©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein-induced Folding of a Group I Intron in Cytochrome b Pre-mRNA (*)

(Received for publication, April 17, 1995; and in revised form, July 5, 1995)

Lynn C. Shaw Alfred S. Lewin (§)

From the Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, Gainesville, Florida 32610-0266

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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(4). 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.


EXPERIMENTAL PROCEDURES

Expression of Cbp2 Protein

Plasmid pET3a-CBP2 was constructed by converting the ATG of the CBP2 gene to an NdeI site by directed mutagenesis and cloning the NdeI-SnaBI fragment between the NdeI and BamHI sites of pET3a(17) . An overnight culture of BL21(DE3) containing this plasmid was used to inoculate 500 ml of LB/ampicillin. Cells were cultured at 37 °C until A reached 0.35. Expression of the Cbp2 protein was stimulated by adding isopropyl-1-thio-beta-D-galactopyranoside to a final concentration of 0.4 mM and continuing incubation for 3 h(17) . Fig. 1shows the results of expression of Cbp2. Phenylmethylsulfonyl fluoride was added to a final concentration of 17 µg/ml, and the cells were harvested by centrifugation, flash frozen, and stored at -70 °C.


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-beta-D-galactopyranoside-induced JM109(DE3) cells containing the pET3a-CBP2.



Isolation of Cbp2 Protein

Frozen cells were thawed and resuspended in buffer I (20 mM Tris-HCl, pH 7.0, 50 mM NaCl, 2 mM EDTA plus protease inhibitors (pepstatin A at 1 µg/ml, aprotinin at 1 µg/ml, phenylmethylsulfonyl fluoride at 17 µg/ml) and dithiothreitol (DTT) (^1)at 5 mM. All procedures were at 4 °C or on ice. The cells were broken open by three passages in a French pressure cell at 18,000 p.s.i. The cells were pelleted at 12,500 rpm in a JA20 rotor in a Beckman J2-21 centrifuge for 15 min. The supernatant was cleared by centrifugation at 30,000 rpm for 30 min in a type 42.1 rotor in a Beckman L5-50 ultracentrifuge. The supernatant was then applied to a DEAE-cellulose column equilibrated in buffer I. The majority of the proteins (including Cbp2) remained on the column in buffer I and were eluted from the column in buffer II (20 mM Tris-HCl, pH 7.0, 500 mM NaCl, 2 mM EDTA plus inhibitors). Fractions containing Cbp2 were detected by SDS-polyacrylamide gel electrophoresis. These fractions were pooled and brought to 30% ammonium sulfate. Insoluble material was removed by centrifugation, and the supernatant was brought to 50% ammonium sulfate. The pellet was recovered by centrifugation at 10,000 rpm for 15 min in a JA-20 rotor in a Beckman J2-21 centrifuge. The pellet was dissolved in 10 mM KHPO(4), pH 7.5, 5 mM DTT and dialyzed extensively against the same buffer. Insoluble material was removed by centrifugation, and the supernatant was applied to a hydroxylapatite column in the same buffer. Proteins were eluted using a linear gradient from 0.3 M to 1 M KHPO(4), pH 7.5. The fractions containing Cbp2 were pooled and dialyzed against three changes of 2 liters of 10 mM Tris-HCl, pH 7.0, 1 mM EDTA, 5 mM DTT, 20% glycerol. The sample was then applied to a heparin-agarose column equilibrated in buffer III (20 mM Tris-HCl, pH 7.0, 7 mM 2-mercaptoethanol, 1 mM EDTA, 20% glycerol plus inhibitors). The proteins were eluted from the column using a linear gradient of buffer III with 0.4-0.8 M NaCl. Fractions containing Cbp2 protein were pooled and dialyzed against two changes of 1 liter each of 10 mM Tris-HCl, pH 7.0, 1 mM EDTA, 5 mM DTT, 20% glycerol, and 1 liter of 10 mM Tris-HCl, pH 7.0, 1 mM EDTA, 5 mM DTT, 50% glycerol. The protein was stored at -70 °C. Fig. 1shows the final Cbp2 product used in these studies.

Splicing Assays

The activity of Cbp2 was determined in splicing assays(16) . 1 µl of P-labeled bI5 transcript (see the next section for preparation) was added to 20 µl of low salt buffer (LSB) (50 mM Tris-HCl, pH 7.5, 50 mM NH(4)Cl, 5 mM MgCl(2)), plus 0.2 mM GTP, and 5 mM DTT. Cbp2 was added (at various dilutions), and the reactions were incubated at 37 °C for 45 min and then stopped by chilling and precipitating with ethanol. The products of splicing were separated by electrophoresis on a 4% polyacrylamide, 8 M urea gel and visualized by autoradiography (Fig. 2). Fig. 2shows that our preparation of Cbp2 was able to convert approximately 65% of substrate into products within 60 min at 37 °C.


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.



Transcription in Vitro

Run-off transcripts were produced using SmaI-digested pSPI5 (containing the entire bI5 intron and flanking exon sequences and intron 4 from the mitochondrial COB gene of Saccharomyces cerevisiae) and T7 RNA polymerase as described by Partono and Lewin(16) .

Hydroxyl Radical Modification of RNA Transcripts

Modifications were done in a final volume of 20 µl based on the method of Hüttenhofer and Noller(18) . The intron-containing precursor RNA (1 µl; final concentration, 7 nM) was added to either (a) LSB, (b) LSB plus 1 µl Cbp2 protein (final concentration, 0.9 µM), or (c) high salt buffer (HSB) (50 mM Tris-HCl, pH 7.5, 50 mM MgCl(2), 500 mM KCl) and the samples were held at 25 °C for 5 min. 4 µl of fresh modification mix (12.5 mM Fe(NH(4))(2)(SO(4))(2), 62.5 mM ascorbic acid sodium salt, 25 mM EDTA, 0.6% H(2)O(2)) was added, and the samples were held at 25 °C for 2 min. The reactions were stopped by the addition of 1 µl of 0.5 M EDTA and 3 µl of 5 M ammonium acetate, and RNA was precipitated with 60 µl of absolute ethanol. Primer extension was performed on the products of the modification.

1-Cylcohexyl-(2-morpholinoethyl)carbodiimide Metho-p-toluenesulfonate (CMCT) Modification of RNA Transcripts

Modifications were done in a final volume of 50 µl based on the methods of Christiansen and Garrett (19) and Stern et al.(20) . The intron containing precursor RNA (1 µl; final concentration, 2.8 nM) was added to either (a) low salt potassium borate buffer (LSPB) (50 mM potassium borate, pH 8.0, 5 mM MgCl(2), 50 mM KCl), (b) LSPB with 1 µl Cbp2 protein (final concentration, 0.34 µM), or (c) high salt potassium borate buffer (HSPB) (50 mM potassium borate, pH 8.0, 50 mM MgCl(2), 500 mM KCl). 10 µg of bovine serum albumin was added, and the samples were held at 25 °C for 5 min, and then held on ice for 5 min. Modification in LSPB required a final CMCT concentration of 2.3 µM, while modification in HSPB required a final CMCT concentration of 2.3 pM. Modification was done on ice for 5 min and stopped by the addition of 5 µl of 0.5 M EDTA, 1 µl of 3 µg/µl tRNA, 5 µl of 5 M ammonium acetate, and RNA was precipitated with 150 µl of absolute ethanol. The products of modification were analyzed by primer extension analysis.

RNase A Modification of RNA Transcripts

Modifications were done in a final volume of 20 µl based on the methods of Christiansen and Garrett (19) and Stern et al.(20) . Precursor RNA was incubated as described above, and the samples were held at 25 °C for 5 min and then on ice for 5 min. 1 µl of RNase A (5 times 10 units) (Boehinger Mannheim) was added, and the sample was held on ice for 30 min. The reaction was stopped by the addition of 1 µl of 3 µg/µl tRNA, 1 µl of 0.5 M EDTA, 3 µl of 5 M ammonium acetate, and RNA was precipitated with 60 µl of absolute ethanol. The products of modification were analyzed by primer extension.

UV Cross-linking of RNA Transcripts

UV cross-linking was done in a final volume of 10 µl. Precursor RNA (1 µl; final concentration, 14 nM) was incubated in either (a) LSB, (b) LSB plus 1 µl Cbp2 (final concentration, 1.7 µM), or (c) HSB at 25 °C for 5 min. The samples were then applied to a Petri dish and placed, uncovered, into a UV-Stratalinker 1800 (Stratagene) and cross-linked at a setting of either 10 or 50 mJ at a wavelength of 254 nm. 2 µl of 5 M ammonium acetate and 2 µl of 1 µg/µl tRNA were added, and the RNA was precipitated with 30 µl of absolute ethanol. Primer extension was performed on the cross-linked products.

Primer Extension

The products of modification were resuspended in 7 µl of buffer A (14 mM Tris-HCl, pH 7.5, 70 mM KCl, 0.14 mM EDTA) containing 10^5-10^6 cpm of P-end-labeled oligomer and held at 80 °C for 2 min and then 25 °C for 15 min. Next, 16.5 µl of buffer B (6.25 mM dNTPs, 250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl(2), 15 mM DTT) and 0.5 µl (100 units) of Superscript RNase H Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) was added. The mixture was incubated at either 37 °C for 30 min or at 50 °C for 20 s. The reaction was stopped by the addition of 2 µl of 5 M ammonium acetate and precipitated with 60 µl of absolute ethanol. Samples were resuspended in dye mix (90% formamide, 20 mM EDTA, pH 8.0, 0.05% bromphenol blue, 0.05% xylene cyanole). Products of reverse transcription were separated by electrophoresis on polyacrylamide-urea gels and visualized by autoradiography. The dried gels weredigitized and quantified on an ImageQuant PhosphorImager, or the autoradiographs were scanned at 170 dots/inch on an Epson 1200C-Pro scanner followed by analysis using NIH Image on a Macintosh IIsi computer. (^2)Bands with differences in intensities between lanes (sites of protection) were confirmed by normalizing to bands of constant intensity just above and below the variable bands. Only reductions (or increases) in intensity that were observed consistently, in at least three experiments were scored.

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).


RESULTS

Strategy

We used three modifying agents to probe the secondary and tertiary structures of bI5. Two of these were chemical modifiers (hydroxyl radicals and CMCT) and the other was enzymatic (RNase A). Hydroxyl radicals have no base specificity and result in strand scission after nucleophilic attack at the ribose sugar ring. Hydroxyl radicals are virtually unaffected by secondary structure, and they make excellent probes for tertiary interactions within bI5(21) . They are potentially useful as probes for quanternary interactions between bI5 and the Cbp2 protein. Because of the small size of hydroxyl radicals, they will be excluded only by those interactions that exclude solvent. CMCT specifically modifies N-3 of uridines, and to a lesser extent N-2 of guanosines, resulting in strand scission and loss of the modified base. CMCT is specific for single-stranded regions and has a molecular weight of 423.6. RNase A specifically cleaves single-stranded RNA on the 3`-side of pyrimidines, generating a 3`-phosphate. RNase A has a molecular weight of approximately 13,700. CMCT and RNase A are good probes for the secondary structure of bI5, particularly since bI5 has a high percentage of uridines. Due to their disparity in size, these reagents provide a convenient way of probing the accessibility of regions within bI5.

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(2), 50 mM NH(4)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(4)(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(2), 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.

Methods of Analysis

Fig. 3Fig. 4Fig. 5show autoradiographs from polyacrylamide-urea gels used to separate the reverse transcription products generated by primer extension on modified RNA templates. The general conditions for each lane were the same for each gel. Lane1 shows the products from primer extension on unmodified RNA. This control was done to show the integrity of the RNA used in the modification assays. In addition to a cDNA corresponding to the full-length RNA, other bands were present. These are attributable to structural constraints in the RNA that prevent elongation by reverse transcriptase or to degradation products of the RNA. For the modification reactions (lanes2-4), a band indicates modification of the nucleotide one position upstream of the position of the band, and the sequences in the figures have been adjusted accordingly. Lane2 shows the products from primer extension on RNA modified in low salt in the absence of Cbp2. This lane gave the base line of modification, and it probed the structure of the intron in solution under conditions that do not permit splicing. Lane3 shows the products from primer extension on RNA modified in low salt in the presence of Cbp2, and lane4 shows the products from primer extension on RNA modified in high salt in the absence of Cbp2. The conditions of incubation represented in lanes3 and 4 permit splicing of the intron. Comparison of the differences in intensity of the bands in lanes3 and 4 with those in lane2 indicated structural transitions that effect a catalytically active structure. Protection of a base from modification was detected if the intensity of the band in lane3 or 4 was less than the intensity of the band in lane2. Hypersensitivity of a base to modification was observed if there was an increase in intensity of the band in lane3 or 4.


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.

Hydroxyl Radical Modification

Fig. 3shows reverse transcription products generated from primer extension on hydroxyl radical modified RNA. Shown are modifications corresponding to most of the intron and some of the exon sequences. Hydroxyl radicals cleaved at nearly every position of the template, although certain positions were highly susceptible. Either Cbp2 (lane3) or high salt (lane4) afforded protection from cleavage at positions throughout the intron. In the substrate domain (panelsA and B), protection was observed in the 5`-exon, in the internal guide sequence, and in the bulged nucleotides in the P2 stem. Of particular note is the reduction in cleavage at position -7 (in the 5`-exon), which appeared to be protein-specific (panelA, lane3). Significant protection was also seen in the catalytic core, especially in P4, P6, and the 5` component of P7 (panelsC, Dand E). A major protection was also seen near the 3`-end of the intron at position 726. Minor reduction in cleavage was seen at many other positions, and digitization of the autoradiograms was essential to quantify differences in intensity.

CMCT and RNase A Modification

Fig. 4and Fig. 5show reverse transcription products generated from primer extension on CMCT and RNase A-modified RNA. We analyzed the intron with CMCT and RNase A to the same extent that we did with hydroxyl radicals, but we only show a limited number of gels from these analyses. Although CMCT can also react with guanosine residues, we found no cleavage of guanosines. We did, however, find apparent cleavage of some of the cytosine residues within the intron. Significant reduction in CMCT cleavage by Cbp2 or high salt was observed in the P1 and P2 helices (Fig. 4A) and in the extended P6 helices (P6a-P6c) (panelB). RNase A modification, while generally confirming the CMCT data, showed less modification of the 5`-exon in the presence of high salt than in the presence of Cbp2. In the IGS and the P2 helices, both Cbp2 and high salt led to a reduction in sensitivity (Fig. 5B).

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.



P1/P10 Helices

Bases within the 5`-splice junction (A-G^2) were resistant to hydroxyl radical cleavage under all conditions. Such resistance may indicate that these bases are buried in the interior of the folded RNA. Some of these same bases within the 5`-splice junction (A-G^2) and the 3`-splice junction (U, G, and U) were also resistant to RNase A and CMCT cleavage under all conditions. Cleavage was found within the internal guide sequence IGS (G-G for hydroxyl radicals; U, C, and U for CMCT and RNase A) under physiological conditions in the absence of Cbp2. Cleavage of these bases within the IGS and other bases of the splice junction (U, U-U for hydroxyl radicals, U and U for CMCT and RNase A) was reduced or eliminated in the presence of Cbp2 and under high salt conditions

Catalytic Core

Portions of the catalytic core were not modified under any conditions. For hydroxyl radical, these were in P4 (U), P7 (U-C), J3/4 (A-U), and J8/7 (A, A-A). Analogous regions of the Tetrahymena ribozyme were resistant to hydroxyl radical modification(25) . For CMCT and RNase A, these were P4 (U), P6 (C and U), P7 (U, C, and C), J3/4 (U), J6/7 (U and C) and J8/7 (U). Cleavage did occur in the following regions of the core: P4 (C and A and reduced cleavage at G for hydroxyl radicals, C for RNase A), P6 (G-G for hydroxyl radicals), P7 (A-U and C and U for hydroxyl radicals, U and U for CMCT and RNase A), J6/7 (C for hydroxyl radicals) and J8/7 (A-G for hydroxyl radicals, C for RNase A). Cleavage at these sites was eliminated or reduced in the presence of Cbp2 and under high salt conditions.

P2/L2/P2a/L2a Stem-Loop Complex

P2 and L2 were modified by all the agents under physiological conditions in the absence of Cbp2. Cleavage was reduced in the presence of Cbp2 and under high salt conditions. Bases in L2 were cleaved to a greater extent than bases in P2 for both hydroxyl radicals and RNase A, but the presence of Cbp2 eliminated cleavage in both. P2a and L2a were modified only slightly, if at all, by all reagents under all conditions. In particular, CMCT did not cleave bases U-U and U (in P2a) and bases U-U (L2a). In contrast, CMCT attacked at positions C and C at or near L2, and the modification at position C appeared to be protein-specific (Fig. 4A).

P6a-c/L6a-c

All modifying agents led to blocks in reverse transcription within the P6a-c/L6a-c stem-loop complex. Little or no protection occurred upon the addition of Cbp2 or under high salt conditions with hydroxyl radicals except at positions in P6a and L6a (C, G-U, and A and A). Protection from CMCT and RNase A cleavage was found throughout this complex, except within L6a, which was not cleaved by either CMCT or RNase A. The amount of cleavage in L6c by RNase A was moderately higher than that for the stem structures, but protection occurred to the same extent within L6c as it did in the rest of the stem-loop complex.

P5 and P8 Helices

Cleavage was generally found in low salt in the absence of Cbp2 with all modifying agents for the P5 and P8 helices. This modification was reduced in the presence of Cbp2 and under high salt conditions.

Other Regions

Our results for the P7.1/L7.1/P7.1a/L7.1a stem-loop complex showed no modification for CMCT at U (L7.1) and U (P7.1a), but cleavage did occur at some positions: U (L7.1a), U (P7.1a), U (L7.1), and U-U (P7.1). Modification was reduced in the presence of Cbp2 and under high salt conditions. Cleavage in J4/5 was found at positions C-G for hydroxyl radicals and at position C for RNase A. Cleavage was reduced in the presence of Cbp2 and under high salt conditions. Cleavage was found at sites within both P9 and P9.1 and J9.1/10 for all modifying agents and was reduced or eliminated in the presence of Cbp2 and under high salt conditions.

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.

UV Cross-linking

Fig. 8shows reverse transcription products generated from primer extension on UV cross-linked RNA. For each panel, lane1 shows the products from primer extension on untreated RNA. For the UV cross-linking reactions (lanes2-4), a band indicates a cross-link of the nucleotide one position upstream of the position of the band. Lane2 shows the products from primer extension on RNA cross-linked in low salt in the absence of Cbp2. This lane shows the background level of cross-linking that occurs within the intron. Lane3 shows the products from primer extension on RNA cross-linked in low salt in the presence of Cbp2, and lane4 shows the products from primer extension on RNA cross-linked in high salt in the absence of Cbp2. Bands unique to lane3 were interpreted as Cbp2-dependent cross-links and were considered to represent RNA-protein cross-links. It is also possible that some of these bands represent RNA-RNA cross-links that are dependent on Cbp2. Bands occurring only in lanes3 and 4 were interpreted as RNA-RNA cross-links, which are not dependent on the presence of Cbp2 but are dependent on the active conformation of the RNA. Fig. 9shows the cumulative results from all UV cross-linking experiments. We found strong stops to reverse transcription at positions C in the upstream exon (exon 5), C in the IGS, C in L2, C and C in P4, C in J4/5, and C in P6a. We found weak to moderate stops at positions U in P1, C and U in P2, U in P3, U in J5/4, and U in P7. With the exception of position C, we interpreted these blocks to reverse transcription to be RNA-protein cross-links because they depended on the presence of Cbp2 and not on the active conformation of the intron. We found no consistent evidence of RNA-RNA cross-links. These would be associated with strong stops both in the presence of Cbp2 and in high salt. The pause at position C in the IGS did appear in both lanes in some of our experiments, suggesting that it might reflect an RNA-RNA cross-link that requires proper folding of the RNA, rather than a protein-RNA cross-link.


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.


DISCUSSION

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. (^3)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.


FOOTNOTES

*
This work was supported by a grant from the National Institute of General Medical Sciences (R01 GM12228). Support facilities were provided by the Interdisciplinary Center for Biotechnology Research and the Center for Mammalian Genetics at the University of Florida. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Genetics and Microbiology, University of Florida, College of Medicine, Box 100266, Gainesville, FL 32610-0266. Tel.: 904-392-0676; Fax: 904-392-3133.

(^1)
The abbreviations used are: DTT, dithiothreitol; CMCT, 1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate; IGS, internal guide sequence; TAR, trans-activating response element.

(^2)
This program was written by Wayne Rashand at the National Institutes of Health and is available by anonymous ftp from zippy.nimh.gov or on floppy disk from NIHS, 5385 Port Royal Rd., Springfield, VA 22161, part number PB93-504868.

(^3)
H. Tirupati and A. S. Lewin, unpublished results.


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