two transesterification reactions
initiated
by nucleophilic
attack of guanosine
at
the 5 splice site (Fig. 1) (4). The remarkable
finding for the
Tetrahymena intron was that splicing
requires
only guanosine
and Mg.
Because
bond formation
and cleavage
are coupled, splicing requires
no external
energy source and is completely reversible.
After excision,
some group I introns
circularize
via an additional
transesterification,
which
may
contribute
to shifting
the equilibrium
in favor of spliced
products
(4).
The ability of group I introns
to catalyze
their own splicing is related
to their highly conserved
secondary
and ter0892-6638/93/0007-0015/$01.50.

FASEB

Center, The Ohio State Universit

Center for Laboratories
and Research,

tiary structures
(Fig. 2) (1, 5, 6). As in protein
enzymes,
the
folding of the intron results in the formation
of an active site
juxtaposing
key residues
that
are widely
separated
in
primary
sequence.
This RNA structure
catalyzes
splicing by
bringing
the 5 and 3 splice sites and guanosine
into proximity and by activating
the phosphodiester
bonds
at the
splice sites (4). Different
group I introns have relatively
little
sequence
similarity,
but all share a series of the short, conserved sequence
elements
P, Q, R, and S, with parts of P/Q
and R/S base pairing
in the conserved
structure
(Fig. 2A).
The boundaries
of group I introns
are marked
simply by a
U residue at the 3 end of the 5 exon and a G residue at the
3 end of the intron (5, 6).
The conserved
group
I intron
secondary
structure
was
deduced
from phylogenetic
comparisons,
and specific
features have been confirmed
by analysis of in vivo and in vitro
mutations
and by structure
mapping
(1, 5, 6). The structure,
shown
in Fig. 2A, consists
of a series of paired
regions,
denoted
P1-PlO,
separated
by single-stranded
regions
(denoted
J) or capped by loops (denoted L). P1 and PlO,
which
contain
the 5 and 3 splice sites, respectively,
are
formed by base pairing
between
an internal
guide sequence
(IGS), generally
located just downstream
of the 5 splice site,
and exon sequences
flanking the splice sites. Group I introns
have been classified
into four major subgroups,
designated
IA to ID (I), based on distinctive
structural
and sequence
features.
Group
IA introns,
for example,
contain
two extra
pairings,
P7.l/P7.la
or P7.l/P7.2,
between
P3 and P7,
whereas
many group lB and IC introns
have a large extension of P5, termed P5abc.
Individual
introns
may contain
additional
sequences,
including
open
reading
frames
(ORF5),
in positions
that do not disrupt
the conserved
core
structure.
The region
of the Tetrahymena intron
required
for enzymatic
activity,
the catalytic
core, consists
of P3, P4, P6,
P7, P8, and P9.0 (1). Studies using Fe(II)-EDTA,
a reagent
that cleaves the sugar-phosphate
backbone,
have shown that
parts of the core are buried
in the structure
inaccessible
to
the solvent,
that Mg2 is necessary
for folding of the intron,
and that individual
RNA domains
fold in a specific order as
Mg2 is increased
(4, 7). All group I introns have fundamentally similar core structures,
but subgroup-specific
structures
such as P7.1, P7.2, and P5abc appear to participate
in addi-

To whom correspondence
should be addressed, at: Department
of Molecular
Genetics,
The Ohio State University,
484 West
Twelfth Ave., Columbus,
OH 43210, USA.
2Abbreviations:
ctDNA, chioroplast
DNA; EBS, exon binding
site; IBS, intron binding site; IGS, internal guide sequence; LTR,
long terminal repeat; mtDNA,
mitochondrial
DNA; ORF, open
reading frame; aaRS, aminoacyl-tRNA
synthetase;
RT, reverse
transcriptase.
To accommodate
a limitation of the number of references, we
have cited reviews wherever possible.
15

Intron

5 Exon

3Exon

muon

5 Exon

GUGCG

3Exon
A

AY

3.j

I
Intron

3Exon

CExon

+
5 Exon

5 Exon
,2wcccc?cclU

3.OH

I,

I,
5 Exon

5 Exon 3Exon

3Exon
U

wmnn

CL

Group H Intron

Group I Intron

Figure L Splicing mechanisms of group I and group II introns.

Conserved nucleotides in introns and flanking exons are indicated.

tional interactions
that stabilize
the core structure
in different ways (1, 8).
A three-dimensional
model of the group I intron catalytic
core has been developed
by Michel and Westhof (1) (Fig. 2B,
C). The underlying
assumption,
first suggested
by Kim and
Cech (9), is that adjoining
helical segments
stack coaxially
to
create
two extended
helices,
P6a-P6-P4-P5
and P8-P3-P7,
which form a cleft containing
the introns active site (Fig. 2B,
C). In the Michel-Westhof
model, the relative
orientation
of
the two helices is constrained
by a previously
proposed
triple
helix involving
parts ofJ3/4-P4-P6-J6/7
and by potential
tertiary interactions
identified
by covariation
of nucleotides
that
are not accounted
for by secondary
structure.
A number
of
these
predicted
interactions
involve
purine-rich
loops or
bulges
engaged
in long-range
interactions
with
double
helices (Fig. 2B). The active site of the intron, formed by the
cleft between
the two helices, contains
binding
sites for the
guanosine
cofactor
and P1 and PlO containing
the 5 and 3
splice sites. The model is designed
so that the disposition
of
these binding
sites accounts
for the known splicing mechanism, which
requires
appropriate
alignments
of guanosine
and the 5 and 3 exons in the first and second steps of splicing (4). Deoxynucleotide
and phosphorothioate
substitution
experiments
suggest that functionally
important
Mg2 ions
are coordinated
at specific positions
around
the active site
(e.g., P1 and J8/7), where they may function
directly in phosphodiester
bond
cleavage
(1, 10). Basic
features
of the
predicted
three-dimensional
structure
have been supported
by mutant
analysis in vitro and by the use of specifically
positioned photochemical
cross-linking
and affinity cleavage reagents (1, 11, 12).
Definition

of splice

sites

and

binding

to the

intron

core

The 5 and 3 splice sites of group I introns

are substrates
that are acted on by the catalytic
core, and they can be recog-

16

Vol. 7

January 1993

nized and cleaved by the core when added on separate

RNA
molecules
(4). The 5 splice site is defined by the P1 pairing
between
the lOS and the 5 exon. Neither
the sequence
nor
length of P1 is fixed, but the conserved
U at the 3 end of the
5 exon always forms a wobble base pair with a 0 residue
in
the IGS (Fig. 2A). Analysis
of in vitro mutants
showed that
the distance
of the UG pair from the bottom
of the P1 helix
is critical
for efficient
cleavage
in the Tetrahymena intron
(4,
13) and that Jl/2 and P2 also play a role in the positioning
of P1 relative
to the core (1, 14, 15).
In the Michel-Westhof
model,
P1 is predicted
to bind to
the core via tertiary
interactions
with J8/7 and J4/5 (Fig. 2B)
(1). Experiments
have shown that the 2 OHs at P1 positions
-2 and -3 contribute
energetically
to binding
P1 to the core,
and that the 2 OH at -3 interacts
with a specific residue
in
J8/7. The latter interaction
is somewhat
different
from that
proposed
by Michel and Westhof (1), but was readily accommodated
by a local revision
of the model (12).
The positioning
of the 3 splice site in group I introns
depends on at least three interactions,
whose relative
importance differs in different
introns
(4). These are the PlO pairing between
the lOS and the 3 exon,
binding
of the
conserved
0 residue
at the 3 end of the intron
to the 0binding
site in the second step of splicing,
and an additional
interaction,
P9.0, which involves
base pairing
between
the
two nucleotides
preceding
the terminal
0 of the intron and
two nucleotides
in J7/9 (Fig. 2A).
Guanosine-binding

site

Group
I introns
have Km values for guanosine
that are as
low as 1 eM and readily
discriminate
between
guanosine
and other
nucleosides
(4). The major
component
of the
guanosine-binding
site corresponds
to a universally
conserved GC pair in P7 (Fig. 2A) (1, 4). Guanosine
was initially
proposed
to interact
with this base pair via formation
of a
base triple, but the contribution
of neighboring
nucleotides
and the binding
of analogs
are also consistent
with a model
in which guanosine
binds axially to the conserved
0 and
flanking
nucleotides
(16). The
guanosine-binding
site of
group
I introns
can also be occupied
by the guanidino
groups
of arginine
or antibiotics,
such as streptomycin,
which act as competitive
inhibitors
of splicing (17). Yarus (18)
noted
that
the
three
nucleotides
that
constitute
the
guanosine-binding
site in different
introns,
AGA/G
and
CGA/G,
correspond
to Arg codons,
and speculated
that
recognition
of amino acids by this and other functionally
important
sites in catalytic
RNAs could have played a role in
the evolution
of the genetic code.
Other

reactions

catalyzed

by group

I introns

In addition
to splicing,
group I ribozymes
can catalyze
a variety of intermolecular
reactions,
including
endonucleolytic
cleavage
of RNA and DNA, RNA polymerization,
nucleotide transfer,
templated
RNA ligation,
and aminoacyl-ester
cleavage
(4, 19). All these reactions
use the same active site
as the splicing
reaction,
and they can be generalized
as
shown in Fig. 3A. In the forward direction,
equivalent
to the
first step of splicing,
the reaction
is thi nucleophilic
attack of
guanosine
on the phosphodiester
b nd succeeding
XXU
(where XX denote exon nucleotides
that pair with the IGS
[XX] to form P1). In the reverse dire tion, equivalent
to the
second step of splicing,
the reaction
is nucleophilic
attack of
the 3OH of XXU on the phosphodiester
bond 3 of the terminal 0 of the intron.
In the intermolecular
reactions,
the
substrates
are oligonucleotides
that are either cognates
of P1

The FASEBJournal

SALDANHA ET AL.

L5

LI

L2

P1

Ill
L8

P8

Figure 2. Group I intron structure. A) Conserved

secondary structure. Arrows indicate splice sites. Conserved sequences P, Q, R, and
S (5, 6) are indicated by heavy lines. Asterisk denotes guanosine-binding
site. Dashes indicate base pairs. Dot indicates wobble base pair
at the 5 splice site. Nucleotides involved in P9.0 pairing are indicated as NN and NN. Nucleotides involved in PlO pairing are indicated
as MMM and MMM.
IGS, internal guide sequence. B) Schematic illustrating some proposed tertiary interactions
in the yeast w intron
(1, 8). Interacting
nucleotides are connected by dashed lines. Circled G shows free guanosine interacting
with conserved GC pair of the
guanosine-binding
site. C) Three-dimensional
structural
model of the Tetrahymena
large rRNA intron from Michel and Westhof (1),
reproduced
by permission of the Journal of Molecular Biology.
or base pair with the lOS to reconstitute
analogs
of P1 or
PlO.
In the site-specific
endonuclease
reaction,
an RNA substrate that resembles
the 5 exon and base pairs to the IGS
is cleaved by guanosine
(Fig. 3B) or OH- (not shown) in a
process analogous
to the first step of splicing.
This reaction
has been modified
in various ways that illustrate
the versatility of the ribozyme.
The rate-limiting
step is the release of
the cleaved RNA products,
so that mutations
in the lOS that
weaken RNA binding
increase
the rate of reaction
(20). The
substrate
specificity
of the reaction
is determined
by the sequence of the IGS, which can be changed
to enable the ribozyme
to cleave
other
RNA
substrates.
DNA
is cleaved
inefficiently,
but ribozymes
having enhanced
DNA cleavage

GROUP

I AND

GROUP

II INTRONS

activity
have been obtained
by iterative
selection
(21, 22).
Recently,
the Telrahymena ribozyme
was shown to catalyze
the
reverse
of an aminoacylation
reaction,
hydrolysis
of an
aminoacyl-ester,
N-formyl-L-methionine,
attached
to the
CCA end of an oligonucleotide
that base pairs to an appropriately
modified
IGS sequence
(Fig. 3E) (19). Although
inefficient,
this reaction
demonstrates
that the ribozyme
can
act on carbon centers,
and supports
the hypothesis
that ancient catalytic
RNAs
could have functioned
as aminoacyltRNA synthetases
in the early evolution
of protein synthesis.
In support
of the idea that primitive
RNAs
could have
been the first self-replicating
molecules,
group I ribozymes
have been shown to act as RNA polymerases.
Initially,
nontemplated
addition
of nucleotides
to oligonucleotides
bound

17

Active Site
Activity

Interactions

X.X.G
EGS

A) Generalized
XXUpN

Forward

G-OH

XXU-OH

GpN

Splicing

Lr
XX

U-OH

mum

XXG

Ts

B)

Reverse

Sequence-SpecificEndonuclease

-CUCUpN-

-CUCU-OH

G-OH---

GpN-

Lra

C) Nucleotidyl Transferase
CCCC C-OH

CCCCC-OH

GpN

CCCCCpN

-.

0) Template-Dependent

1mmmm

G-OH

rGGAGG
I
OS

Ligation
-

-M

GpN-

E) Aminoacyl

-MpN-

MOH NI II

I I

Esterase
CAACCA

CAACCAfMet + OH

CAACCA-OH

fMet

IMet

mm

rGU U000
3$

Figure
3. Reactions
catalyzed
by group
I ribozymes.
The
guanosine-binding
site of the intron core is indicated by an indentation. This site may be occupied by a free guanosine in the first step
of splicing or by the conserved guanosine at the 3 end of the intron
in the second step of splicing. The figure is based on Cech (4).
to the introns
lOS was demonstrated
by intron-catalyzed
disproportionation,
nucleotidyl
transferase,
or ligation
reactions (4). The latter are analogous
to the second step of splicing in that short oligonucleotides
bound
to the lOS can attack
dinucleotides
GpN
or oligonucleotides
OpN(N),
where the 0 residue of the di- or oligonucleotide
is analogous
to the conserved
0 at the 3 end of the intron (Fig. 3C). Subsequently,
template-dependent
addition
of oligonucleotides
was demonstrated
in a reaction
analogous
to reversal of the
first step of splicing (Fig. 3D) (23). Fortuitously,
the addition
of spermidine
suppressed
the need for the UG pair ordinarily required
at the ligation junction,
enabling
the reaction
to
tolerate
a Watson-Crick
pair at this position
(23). A recent
breakthrough
toward achieving
self-replicating
RNA was the
demonstration
that different
segments
of a group I ribozyme
could assemble
to form a multisubunit
ribozyme
that replicated one of its segments
by template-directed
ligation
of
oligonucleotides
(24).
GROUP

II INTRONS

Thus far, group II introns

have been found only in fungal
and plant mitochondria
and in chloroplasts.
Most of these
introns are in protein-coding
genes, with a few in tRNA and

18

Vol. 7

January

1993

rRNA genes (25). As in the case of group I introns,

the variable distribution
of group II introns
suggests
that they were
dispersed
recently
as mobile elements,
but unlike group I introns, group II introns
have not been found outside
of organelles (2, 25). Group
II introns
are of particular
interest
because of their possible evolutionary
relationship
to nuclear
mRNA
introns,
which was suggested
initially
by similarities
in their splicing
mechanisms
(25-28).
mechanism

and

structure

The splicing mechanism

of group II introns
is shown in Fig.
1. As in nuclear
mRNA
introns,
splicing
is initiated
by the
formation
of an intron lariat in which the 5 end of the intron
is linked
by a 2-5 phosphodiester
bond to a nucleotide
residue,
usually an A, near the 3 end of the intron (25). A
few group II introns
have been shown to self-splice
in vitro,
but only at slow rates (11,2 = 10 mm) under nonphysiological conditions
(e.g., 45#{176}C,
100 mM Mg),
which
suggests
that additional
trans-acting
components
are ordinarily
required
for efficient splicing.
In contrast
to the situation
for
nuclear
mRNA
introns,
branching
is not obligatory
for the
splicing of group II introns,
and at least under in vitro conditions H2O or OH- are able to substitute
for the branch
point
2 OH in the initial nucleophiic
attack at the 5 splice site
(29).
Like group I introns,
group II introns have little sequence
similarity,
but share a conserved
secondary
structure
required
for catalytic
activity.
This
structure
is generally
depicted
as six helical domains
(I to VI) radiating
from a
central wheel (Fig. 4) (25). Two major subclasses
of group II
introns
(IIA and IIB) have been distinguished
based
on
structural
features,
and like group
I introns,
individual
group II introns may contain
additional
sequence,
including
ORFs,
in positions
that do not disrupt
the conserved
structure. Group
II introns
have conserved
5- and 3-boundary
sequences
(GUGYG
and AY), which
somewhat
resemble
those in nuclear
mRNA
introns
(25).
Although
there is no three-dimensional
model for group II
introns,
the function
of some domains
has been established
(25, 30, 31). Domain
I contains
binding
sites for the 5 and
3 exons (see below).
Domain
VI is a helix containing
the
branch
site, usually a bulged A residue.
Domain
V, the most
highly conserved
substructure,
is required
for catalytic
activity and binds to domain
I to form the catalytic
core. A
derivative
of yeast intron
aI5-y containing
domains
I, III,
and V splices, albeit poorly, and a derivative
containing
only
domains
I and V cleaves at the 5 splice site (29). Consistent
with the view that group
II intron
domains
could have
evolved into trans-acting
snRNAs,
domain V and subdomain
ICI can function
in trans to facilitate
in vitro splicing of mutant introns
lacking
these domains
(31, 32).
Definition

of splice

sites

As in group I introns,
the definition
and binding
of the 5
splice site in group
II introns
depends
on interaction
between the intron and sequences
in the 5 exon (25, 28, 30).
The most critical interaction
is base pairing
of exon binding
site I (EBS1), a short sequence
in domain
I, with exon sequences
immediately
upstream
of the 5 splice site (intron
binding
site 1 [IBS1J; Fig. 4). Like the P1 pairing
in group
I introns,
the EBSI-IBS1
pairing
is not conserved
in sequence,
but the 5 splice site is always after a specific base
pair in the structure.
Two additional
base-pairing
interactions, EBS2-IBS2
and c-c, also contribute
to positioning
the
5 splice site.

The FASEBJournal

SALDANHA

ET AL.

vI
5_

Figure 4. Group II intron conserved

secondary
structure.
The
drawing is based on yeast intron coxl-15y (28). I to VI represent
group
II intron
domains.
Dotted
lines indicate
interactions
described in the text. EBS, exon binding site; IBS, intron binding
site.

The definition
of the 3 splice site in group II introns
involves contributions
of at least four interactions:
1) docking
of domain
VI to the core, 2) base pairing
of the terminal
nucleotide
of the intron and a nucleotide
between
domains
II and III (-y--y), 3) base pairing
of the first nucleotide
of the
3 exon and the nucleotide
preceding
EBS1 in a guide interaction, and 4) another,
undefined
interaction,
also involving
the first nucleotide
of the 3 exon (25, 30). Under
in vitro
conditions
some of the 5 and 3 splice site interactions
in
group
II introns
are dispensable,
but they may all be required
for efficient
splicing
in vivo (30).

Degenerate

and

trans-spliced

group

II introns

Degenerate
group II introns that are functional
despite lacking some domains
have been found in plant mitochondria
and chloroplasts
(25). Euglena ctDNA,
for example,
contains
a large number
of relatively
short group II introns
(277-618
nt compared
with
1 kb), which sometimes
lack recognizable cognates
of domains
I, II, III, or IV, and it also contains a potentially
related class of short (100 nt) AT-rich
introns
termed
group III introns (25, 33). The latter have 5
boundary
sequences
that are degenerate
versions of the conserved
group II intron
sequence,
and some have potential
cognates
of domains
I and VI. The degenerate
group II and
group III introns
may be akin to evolutionary
intermediates
between
group
II introns
and nuclear
pre-mRNA
introns
(34, 35). They presumably
require
trans-acting
factors
for
splicing,
and it is possible that missing RNA domains
are encoded elsewhere
and function
in trans.
The ability
of group
II intron
domains
to reassociate
specifically
in vivo is evidenced
by trans-spliced
group II introns, which have been found in the rps-12 gene of higher
plant
ctDNA,
the psaA gene in Chiamydomonas
reinhardtii
ctDNA,
and the nadi and nad5 genes in higher plant mtDNA

GROUP

I AND GROUP

II INTRONS

(25, 26). These

genes consist
of widely
separated
exons
flanked
by 5- or 3-segments
of group II introns
split in
either domains
III or IV. The exons at different
loci are transcribed
into separate
precursor
RNAs,
which
are transspliced, presumably
after the association
of the two segments
of the group II intron.
In all these cases, continuous
versions
of the same gene or closely related
genes are present
in bacteria or in organelles
of other species,
suggesting
that transspliced genes reflect genomic
rearrangements
that occurred
within
group II introns.
Genetic
analysis of trans-splicing
of the Chiamydomonas
rein/zardtii psaA gene showed
that a number
of nuclear
gene
products,
presumably
proteins,
are required.
Additionally,
intron 1 of this gene is split into three segments.
The 5 exon
is flanked by parts of domain
I and the 3 exon by parts of
domains
IV to VI, respectively.
The middle
segment
of the
intron is encoded
at a remote locus, tscA, and consists of the
remainder
of domains
I to IV. This tscA segment
can apparently associate
with the other two intron segments
to reconstitute the intron (36). The findings for tscA RNA bolster the
idea that group II intron domains
might have evolved into
snRNAs
in the evolution
of nuclear
pre-mRNA
introns.
An evolutionary
relationship
and nuclear
mRNA
introns?

between

group

II introns

The similar
splicing
mechanisms
of group
II and nuclear
mRNA
introns
immediately
suggested
a possible evolutionary relationship,
and this belief has been reinforced
by
findings
of degenerate
group II introns
in some organisms
and the ability
of group II intron
domains
to function
in
trans. The evolution
of group
Il-like
introns
into nuclear
mRNA
introns
is thought
to have occurred
by the progressive loss of internal
RNA structures,
which were assimilated
by the host organisms
and evolved into snRNAs
(26-28).
Indeed, it seems difficult
to imagine
anything
other than an
evolutionary
rationale
for the existence
of snRNAs,
as splicing could be carried
out simply by using protein
enzymes,
as for nuclear
tRNA
introns.
Cavalier-Smith
(37) and Palmer
and Logsdon
(2) noted
that nuclear
mRNA
introns
are limited
to recently
evolved
eukaryotes
and thus far appear
to be lacking
in Giardia and
other
primitive
eukaryotes.
Because
of this
restricted
phylogenetic
distribution,
they argue that nuclear
mRNA
introns
arose late in eukaryotic
evolution
and suggest
that
this might have involved
transfer
of organellar
group II introns to the nucleus.
The required
process of DNA transfer
from organelles
to nudear
genomes
is well documented.
Once in the nucleus,
the separation
of transcription
from
translation
would lessen the selective pressure
for rapid splicing and permit evolution
of the slower spliceosomal
mechanism. Nuclear mRNA
introns could then disperse by a variety
of processes,
including
exon shuffling,
insertion
into protosplice sites, e.g. via reverse splicing,
and molecular
mimicry
by certain
transposable
elements
containing
splice sites near
their boundaries
(2).
Although
the structures
and functions
of group II intron
domains
and snRNAs
are not as yet sufficiently
defined
to
permit
strong conclusions
about evolutionary
relationships,
a number
of potential
correspondences
have been noted.
These include 1) the EBS1-IBS1,
guide and c-c interactions
involved
in defining
the 5 and 3 splice sites, which appear
analogous
to interactions
involving
Ui and U5 snRNAs
(38), 2) domain
VI, whose structure
resembles
that formed
by binding
of U2 snRNA
to nuclear
mRNA
introns
in that
the branch
point nucleotide
is bulged from a helix (28), and
3) domain
V, whose structure
and disposition
relative to the

19

branchpoint
may resemble
those resulting
from a newly
described
interaction
between
U2 and U6 snRNAs
(39).
Further
biochemical
analysis
should provide
insight into the
extent to which the group II intron and nuclear
mRNA
intron splicing
mechanisms
are related.

INVOLVEMENT
GROUP
I AND

OF PROTEINS
IN SPLICING
GROUP
II INTRONS

Group
I and group II introns
are presumed
to have been
self-splicing
initially,
but many of these introns
now require
proteins
for efficient splicing in vivo, presumably
in order to
compensate
for structural
defects
that have accumulated
during evolution
(1, 6, 40). Genetic
analysis of mitochondrial
RNA splicing in Neurospora and yeast has shown that some of
the proteins
required
for splicing
group I and group II introns
are encoded
by host chromosomal
genes,
whereas
others are encoded
by the introns
themselves.
Maturases
Several group I and group II introns in yeast mtDNA
encode
maturases
that function
in splicing
the intron that encodes
them. These include group I introns cob-12, -13, and -14, and
group
II introns
coxl-I1 and -12 (6, 40). The cob-I4 and
coxl-I1 proteins
are shown schematically
in Fig. 5. In all the
cases indicated
above, maturase
function
has been demonstrated
genetically
by showing that mutations
in the introns
ORF
result
in defective
splicing,
which
can be complemented
by the wild-type
protein
in vivo. Thus far, there
are no biochemical
assays
for maturases,
and although
related
ORFs
are present
in other
organisms,
the only
confirmed
maturases
remain
those defined
genetically
in
yeast.
Characterization
of mutants
has shown that all the yeast
mtDNA
maturases
primarily
function
only in splicing the intron that encodes
them,
except
for the cob-14 maturase,
which splices both cob-14 and another
closely related
group
I intron, coxl-14 (6, 40). The coxl-14 intron encodes a protein
that is structurally
related to the cob-14 maturase
and has a
latent maturase
activity
that can be activated
by mutation.
However,
the coxl-14 protein
does not ordinarily
function
in
splicing
and instead
has a site-specific
endonuclease
activity
that functions
in intron mobility
(40) (see below). The intron
specificity
of maturases
suggests
that they function
in splicing by recognizing
unique
structural
features
of the introns
that encode
them.
All the yeast mtDNA
group I and group II maturases
are
in frame with the upstream
exons, and the active maturase
may be generated
by proteolytic
cleavage downstream
of the
5 splice site (6, 40). This mode of synthesis
presumably
results in a feedback
regulation
in which a decreased
rate of
splicing
leads to an increased
amount
of maturase
and vice
versa. The maturases
encoded
by group I introns are characterized by two repeats of a sequence
motif variously
referred
to as P1 and P2, dodecapeptide,
or LAGLI-DADG
(Fig. 5).
This same motif is characteristic
of a larger family of proteins, which have site-specific
endonuclease
activities
that
mediate
group
I intron
mobility
(41). Group
II intron
maturases,
on the other hand,
are structurally
related
to
reverse transcriptases,
which may also function
in intron mobility (Fig. 5) (42). As discussed
elsewhere,
it seems likely
that
the mobility
functions
of group
I and
group
II
maturases
evolved
first and the splicing
function
evolved
secondarily
as a result of ability of the proteins
to recognize
specific sequences
or structures
within the intron or flanking
exons (40, 43).
20

Vol. 7

January 1993

The

FASEB

Nuclear-encoded

proteins

Nuclear-encoded
proteins
required
for splicing
mitochondrial
introns
have
been
identified
by
screening
of
cytochrome-deficient
mutants
(per in yeast or cyt in Neurospora) or by isolating
nuclear
suppressors
of splicing
mutants (6, 40). In Neurospora, the products
of three nuclear
genes (cyt-18, cyl-19, and cyt-4) have been implicated
in splicing the mt large rRNA intron and a number
of other group
I mitochondrial
introns.
By contrast,
most of the yeast proteins function
in splicing
only a single intron
(e.g., CBP2
functions
in splicing cob-I5 and MSS18 functions
in splicing
of coxl-I513).
As reviewed elsewhere
(40), the proteins
required
for splicing group
I introns
include
aminoacyl-tRNA
synthetases
(aaRSs)
and other proteins
that have some additional
function in their host cells. The Neurospora cyt-18 gene, for example, has been shown to encode the mt TyrRS.
Likewise,
the
yeast mt LeuRS,
which is encoded
by nuclear
gene NAM2,
functions
in splicing
the two closely related group I introns
cob-14 and coxl-14 (see previous
text), apparently
by acting in
concert
with one or both of the intron-encoded
proteins.
Studies
with protein-dependent
in vitro splicing
systems
have shown
that
the group
I intron
splicing
reactions
promoted
by the Neurospora CYT-18 and the yeast CBP2 proteins proceed by the same guanosine-initiated
transesterification mechanism
used by self-splicing
group I introns and remain dependent
on the conserved
group I intron
structure,
suggesting
that they are still essentially
RNA catalyzed
(40).
The CYT-18 protein,
which functions
in splicing many different group I introns,
was shown to suppress
structural
mutations in different
regions of the phage T4 Id or yeast a introns.
From
the spectrum
of mutations
that
could
be
suppressed,
it was inferred
that the CYT-18
functions
in
splicing by stabilizing
the catalytically
active structure
of the
group
I intron
core (44). The CYT-18
protein
binds
to
P4-P6,
a highly conserved
structure
of the group I intron
catalytic
core, and may additionally
contact
P7-P9 to stabilize the two major helices of the core in the correct
relative
orientation
to form the introns active site (45). The ability
of the CYT-18 protein,
the mt TyrRS, to bind specifically
to
the group I intron catalytic
core suggests
that the core may
have structural
features
that resemble
those in tRNAs,
which

Intron-Encoded Proteins
386 aa

Group I:
S.c. cob-14

#{149}
Il5aa,

Exon4

Exon 5

LAGLIDGDG

T4 td-11

245 ax

GroupI:

FIGFFDADG
-

16 aa
.

Exoni

fl\

Exon 2

ISV VVG

Group II:
S.c. coxl-I1

260aa

I
Exon

I:::
Z

75aa

53aa

..

RI-Homology

DomainX Zn linger Exon 2

Figure 5. Representatives
of three types of intron-encoded
proteins.
Demarcated
areas are those containing
conserved amino acid sequences shared by other members of the same protein family. Protein sequences that match consensus sequences described in the text
are shown below each protein. RT, reverse transcriptase.
Journal

SALDANHA ET AL.

could reflect convergent

evolution
or an ancestral
relationship between
group
I introns
and tRNAs
(45). The yeast
CBP2 protein
also requires
an intact catalytic
core for binding, but in this case, binding
is dependent
on structural
features that are specific to cob-15 (46).
As discussed
elsewhere,
synthetases
and other pre-existing
cellular RNA-binding
proteins
may have adapted
to function
in splicing
by fortuitously
recognizing
sequences
or structures in group I introns
that resemble
their normal
cellular
RNA targets (40). The finding that many of the proteins
required
for splicing
group I introns
differ between
Neurospora
and yeast suggests
that this adaptation
occurred
after the
divergence
of fungal species, possibly reflecting
the relatively
recent dispersal
of the introns
as mobile
elements
(40). In
some cases, protein-dependent
splicing may provide a means
of regulating
RNA
catalyzed
reactions.
The Neurospora
CYT-4 protein,
for example,
shows significant
similarity
to
Saccharomyces
cerevisiae and Schizosaccharomyces
pombe
gene
products
involved
in the function
of cell cycle protein
phosphatases,
and may play a role in coordinating
mitochondrial
RNA splicing
and processing
reactions
with cell cycle and
other aspects
of cellular
metabolism
(47).
If group II introns
are evolutionarily
related
to nuclear
mRNA
introns,
then one might expect similar proteins
to be
involved
in their
splicing.
Unfortunately,
no proteindependent
in vitro splicing systems have been developed
for
group II introns.
The number
of group II intron splicing factors identified
genetically
is small, and some of these may
affect splicing indirectly.
For example,
the MRS3 and MRS4
genes, which were identified
by suppression
of a group II intron splicing defect when expressed
from a multi-copy
plasmid, are homologous
to ion-carrier
proteins
and may affect
splicing
by altering
the ionic environment
in mitochondria
(48). Two genetically
identified
proteins
that are likely to
function
directly
in group II intron splicing
are MRS2 and
MSSJJ6
(49, 50). Analyses
of mutants
suggest that MRS2
functions
in splicing
all group II introns
(coxl-II,
-12, -ISy
and cob-Il) and is relatively
specific for these introns,
whereas
MSS1I6 is involved in splicing
group II introns
(coxl-II and
cob-Il) and some group I introns.
Both MRS2
and MSSII6
appear
to have some additional
function
besides splicing,
as
gene disruptions
result in a respiratory-deficient
phenotype
in yeast strains
whose mtDNA
contains
no introns.
MRS2
has no reported
similarity
to other proteins,
but the MSS1I6
protein
has a DEAD-box
motif characteristic
of RNA helicases, which do indeed function
in splicing
nuclear
mRNA
introns
(49, 50).
GROUP
GENETIC

I AND GROUP
ELEMENTS

II INTRONS

ARE

MOBILE

Both group I and group II introns

have been shown to be
mobile genetic elements
that have developed
mechanisms
for
inserting
into intronless
genes (41, 43, 51, 52). Because
group
I and group II introns
carry their own splicing
apparatus,
they could in principle
spread to different
cellular
compartments
or organisms,
with their insertion
having
minimal
effects on gene expression
as long as they can be spliced
efficiently.
As discussed
previously,
the splicing of both group
I and group II introns
is dependent
on specific pairings
between
the
intron
and
flanking
exons
(e.g.,
P1 and
EBS1/IBS1).
Because
the sequences
involved
in these pairings differ in individual
introns,
mobile introns
must be inserted in the appropriate
sequence
context in order to splice
efficiently.

GROUP

I AND

GROUP

II INTRONS

Group

I intron

mobility

via site-specific

endonucleases

A number
of group I introns achieve such site-specific
insertion by using site-specific
endonucleases
encoded
within the
intron (41, 43, 51, 52). Remarkably,
each intron-encoded
endonuclease
cleaves at a different
asymmetric
target sequence,
generally
spanning
20 bp, which is located at or near the
site of intron insertion.
In crosses between
strains containing
intron
and intron
alleles, the endonuclease
promotes
high
frequency
transfer
of the intron or homing
by generating
a double-stranded
break in the intron
allele. The resulting
DNA
ends invade
the intron
allele to prime
replicative
transfer
of the intron
by a double-stranded
break
repair
process.
Because
formation
of the initial heteroduplex
depends on homology
of flanking exons and there is nucleolytic
degradation
of the cleaved recipient,
transfer
of the intron is
accompanied
by coconversion
of flanking
genetic markers,
a
hallmark
of this mechanism.
Sequence
comparisons
show that group I intron-encoded
endonucleases
include
two major
structural
classes,
one
characterized
by the LAGLI-DADG
motif also found
in
group I intron maturases
and the other by some variation
of
the motif GIY-(1O/ll
aa)-YIG
(41, 51) (Fig. 5). Members
of both the LAGLI-DADG
and OIYYIG
classes have been
found outside of introns,
and the former include the wellknown HO endonuclease,
which is involved in yeast mating
type switching
(41, 53). In two cases, LAGLI-DADO
polypeptides,
which are not associated
with conventional
introns,
have their coding
sequences
inserted
directly
in those of
other proteins - the yeast vacuolar
W-ATPase
and the archaebacterial
Therrnococcus
litoralis
DNA
polymerase.
Remarkably,
these inserted LAGLI-DADG
polypeptides
not
only have site-specific
endonuclease
activity,
which
could
promote
mobility
of the insertion,
but are also associated
with protein-splicing
events that excise them and join the
flanking
protein
sequences
(53, 54). The finding
of mobile,
non-intron-encoded
endonucleases
strongly
supports
the
proposal
that ORFs
encoding
such endonucleases
are independent
genetic elements
that inserted
into previously
existing group I introns
(51, 55). Further,
the association
of
some LAGLI-DADO
proteins
with protein
splicing
events
raises the possibility
that the related group I intron proteins,
which are translated
in-frame
with upstream
exons, catalyze
their own proteolytic
processing
(53).

Reverse splicing
intron mobility

and other possible

mechanisms

for

Because
the double-stranded
break repair
process
depends
on exon homology,
it does not favor transposition
of introns
to other locations,
and there are indications
that other types
of processes
contribute
to the mobility
of group II introns
(see below). A second possible
mechanism
for intron insertion, reverse
splicing,
has been demonstrated
in vitro for
both group I and group II introns (56). For both types of introns, reverse splicing
requires
only a short RNA target sequence,
which corresponds
to the 5 exon and suffices to form
the P1 pairing
in the case of group
I introns
and the
EBS1/IBS1
pairing
in the case of group
II introns.
The
recombined
RNA could in principle
reintegrate
into the genome after reverse transcription
(57, 58). Although
reverse
splicing is inefficient
in vitro, proteins
that function
in splicing of the intron should also accelerate
reverse splicing
and
may thus contribute
to intron mobility
in vivo (59).
Group I and group II introns both have some activity with
DNA substrates
(21, 22, 60) and, in principle,
the excised in-

21

tron RNAs could integrate

directly
by reverse splicing
into
single-stranded
DNA or into RNA primers
at replication
forks (57, 58). In addition,
autonomous
DNA elements
have
been described,
which may either be circular
cDNA copies
of excised group I or group II introns
or evolved from them
(43). cDNA copies of excised introns could conceivably
contribute
to mobility
by integrating
directly
into genomic
DNA,
perhaps
facilitated
by an integration
activity
as for
retroviral
proviruses.
Possible evolutionary
relationships
between autonomous
forms of introns
and infectious
elements,
such as RNA viruses
or retroviruses,
have been discussed
elsewhere
(43).
Group II introns
transcriptase-like

are mobile
proteins

and

encode

reverse

The first indication

that group II introns
might be mobile
elements
was the finding that a number
of these introns contain ORFs
with significant
similarity
to reverse transcriptases, particularly
those of the non-LTR
class of retroid
elements,
i.e., those lacking long terminal
repeats
(LTR5) (42,
61, 62). The yeast coxl-Il
intron,
which encodes
such an
ORF, is shown in Fig. 5. Although
reverse transcriptase
activity
has not been
demonstrated
biochemically
for any
group II intron-protein,
most ORFs have good matches
for
the seven conserved
sequence
blocks found in all functional
reverse transcriptases.
The group II ORFs generally
contain
an additional
upstream
conserved
sequence
(Z) characteristic of non-LTR
reverse transcriptases;
some also have a Zn2
finger-like
motif at their COOH-terminus,
and a few, e.g.,
the yeast coxl-I1 and -12 ORFs,
contain
weak matches
to
retroviral
protease
domains.
Another
conserved
domain,
which we denoted
X, is always found between
the reverse
transcriptase
and Zn2 finger domains,
but is also found in
group II proteins
that lack the latter domains.
As discussed
previously,
the reverse transcriptase-like
proteins
encoded
by
yeast coxl-I1 and -12 function
as maturases
in splicing the introns that encode them.
Three group II introns that encode reverse transcriptaselike proteins,
S. cerevisiae coxl-II and -12 and Kluyveromyces Instis coxl-I1, a cognate
of S. cerevisiae coxl-12, have been shown
to transfer
at frequencies
approaching
100% to intronless
alleles during crosses (63, 64). Transfer
of the yeast coxl-I1 and
-12 introns
is accompanied
by coconversion
of distal group
I introns but is not dependent
on group I introns,
as it occurs
in strains from which all group I introns
have been deleted.
Significantly,
the transfer
of group II introns
was shown to
be inhibited
in two splicing-defective
mutants;
one, a cis mutation that affects intron structure,
and the other a trans mutation that affects maturase
activity of the intron ORF (63).
The requirement
for splicing
is clearly different
from the
situation
for group I intron mobility
and strongly
suggests
that excision of the intron is required.
In general,
such a requirement
could reflect either that the excised intron
is an
intermediate
in mobility
(e.g., via reverse splicing
or direct
integration)
or that the excised intron
acts as a mRNA
or
cofactor
for an activity
that functions
in mobility
(e.g.,
reverse
transcriptase
or site-specific
endonuclease).
The
coconversion
of flanking
markers,
on the other hand, could
result either from recombination
with reverse transcripts
of
all or part of the donor pre-mRNA
or from double-stranded
break repair promoted
by an endonuclease.
The R2 element
of Bombyx mon encodes
a reverse transcriptase-like
protein
that is related to the group II intron proteins
and has a sitespecific endonuclease
activity, which cleaves at the insertion
site of the element
(65).
Instances
where
group
II introns
have transposed
to
another
location
have been found in EuIena ctDNA.
These
22

Vol. 7

January 1993

are composite
introns,
termed twintrons,
in which a group
II intron has inserted
into another
group II or group III intron (34, 35). In two cases described
in detail, the insertion
appears
to have occurred
into a region essential
for splicing,
so that the internal
intron must be spliced first to reconstitute
the external
intron.
For both twintrons,
it was possible
to
identify
potential
EBS1-IBS1
and EBS2-IBS2
interactions
between
the internal
intron and the external
intron,
leading
to the suggestion
that insertion
occurring
by reverse splicing
followed by reverse transcription
and reintegration
into the
genome.
The reverse transcriptase-like
proteins
encoded
by group
II introns
may also contribute
to various
site-specific
deletions resulting
from recombination
of genomic
DNA with
cDNA copies of spliced or misspliced
RNAs (52). A cogent
example
is the phenomenon
of precise
intron
deletion
in
yeast mtDNA,
which presumably
results
from recombination with a cDNA copy of spliced mRNA.
The phenomenon
was reported
first by Slonimski
and co-workers
(reviewed
in
ref 52), who found that a number
of splicing
defective
mutants revert by precise deletion
of the impaired
intron
from
mtDNA.
Both group I and group II introns could be deleted
in this way, and the deletion
of the impaired
intron was frequently
accompanied
by deletions
of neighboring
upstream
or downstream
introns,
which were not under selection.
In
addition,
it was found that the initial intron mutation
had to
be somewhat
leaky, presumably
in order
to generate
the
spliced
mRNA
intermediate.
By using
yeast strains
with
different
combinations
of mtDNA
introns,
it was shown that
precise
intron
deletion
was dependent
on the presence
of
coxl-I1 and/or
coxl-12 in the mtDNA,
consistent
with a requirement
for a reverse transcriptase
activity encoded
by one
of these introns.
The ability to precisely
delete introns
from
yeast mtDNA
is presumably
counterbalanced
by efficient
mechanisms
for intron insertion,
but this process could have
contributed
significantly
to intron loss in the course of evolution.

PROSPECTS

FOR

FUTURE

RESEARCH

Research
on group I and group II introns
should continue
to provide fundamental
insights into RNA chemistry
and the
evolution
of proteins
to assist and regulate
RNA-catalyzed
splicing reactions,
as well as into the amazing
adaptations
of
the introns
to function
as mobile
genetic
elements.
The
research
also seems poised
to address
a number
of longstanding
evolutionary
questions
about the feasibility
of selfreplicating
RNAs, the evolution
or protein synthesis,
and the
origin of introns.
Yet it is worth keeping
in mind that contemporary
group
I and group
II introns
are themselves
highly
evolved.
They have a demonstrated
propensity
for
rapid evolutionary
change and may be only distantly
or not
at all related
to molecules
in the primordial
world. Studies
of group I and group II introns,
as well as other catalytic
RNAs,
will undoubtedly
provide
insights
into evolutionary
mechanisms,
leading
to logically
compelling
scenarios.
However,
if what we have seen so far is any indication,
we
will probably
still be surprised
by the real answers.

We thank our laboratory colleagues and Drs. Donald Copertino

(U. Arizona), Daniel Herschlag (Stanford), and Philip Perlman (U.
Texas) for their comments
on the manuscript.
Work in the authors
laboratories
was supported by grants GM39422 and GM44844 to
M.

B. and grants

The FASEBJournal

GM37949

and GM37951

to A. M.

L.

SALDANHA FT AL.

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