(Received for publication, April 17, 1995; and in revised form, July 5, 1995)
From the
Some group I introns have been shown to be self-splicing in vitro, but perhaps all require proteins for splicing in vivo. Sequence differences affect the stability of secondary structures and may explain why some group I introns function efficiently without protein cofactors while others require them. The terminal intron of the cytochrome b pre-mRNA from yeast mitochondria needs a nucleus-encoded protein for splicing, even though it splices autocatalytically in high salt in vitro. This system has the advantage that the protein is specific for this intron, and yet the structure of the catalytically active RNA can be studied in its absence. We have modified the intron by chemical and enzymatic treatment in the presence and absence of the protein to determine the impact of the protein on the secondary and tertiary structures of the intron. We found protein-induced formation of secondary and tertiary structures within the intron, and the same structures also form in high salt autocatalytic conditions. We have also studied UV cross-links to determine those bases of the intron that interact directly with the protein and found that the protein contacts the intron most intimately at the structures denoted P1, L2, P4, and P6a.
Group I introns have been shown to be self-splicing in
vitro. The reaction requires Mg, which has at
least a structural role and perhaps a catalytic role in
splicing(1, 2) . Guanosine is required to initiate the
first of two transesterfication reactions that yield the ligated
exons(3) . The group I intron of the large subunit rRNA of Tetrahymena requires no protein for splicing, but many group I
introns do require protein cofactors for splicing in
vivo(4) . Consequently, a comprehensive view of group I
introns must explain the role of ancillary proteins. In addition,
protein-assisted splicing of group I introns may serve as a model for
the function of small ribonucleoproteins, which are thought to have a
catalytic function in the splicing of introns in nuclear pre-mRNA.
Simple systems exist for the study of a few group I introns that
require at least one protein for splicing. The best studied of these
proteins is CYT-18 from Neurospora crassa, which can function
in the splicing of several group I introns(5) . CYT-18, which
is a mitochondrial tRNA synthetase, cannot enhance the splicing of all
group I introns, but it recognizes a subset of structural elements
common to a class of group I introns(6) . CYT-18 is believed to
stabilize the correct tertiary folding of the catalytic core of the
introns it stimulates(7, 8) . Proteins from Escherichia coli, especially ribosomal protein S12, have been
shown to stimulate splicing of group I introns from bacteriophage
T. Coetzee et al.(9) have shown that
these proteins facilitate splicing by relatively weak, nonspecific
binding to intron RNA. They suggest that S12 acts as an RNA chaperone,
preventing formation of inhibitory structures and facilitating helix
formation and perhaps tertiary interactions. Group I introns share
common secondary and tertiary structures that have been analyzed based
on phylogenetic and mutagenic
analysis(10, 11, 12) . The catalytic core of
the intron contains the guanosine binding site and the sites of primary
sequence homology common among group I introns. Other conserved
structures are the P1/P10 helices, which consist of the internal guide
sequence base paired with the 5`- and 3`-splice junctions,
respectively.
It is not clear why a group of such structurally similar introns have a diverse requirement for protein cofactors when the basic chemical reaction mechanism of splicing is identical for all group I introns. Despite conservation of secondary structure, these introns vary considerably in sequence(13) . Sequence differences that affect the stability of RNA helices may explain why some group I ribozymes function efficiently without protein cofactors while others require them.
We have studied an intron that needs a single protein for activity in vivo. Splicing of the fifth intron of the cytochrome b pre-mRNA (bI5) from yeast mitochondria requires the nucleus-encoded Cbp2 protein(14, 15) , but in high salt this intron is self splicing(16) . This system has the advantage that the protein is specific for this intron and yet the structure of the catalytically active RNA can be studied in its absence. We have modified the intron by chemical and enzymatic treatment in the presence and absence of the protein to determine what impact Cbp2 has on the secondary and tertiary structures of bI5. We have also performed UV cross-linking experiments to help determine those bases that interact directly with Cbp2. This is an effort to probe the RNA-protein interactions required for group I intron splicing. Our results suggest that Cbp2 stabilizes AU-rich helices and also forces the intron to assume the same tertiary structure under physiological conditions as it does in the high salt, autocatalytic conditions.
Figure 1:
Expression and
purification of Cbp2 protein. Protein samples were separated on a 10%
SDS-polyacrylamide protein gel. These proteins were visualized by
silver staining. Lane1 is Cbp2 protein (74,000 Da)
purified as described. Lane2 is a low molecular
weight standard from Bio-Rad. The standard consists of phosphorylase b
(97,400 Da), bovine serum albumin (66,200 Da), ovalbumin (45,000 Da),
carbonic anhydrase (31,000 Da), soybean trypsin inhibitor (21,500 Da),
and lysozyme (14,400 Da). Lane3 is a total protein
isolate from noninduced JM109(DE3) cells containing the pET3a-CBP2
plasmid. Lane4 is a total protein isolate from
isopropyl-1-thio--D-galactopyranoside-induced JM109(DE3)
cells containing the pET3a-CBP2.
Figure 2:
Splicing assays on P-labeled
RNA using purified Cbp2 protein. Panels A and B,
autoradiographs from a 4%-polyacrylamide-8 M urea gel used to
separate products of splicing assays. Panel A,lane1 is products of transcription; lane2 is a RNA incubated in low salt buffer; lane3 is
RNA incubated in low salt buffer with purified Cbp2 protein; lane4 is RNA incubated in high salt buffer. The identity of
the bands is as indicated. The openbox represents
the 5`-exon, the blackbox represents the 3`-exon,
and the blackline represents the intron. Panel
B, splicing reactions were done in low salt splicing buffer using
P-labeled RNA transcript (2.5 µM) as
substrate. Cbp2 protein (10 µM) was added, and incubations
were done at 37 °C with time points taken at 0, 30, and 60 min. The
reaction products were separated on a 4% polyacrylamide-8 M urea gel. This gel was dried and digitized on an Image-Quant
PhosphorImager to allow analysis of the bands. Panel C, the leftverticalaxis is the percent of
unspliced RNA substrate remaining. The data were derived from the bands
indicated on the gel in panelB. The rightverticalaxis is the relative amount of spliced
intron products (shown in pixels). The data were derived from the bands
indicated on the gel in panelB.
Oligomers used were E6, 5`-TAAAATAGTATAGAATGG-3` (position 21-38); AL148, 5`-CTCCTTTCGGGGTTCCGGCT-3` (position 562-582); AL72, 5`-AATTAATTGGTTTTCGTGGAA-3` (position 484-496); AL212, 5`-GTGGAAAGGTATTACTATCTCAGT-3` (position 458-481); AL157, 5`-CTAGTCGTTGAACGTTTT-3` (position 433-450); AL70, 5`-AGATGATATAAACTTCGCTGCAAGT-3` (position 345-371); AL169, 5`-CTTGACAATTAACCAATTTT-3` (position 287-306); and AL213, 5`-ATATAAAATAAATAAGATTAAATAAT-3` (position 83-109).
We also UV irradiated bI5 in the absence and presence of Cbp2 in order to identify bases of the intron that interact directly with the protein. Irradiation of protein-RNA complexes results in covalent linkages between amino acid residues and nucleic acid bases (22) . Photo excitation of a base generates a free radical followed by the abstraction of a hydrogen atom from a proximate amino acid residue to form another free radical. A stable covalent linkage is formed by recombination of the purinyl or pyrimidinyl radical with the amino acid residue. UV irradiation produces cross-links at the interface of the protein-nucleic acid complex.(23) . For ribouracil and cysteine, this interface is probably about 2 Å since cross-linking results in a covalent linkage of 1.75 Å(24) . Therefore, any RNA-protein cross-links we found were considered to represent an intimate contact between the base and an amino acid residue of Cbp2.
While loops L1 and L8 are not
required for splicing in vitro(25) , it is possible
that these regions play some role in vivo. Therefore we chose
to study the wild-type version of the intron. All treatments of bI5 were done in the absence of GTP so that no cleavage
occurred at the 5`-splice site. We looked at the modification of bI5 under three conditions. First, we modified bI5 in
low salt (5 mM MgCl, 50 mM NH
Cl), which mimics physiological ionic strength and
intramitochondrial magnesium. These conditions yield information about
the secondary and tertiary structure of bI5 in the absence of
Cbp2 in a situation where bI5 is catalytically inert.
Nevertheless, this level of mono- and divalent salt is sufficient to
stabilize the active structures of other group I introns such as the
large subunit intron of Tetrahymena(3) , the td intron of bacteriophage T
(7) and even the
intron of yeast mitochondria(26) . Second, we modified bI5 in low salt in the presence of Cbp2. Cbp2 stimulates
efficient splicing under these conditions (Fig. 2). From this
incubation, we obtained information about the changes in secondary and
tertiary structure that occur within bI5 as a consequence of
the interactions between bI5 and Cbp2. Third, we modified bI5 in high salt conditions (50 mM MgCl
,
500 mM KCl), which permit autocatalytic splicing. Presumably,
high salt conditions provide ionic shielding that permits bI5
to assume the correct conformation required for splicing. Modification
in high salt provides a confirmation of results obtained in low salt
with Cbp2 and allows us to discriminate between changes in modification
that result from changes in RNA structure and those that are a direct
consequence of protection of the RNA by the protein.
Figure 3: Hydroxyl radical modification on in vitro transcribed bI5. Autoradiographs from 10% polyacrylamide-8 M urea gels used to separate products from primer extension on hydroxyl radical modified RNA. In each panel, lane1 is primer extension on unmodified RNA, lane2 is primer extension on RNA modified in low salt, lane3 is primer extension on RNA modified in low salt in the presence of Cbp2 protein, and lane4 is primer extension on RNA modified in high salt. A, results of primer extension using the AL213 primer, which anneals downstream of the 5`-splice junction (The 3`-ends of primers are indicated by arrows in all figures.). B, results of primer extension using the AL169 primer, which anneals adjacent to the P2 stem-loop complex and the IGS. C, results of primer extension using the AL70 primer, which anneals adjacent to the P4 and P5 region. D, results of primer extension using the AL72 primer, which anneals adjacent to P7.1, P7, and P6. E, results of primer extension using the E6 primer, which anneals downstream of the 3`-splice junction.
Figure 4: CMCT modification on in vitro transcribed bI5. Autoradiographs from 10% polyacrylamide-8 M urea gels used to separate products from primer extension on CMCT modified RNA. Lanes are the same as in Fig. 3. A, Results of primer extension using the AL169 primer, which anneals adjacent to the P2 stem-loop complex and the IGS. B, results of primer extension using the AL72 primer which anneals adjacent to P7.1, P7, and P6.
Figure 5: RNase A modification on in vitro transcribed bI5. Autoradiographs from 10% polyacrylamide-8 M urea gels used to separate products from primer extension on RNase A modified RNA. Lanes are the same as in Fig. 3. A, results of primer extension using the AL213 primer, which anneals downstream of the 5`-splice junction. B, results of primer extension using the AL169 primer, which anneals adjacent to the P2 stem-loop complex and the IGS.
A problem frequently encountered with primer extension is that the reverse transcriptase sometimes adds a base to the 3`-end of the cDNA at 37 °C (equivalent to one base beyond the actual 5`-end of the modified RNA). This problem was circumvented by performing the primer extension reaction at 50 °C.
Fig. 6and Fig. 7show the cumulative results of all modification assays for all three modification agents. Detailed below are a comparison of results for the major structures of bI5.
Figure 6: Bases of intron bI5 protected from modification in the presence of Cbp2 protein and high salt. The proposed secondary structure for bI5 showing sites of protection from cleavage. Lowercaseletters represent the exons, uppercaseletters represent the intron. Arrows indicate the position of the 5` and 3`-splice junctions. Boxed bases show the potential base pairing that forms P10. Sequences for L1 and L8 are not shown. Shorthorizontal and verticallines reflect hydrogen bonds. A, hydroxyl radical cleavage results; B, CMCT cleavage results; C, RNase A cleavage results. Opencircles indicate bases protected from cleavage in the presence of Cbp2 and in high salt. Intensity of cleavage is indicated by the thickness of the circle, with thicker circles indicating a 2-3-fold increase in cleavage at those positions. These figures do not indicate bases that are not sensitive to the modifying agents under physiological conditions in the absence of Cbp2. Nor do they indicate bases that are cleaved but not protected in the presence of Cbp2.
Figure 7: Bases of L1 protected from RNase A modification in the presence of Cbp2 protein and high salt. A secondary structure of loop L1 generated by Fold (GCG sequence analysis software) showing sites of protection from RNase A cleavage. Lowercaseletters represent exon 5; uppercaseletters represent the intron. The arrow indicates the position of the 5`-splice junction. Opencircles indicate bases protected from cleavage in the presence of Cbp2 in high salt. Shorthorizontal and verticallines reflect hydrogen bonds. Protection between positions 20 and 116 was not determined.
Gampel and Cech (25) have shown that loops L1 and L8 are not required for splicing of bI5. Our results for L1 (Fig. 7) showed patterns of CMCT and RNase A cleavage and protection within L1 consistent with the formation of stem-loop structures in the presence of Cbp2 and in high salt. Regions that are susceptible to these reagents and are therefore probably single-stranded and become protected in the presence of either high salt or Cbp2. We found, but do not show, similar patterns of protection for L8.
Figure 8: UV cross-linking on in vitro transcribed bI5. Autoradiographs from 10% polyacrylamide-8 M urea gels used to separate products from primer extension on UV cross-linked RNA. In each panellane1 is primer extension on noncross-linked RNA; lane2 is primer extension on RNA UV cross-linked in low salt; lane3 is primer extension on RNA UV cross-linked in low salt in the presence of Cbp2 protein; and lane4 is primer extension on RNA UV cross-linked in high salt. A, results of primer extension using the AL72 primer, which anneals adjacent to the P2 stem-loop complex and the IGS; B, results of primer extension using the AL70 primer, which anneals adjacent to P7.1, P7, and P6.
Figure 9: RNA-protein UV-induced cross-links in bI5. The proposed secondary structure of bI5 showing bases (opencircles) that cross-link after exposure to UV radiation. Thickness of the circle indicates relative intensity of the band found on the polyacrylamide-urea gels. Thick circles indicate bands with an intensity 5-10-fold greater than bands indicated with thin circles. Shorthorizontal and verticallines reflect hydrogen bonds.
The cross-linking data suggest that Cbp2 contacts conserved elements of the catalytic core, especially P4, but also nonconserved structures such as L2 and L6a as well as the 5`-exon and possibly the internal guide sequence.
This intron, bI5, absolutely requires Cbp2 for splicing in vivo(13, 14) . In vitro, under high salt conditions, splicing is autocatalytic(14) , showing that the protein plays a structural role and does not contribute directly to catalysis. We found that, in the absence of Cbp2, bI5 assumes a conformation with secondary and tertiary elements similar to those previously proposed and experimentally verified for other group I introns. However, in bI5, some of these elements exist in transitional states in which the conserved structures, if formed, are not favored. This is the case in particular for P1, P2, P5, P6, P10, and P11. Our results show that addition of Cbp2 to the intron in low salt, or incubation in high magnesium and monovalent salt without Cbp2, promotes the formation of these structures to generate a catalytically active intron (Fig. 10).
Figure 10: Cbp2 facilitated formation of secondary and tertiary structures within bI5. The top structure indicates the extent of secondary structure in bI5 in the absence of Cbp2. Shorthorizontal and verticallines reflect hydrogen bonds. The bottom structure shows the secondary and tertiary structure changes that occur within bI5 in the presence of Cbp2. The brackets joined by the dottedline indicate the potential tertiary interaction between L6a and L7.1a reported by Michel and Westhof (10) .
The P1/P10 helices are the sites of phosphoryl exchange that yield the ligated exons as product. The P1 helix consists of the 5`-splice junction paired with the 3`-half of the IGS, and the P10 helix consists of the 5`-component of the IGS paired with the 3`-splice junction. We found that the splice junctions are resistant to modification under low salt conditions in the absence of Cbp2 and that they become even less accessible in the presence of Cbp2 and in high salt. The IGS is, however, quite accessible to cleavage by all modifying agents under physiological conditions, and becomes protected by the addition of Cbp2 or by incubation in high salt. These asymmetric cleavage patterns within the P1/P10 helices can be attributed to the tertiary interactions within bI5 rather than just simple secondary structural changes (i.e. formation of the helices). This conclusion is substantiated by the lack of cleavage by hydroxyl radicals within the splice junctions, but significant cleavage in the IGS, indicating that the splice junctions are less accessible to solvent than is the IGS. One such tertiary interaction was suggested by Wang et al.(27) , who demonstrated a site-specific cross-link between the 5`-end of the IGS in the Tetrahymena ribozyme and phylogenetically conserved adenosine residues in J4/5 near the core. Interestingly, we see a reduction in hydroxyl radical cleavage in analogous positions in J4/5 of bI5 under conditions in which the intron becomes active and the IGS becomes protected. Consideration of our results in conjunction with those from Tetrahymena, and the proximity of these regions in the tertiary structure proposed for group I introns, suggests an interaction between the IGS and J4/5 in bI5. The three-dimensional structure proposed for the Tetrahymena intron by Michel and Westhof (11) also shows that the P1/P10 helices are held within the catalytic core positioning the IGS on an outward face, somewhat protecting the splice junctions. This model is consistent with our results.
The catalytic core of group I introns consists of helices P3, P4, P6, and P7 and joining regions J3/4, J4/6, J6/7, and J8/7. We found a gradient of accessibility to modification within the catalytic core. Accessibility decreases with the increasing size of the modifying agent, with RNase A having the least access and hydroxyl radicals having the most. The inaccessibility of regions within the catalytic core, especially to hydroxyl radicals, indicates that they are deep within the tertiary structure of bI5 and have no exposure to solvent. These results are similar to those of Celander and Cech (28) and Hershlag et al.(29) for the structure of the core of the Tetrahymena intron. Other bases, while still within the catalytic core, do have limited access to solvent since they are cleaved by hydroxyl radicals. The lack of cleavage at these positions by CMCT shows that they are engaged in base pairing or are inaccessible to even moderately sized molecules. We show that some positions within the catalytic core were cleaved by all modifying agents. In each of these instances, cleavage was reduced or eliminated in the presence of Cbp2. This shielding from cleavage may represent a transition in secondary structure. For example, RNase A and CMCT cleavage at the ends of the P7 helix was reduced in the presence of Cbp2 showing that the addition of Cbp2 facilitates the formation of the helix. Stabilization was also observed in the P4 and P6 helices. However, as in the P1/P10 helices, the cleavage patterns within P4 and P6 were asymmetric, indicating that these helices were only partially exposed. The inaccessibility of these sites might be explained in part through blockage by the P1/P10 helices, which are seen to contact P4 and P6 in the model of Michel and Westhof(11) . In addition, Cbp2 may directly block sites within the core. Our results show that some structures that make up the catalytic core exist in the absence of Cbp2, but only in the presence of Cbp2 does a catalytically active core form (Fig. 10). We found four UV-induced cross-links within P3, P4, and P7. This result suggests that Cbp2 contacts bI5 at, or near, these positions to stabilize the core and yield a catalytic intron.
P2 and L2 are
protected from CMCT and RNase A modification in the presence of Cbp2
and under high salt conditions. Protection of P2 probably represents
the formation of the helix, but protection of L2 must represent a
higher level interaction between L2 and another structure in the intron
dependent on Cbp2. We found a strong UV-induced cross-link within L2
and weak cross-links within P2, suggesting that Cbp2 interacts directly
with the P2/L2 stem-loop. The P2 stem-loop complex is structurally
similar to the TAR RNA, which is the human immunodeficiency virus Tat
protein binding site (Fig. 11). TAR has two A-form helices, a
six-nucleotide loop, and a three-nucleotide pyrimidine
bulge(30) . Bases in the pyrimidine bulge are important for the
recognition of the Tat protein. In bI5, L2 is a four-base
bulge that includes three Cs. Our cross-linking data suggest that Cbp2
contacts this bulge at C. We also show CMCT protection
data suggesting direct protection of position C
in P2a by
Cbp2. These data demonstrate that the P2 stem-loop complex may be a
protein binding site similar to the TAR RNA Tat binding site. The lack
of cleavage in P2a can be explained by a persistent helix that is
present in the absence of Cbp2, but this cannot be the case for the
lack of cleavage in L2a. It is likely that this stem-loop structure is
protected from cleavage by some tertiary interaction that does not
appear to be dependent on Cbp2. In the large rRNA intron from Neurospora mitochondria, deletion of the analogous P2
stem-loop complex inactivates splicing (31) just as it does
with the Tetrahymena large rRNA intron(32) .
Figure 11:
The human immunodeficiency virus Tat
protein binding site. A, TAR RNA the human immunodeficiency
virus Tat protein binding site(30) . Boxedbases indicate nucleotides important for recognition by Tat protein. Arrows indicate phosphates whose ethylation interfere with
binding of Tat protein. B, the P2 stem-loop complex
from bI5. The boxedbase (C)
is the position of a strong UV-induced cross-link suggesting Cbp2
contact. The circled base (C
) shows possible
protection by Cbp2 from CMCT digestion.
Our results confirm the proposed secondary structure for helices P3, P6a-c, and P8. These helices are accessible to all the modifying agents under conditions that mimic physiological ionic conditions in the absence of Cbp2. For CMCT and RNase A, this cleavage is reduced in the presence of Cbp2 and in high salt. Cleavage by hydroxyl radicals is generally not affected except in P6a. This result shows that these helices are not positioned deep within bI5, but perhaps contribute to the structural components of bI5 encasing the catalytic core.
The five-nucleotide bulge designated L6a is not cleaved by CMCT or RNase A under any conditions. This is in marked contrast to P6a and P6b, which are cleaved by both CMCT and RNase A. This finding suggests that L6a is involved in tertiary pairing. Michel and Westhof (11) have demonstrated the potential for such an interaction between L6a and L7.1a. The P7.1 stem-loop complex shows an asymmetric cleavage pattern. What cleavage does occur is reduced in the presence of Cbp2 and high salt. It does appear that this stem-loop structure is involved in a tertiary interaction, but whether or not there is an interaction between this structure and L6a cannot be determined from these data. Our results also suggest that Cbp2 is involved in the potential tertiary interactions that include L6a and L7.1a. We found a strong UV-induced cross-link in P6a one base upstream of L6a, suggesting that Cbp2 binds to this region and forces L6a into a structure required for catalysis.
Our results indicate that Cbp2
contacts bI5 most intimately at P1, L2, P4, and P6a,
demonstrating that there are multiple bI5-contact points on Cbp2. By
comparison, bI5 was found to cross-link to at least two distally
located peptides of Cbp2. ()Our experiments show that the
secondary and tertiary structures of bI5 in the presence of
Cbp2 are consistent with those found in other group I introns. We have
shown that Cbp2 facilitates the formation of helices within bI5, as evidenced by the increased protection of these
structures in the presence of Cbp2. We have also provided evidence that
indicates the Cbp2-induced formation of tertiary structures within bI5. Finally, our results support the hypothesis that the
binding of Cbp2 alters the structure of the intron and stabilizes its
active form.