THE JOURNAL OF BIOLOGICAL CHEMISTRY ( c r 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 5, Issue of March 10, pp. 3108-3115,1985 Printed in U. S.A.

Transfer RNA Splicing in Saccharomyces cerevisiae SECONDARY AND TERTIARY STRUCTURES OF THE SUBSTRATES*

(Received for publication, June 11, 1984)

Ming-Chou Lee and Gayle KnappS From the Department of Microbiology, University of Alabama in Birmingham, Birmingham, Alabama 35294

Secondary and tertiary structures of four yeast tRNA precursors that contain introns have been elu- cidated using limited digestion with a variety of single- strand- and double-strand-specific nucleases. The pre- tRNAs, representing the variety of intron sizes and potential structures, were: pre-tRNAEAA, pre-tRNAe&, pre-tRNApAu, and pre-tRNApzc4. Conventional tRNA cloverleaf structure is maintained in these precursors except that the anticodon loop is interrupted by the intron. The intron contains a sequence which is com- plementary to a portion of the anticodon loop and al- lows the formation of a double helix often extending the anticodon stem. The 5’ and 3’ splicing cleavage sites are located at either end of this helix and are single-stranded. The intron is the most sensitive region to nuclease cleavage, suggesting that it is on the surface of the molecule and available for interaction with the splicing endonuclease. Absence of Mg2+ or spermidine renders the dihydrouridine and T$C loops of these precursors highly sensitive to nuclease digestion. These ionic effects mimic those observed for tRNAPh” and suggest that the tRNA portion of these precursors has native tRNA structure. We propose consensus sec- ondary and tertiary structures which may be of signif- icance to eventual understanding of the mechanism of yeast tRNA splicing.

The interruption of the gene by noncoding sequences (com- monly referred to as intervening sequences, IVS, or introns) occurs in genes of each of the major classes of RNA: transfer RNA, ribosomal RNA, and messenger RNA (reviewed in Refs. 1-3). These intervening sequences are removed from tran- scripts by the process of RNA splicing, defined as the excision of the intervening sequence from a RNA precursor and sub- sequent ligation of the coding fragments to yield the mature- sized RNA. The molecular mechanism by which this process occurs is not well understood. It differs among the several classes of RNA and may also vary for the same class of RNA in different organisms (e.g. the ligation step of tRNA splicing appears to differ for yeast and HeLa cells, although only the yeast mechanism was studied using a homologous system while the HeLa cell studies were heterologous (4, 5)). In the case of the precursor of 26 S ribosomal RNA of Tetrahymena thermophila, no proteinic enzyme appears to be required in that this RNA undergoes autocatalytic excision and ligation

* This research was supported by a grant from the American Cancer Society (MV-138). The costs of publication of this article were de- frayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 LJ.S.C. Section 1734 solely to indicate this fact.

$ T o whom reprint requests and correspondence should be ad- dressed.

in vitro (6, 7). The other splicing systems that are currently under investigation are more “classical” in their mechanisms, proteinic enzymes having been demonstrated or implicated in the splicing reaction. However, in all of these systems, RNA structure appears to be a key element of the splicing event.

Recent analysis of available nucleotide sequence and ge- netic data for fungal mitochondrial mRNA genes suggests that a common secondary structure can form in which a conserved sequence observed in seven introns brings two splice junctions together through the formation of hydrogen bonds with exon bases (8). Similarly, a conserved RNA struc- ture has been proposed as one model for metazoan nuclear mRNA splicing (9,lO). In this model a separate RNA molecule (a small nuclear RNA taking the place of the intron-specified sequences of fungal mitochondrial mRNA precursors) pro- vides a scaffold which brings splice junctions in close prox- imity for intron excision and exon ligation (9, 10). In Saccha- romyces cerevisiae, nine tRNAs have been found to be derived from intron-containing tRNA precursors (pre-tRNAs) (11, 12)’ that are cleaved by the same partially purified splicing endonuclease (13). Substrate recognition in tRNA splicing cannot be sequence-specific since no consensus sequence can be found. However, certain common features of secondary structure can be inferred from the sequences of these pre- tRNAs (14),’ suggesting that the selection of cleavage sites is probably directed by the RNA structure.

To elucidate the mechanism of intron excision during tRNA splicing it is important to establish the native secondary and tertiary structures of these tRNA precursors. Using limited ribonuclease digestion (15), we have probed the native struc- tures of yeast pre-tRNAs which cover the spectrum of intron sizes and complexities. We have also investigated conforma- tional changes of these pre-tRNAs induced by varied ionic environments. Divalent cations and polyamines have been shown by NMR to have profound effects on the stability of the tertiary structure of yeast tRNAPhe (16). Conformational changes in tRNAs induced by varying Mg2+ concentrations have also been detected by the structure-probing method (17). Using a variation of this methodology we have investigated the effects of both M$+ and spermidine on the conformation of four yeast pre-tRNAs. By comparing these results with those obtained for mature tRNAs we have predicted a prob- able common tertiary structure for yeast pre-tRNAs.

EXPERIMENTAL PROCEDURES

Enzymes and Chemicals-Ribonuclease A and RNase T1 were Sankyo products purchased through Calbiochem-Behring. Ribonu- clease Phy M and mung bean nuclease were obtained from P L Biochemicals. Human placental RNase inhibitor, RNase U2, and CTP were obtained from Sigma. Duplex-specific RNase V1 was the gift of Dr. Donald Lightfoot. Adenosine 5 ’ - [ ~ ~ ~ P ] t r i p h o s p h a t e

Ogden, R. C., Lee, M.-C., and Knapp, G., (1984) Nucleic Acids Res. 12.9367-9382.

3108

Struc ture of Yeast Intron-containing tRNA Precursors 3109

(-2000 Ci/mmol) was obtained from ICN. Yeast carrier RNA was prepared from commercial bakers' yeast (Fleischman, lyophilized dry yeast) by phenol extraction and hatch elution from DEAE-cellulose (11). Commercial yeast (Budweiser, pressed wet cakes) was also used in the preparation of nucleotidyl transferase (see below). Autoradiog- raphy was performed a t -70 "C using DuPont x-ray film (Cronex I v ) and lightning-plus intensifying screens.

Isolation and 3' End-labeling of Yeast tRNA Precursors-Nucleo- tidy1 transferase was prepared from 130 g (wet weight) of yeast which was crumbled into 450 ml of toluene that was maintained a t -40 "C with dry ice for 4 h. The frozen cell lysate was separated from the toluene by filtration through cheesecloth and allowed to thaw follow- ing the addition of 20 ml of Buffer A (100 mM Tris.HC1 (pH 8.0), 10 mM MgCI2, 1 mM NaZEDTA, 2 mM a-toluenesulfonyl fluoride, and 10 mM 2-mercaptoethanol). Residual toluene was removed by aspi- ration. The thawed lysate was further diluted with 25 ml of Buffer A and fractionated by a modification of the method of Sternbach et al. (18) described by Rosa (19). Terminally labeled pre-tRNAs were generated using unlabeled RNA which was prepared from the rnal mutant of S. cereuisiae, strain ts136, by a modification of the com- bined methods of Rubin (20) and Knapp et al. (11). Cell cultures grown at 23 "C to a density of 3 X lo7 cells/ml (Am -2.0) in complete medium (1% yeast extract (Difco), 2 % peptone (Difco), 2% glucose) were shifted to 35 "C for 30 min. Cells were rapidly harvested and RNA was fractionated by phenol extraction and DEAE-cellulose chromatography as described previously (11). The tRNAs and pre- tRNAs were end-labeled a t their 3' termini using yeast nucleotidyl transferase. The end-labeling reaction mixture (10 p l ) contained 4 pg/ml of ts136 RNA, 10 mM Tris.HC1 (pH 8.7), 10 mM MgC12, 0.06 mM CTP, 250 pCi of [n-32P]ATP (2000 Ci/mmol), 0.4 units of RNasin, and 1 X unit of partially purified nucleotidyl transferase. After incuhation at 24 "C for 1 h, the end-labeled RNAs were separated in a two-dimensional polyacrylamide gel electrophoresis system. This two-dimensional system consisted of a first dimension 10% (w/v) polyacrylamide (30:l; acrylamide/bisacrylamide) gel containing 4 M urea, 1 mM EDTA, and 90 mM Tris.borate (pH 8.3) and a second dimension 20% (w/v) polyacrylamide (30:l; acrylamide/bisacrylam- ide) gel containing 7 M urea, 1 mM EDTA, and 90 mM Tris.borate (pH 8.3). The RNAs were eluted by diffusion at 37 "C from crushed gel slices into 1% phenol, 0.3 M NaCI, 1 mM EDTA, and 10 mM Tris. HC1 (pH 7.4). The RNAs were precipitated with ethanol and dissolved in distilled water.

Structureprobing of tRNA Precursors-The RNA sequences of the 3' end-labeled tRNA precursors were determined using alkali or base- specific RNases: RNase U2 (adenosine-specific), RNase Phy M (adenosine- and uridine-specific), RNase A (pyrimidine-specific), and RNase T1 (guanosine-specific) as previously described (21, 22). The amounts of RNase per pg of RNA were determined empirically for each pre-tRNA and enzyme. Our standard structure-probing reac- tions using single-strand- and base-specific RNase U2, RNase A, and RNase T1 and duplex-specific RNase V1 were done a t 37 "C for 20 min in 10 mM Tris.HC1 (pH 7.0), 10 mM MgCI2, and 100 mM KCI. Structure probing using single-strand-specific mung bean nuclease was done at 37 "C in 30 mM sodium acetate (pH 7.0), 50 mM NaC1, 1 mM ZnC12, and 5% (v/v) glycerol. Serial enzyme/RNA ratios were performed under these conditions to select an optimal RNase/RNA ratio that generated a primary cleavage product from intact 3' end- labeled pre-tRNA molecules. This ratio was then used in subsequent reactions varying in MgCL and/or spermidine concentrations as defined under "Results." Multiple cleavages, which provided infor- mation mainly about secondary structure, were obtained by using each probing enzyme a t a 10-fold higher RNase/RNA ratio for a shorter incubation time (1 to 10 min). The structure-probing reactions ( 2 p l ) were terminated by the addition of 2 p1 of a solution containing 9 M urea, 10% (v/v) glycerol, 0.05% (w/v) xylene cyanol, and 0.05% (w/v) bromphenol blue and freezing immediately in dry ice. The structure probing and sequencing reactions were thawed and imme- diately electrophoresed through polyacrylamide-urea slab gels accord- ing to the methods described previously (21, 23). Slab gels of different polyacrylamide concentrat.ions (10% (w/v) polyacrylamide (7 M urea) or 20% (w/v) polyacrylamide (7 M urea)) and different lengths (35 or 85 cm) were used to resolve various regions of the RNA molecules.

RESULTS

The temperature-sensitive rnal mutant of S. cereuisiae accumulates intron-containing precursors of nine distinct

tRNAs (11, 24).' The introns in these pre-tRNAs range in size from 14 to 60 nucleotides and, on the basis of computer or hand calculations of potential lowest free energy structures (25), may assume secondary structures of varying complexity. We selected for this study pre-tRNAs which cover the spec- trum of sizes and possible complexities of introns as well as different tRNA structure classes (26). These four pre-tRNAs are pre-tRNA@;& pre-tRNA&<, pre-tRNA&A, and pre- tRNA& which contain introns of 19, 28, 32 (or 33), and 60 nucleotides, respectively. The native structures of these four uniquely end-labeled pre-tRNAs were probed using limited RNase digestion which allowed the determination of regions in the RNA molecule that were most accessible to RNase cleavage and,therefore, least involved in secondary or tertiary structure (15, 22, 27).

Aspects of the Secondary and Tertiary Structures of Yeast Pre-tRNAs-Conditions which allowed multiple cleavages of pre-tRNA&A by single-strand-specific RNases (see "Experi- mental Procedures"; data not shown) identified the single- stranded regions in this precursor. These regions can be observed in the secondary structure of pre-tRNAeA shown in Fig. 4A. The observed secondary structure demonstrates that pre-tRNAgA retains tRNA structure except in the an- ticodon loop area which is interrupted by the intron. In this region, two additional hairpin structures are formed. A helical stem is formed by part of the anticodon loop and a comple- mentary sequence in the 5"portion of the intron (we will call it the "anticodon-intron helix"). The 5' splicing cleavage site is included in the loop of this hairpin structure. The other hairpin structure is formed by the middle portion of the intron (we call it the "extra arm"). The six nucleotides at the 3' terminus of the intron and the 3' terminal nucleotide of the anticodon loop (see Fig. 4A) form a single-stranded interior loop which includes the 3' splicing cleavage site. Thus, both splicing cleavage sites are in single-stranded regions of the molecule.

The primary cleavage sites in pre-tRNAZA for different probing enzymes are shown in Fig. 1 and summarized in Fig. 4 A . As these figures demonstrate, primary cleavage sites for RNase U2 (A4' and A41), RNase A (U"'), and mung bean nuclease (A4' and A'') are all located in the single-stranded loop containing the 5' cleavage site of the splicing endonucle- ase. The primary cleavage site of RNase T1 is G57 (Figs. 1 and 4A). Residue 57 of pre-tRNAkA was recognized by both RNase A (uridine) and RNase T1 (guanosine), confirming the sequence variation (represented by the double-headed arrows in Fig. 4A) found in two published tRNA&A gene sequences (28, 29). This residue is in the loop of the extra arm. Since the other loop which contains the primary cleavage sites for RNase U2, RNase A, and mung bean nuclease does not have a guanosine residue, the fact that the extra arm is cleaved under primary cleavage conditions by RNase T1 but not by RNase A, RNase U2, or mung bean nuclease suggests that this region is exposed on the surface of the molecule but may not be the most accessible single-stranded region. Digestion of pre-tRNAZA with duplex-specific RNase V1 resulted in cleavage at residues G3' and G5' (Fig. 1) with the predominant cleavage at residue G5' in the stem of the extra arm. This cleavage confirms the existence of the extra arm and its relative availability on the outside of the pre-tRNA molecule. These data indicate that the intron-anticodon region of pre-tRNA&* is the most accessible portion of the precursor molecule. Furthermore, the D2 and T$C loops, detected in probes of secondary structure (data not shown) but not under

The abbreviations used are: D, dihydrouridine; $, pseudourine; T, thymidine.

3110 Structure of Yeast Intron-containing tRNA Precursors

D LOOP[

CLEAVAGE SITE -8 5"SPLICING

EXTRA )

LOOP [

CLEAVAGE SITE" 3"SPLICING

2 SEQUENCF STRUCTURE PROBING

U2 A TI MB V I U2 A TI MB V I CLEAVAGE SITES

(A) (U)(G)(SS) (DS)

VARIABLE REGION

31

41 42 41 40 40

52

57

d

r TYC LOOP1

L - *

.I 0

B e-'

FIG. 1. Primary 'cleavage sites of structure-probing en- zymes in pre-tRNA&A. The enzyme/RNA ratios in the structure- probing reactions which produced primary cleavages were: RNase U2, 1 X lo-* unitslpg of RNA; RNase A, 1 X lo-' units/pg of RNA; RNase T 1 , l X unitslpg of RNA; and mung bean (MB), 1 X 10" units/pg of RNA. RNase V1 was used at an appropriate dilution of the stock solution. The reactions contained approximately lo4 cpm of 3' end-labeled pre-tRNA& and were incubated at 37 "C for 20 min, except for RNase U2 which was incubated for 1 h. Reaction mixtures were electrophoresised on a 85-cm long 10% polyacrylamide gel which was exposed using two x-ray films to cover the whole gel length. Primary cleavage sites are indicated in the right panel; the numbers refer to nucleotide positions in the pre-tRNA sequence (see Fig. 4A). Sequence variation is seen at nucleotide position 57 and is explained in the text. The portion of the gel containing the sequencing reactions was exposed for twice the time that was necessary for the structure-probing reactions to allow clear reading of the sequences. The symbols X and B indicate the positions of marker dyes, xylene cyano1 and bromphenol blue, respectively. The left panel shows the positions of features of the secondary structure with respect to the cleavage ladders.

primary cleavage conditions (Fig. l ) , appear to be involved in tertiary structure. This observation would be consistent with the tRNA moiety of the pre-tRNA, assuming a tertiary struc- ture like that observed for x-ray crystal structure of tRNAPh' (30, 31) and inferred for tRNA solution structure in crystal- solution comparisons (32-34).

Other yeast pre-tRNAs were also studied to establish whether the structure observed for pre-tRNAeA could be a common feature. The secondary and tertiary structures of pre-tRNAFzG, pre-tRNAeAu, and pre-tRNAF62 were probed using the same approach as for pre-tRNA&A. Fig. 4, B to 0,

summarizes these data and shows the secondary structures and primary cleavage sites of different structure-probing en- zymes in these preARNAs.

Pre-tRNAF& contains a class I11 tRNA (26) similar to pre-tRNA&A; however, unlike the latter it has one of the shortest introns among the yeast pre-tRNAs (19 us 32 or 33 nucleotides, respectively). The structure-probing data (not shown) demonstrate that pre-tRNAp& (Fig. 4B) has a struc- ture much like pre-tRNAKA except that the extra arm of the intron is not present in this molecule. There is, however, a short single-stranded region which includes the 3' splicing cleavage site. Helix-specific RNase V1 cleaved primarily at the stem formed by the anticodon loop and complementary sequences in the intron, confirming the presence of the anti- codon-intron helix.

Fig. 4C shows the secondary structure of pre-tRNA&. Although the large size of the intron may seem to contribute to a complex structure, the structure-probing results (data not shown) summarized here show that this pre-tRNA retains features seen in both pre-tRNA&* and pre-tRNA@&. The tRNA portion of the precursor has the D, T+C, acceptor, and anticodon helices. The D and T+C loops are not cleaved under primary cleavage conditions. Nucleotides of the anticodon loop are base-paired with a complementary sequence in the intron although the anticodon-intron helix thus formed con- tains a mismatch of the central base in the anticodon. The majority of the sequence of this large intron is found in two hairpin stem-loop structures located in place of the lower loop seen in pre-tRNAgA. The 5' and 3' splice junctions are both single-stranded in pre-tRNAgAU.

One pre-tRNA from a population of precursors which con- tain the same tRNA& sequence' was also studied. The precursor, pre-tRNAFzg, has 28 nucleotides in its intron. The intron sequence was obtained from the sequencing reactions which accompanied the structure-probing experiments (data not shown). It differs at several nucleotide positions from intron sequences previously reported.' The structure-probing data (not shown) for this precursor revealed similar secondary and tertiary structure (Fig. 40) as has been previously de- scribed for the other three pre-tRNAs. A hairpin stem-loop structure, found in the 5' portion of the intron, is very sensitive to single- and double-strand-specific nucleases. Both splice junctions are single-stranded and the 5' splice site in this precursor is the primary cleavage site of RNase T1. One potential difference was observed. Only part of the anticodon (2 out of 3 nucleotides) can form a traditional Watson-Crick helix with the complementary sequence in the 3' portion of the intron. Additionally, the anticodon portion of the precur- sor is very susceptible to RNase T1 cleavage including the central nucleotide of the anticodon (shown as a Watson-Crick base pair in Fig. 40). The potential for a nontraditional G . A base pair between the third anticodon nucleotide (a guano- sine) and the adenosine opposing it in the secondary structure must be considered. Deoxyguanosine-deoxyadenosine pairing has been observed and produced helices with lower stability and potentially deformed backbone structure (35). The exist- ence of such structures in RNA and their susceptibility to RNase T1 have yet to be demonstrated but could explain our observation.

Effects of Mg2+ and Spermidine on Pre-tRNA Structure- In order to further substantiate the observation that the pre- tRNA assumes a tRNA-like tertiary structure, we looked at the nuclease sensitivity of pre-tRNAs at different concentra- tions of divalent cation (Mg2f) and polyamine (spermidine). I t is well known that tRNA structure is highly dependent on its ionic environment and that the tertiary structure is espe-

Structure of Yeast Intron-containing tRNA Precursors 3111

cially sensitive to changes in Mg2+ and spermidine concentra- tions (16, 34). We postulated that if the pre-tRNA has ele- ments of tRNA structure, stabilization of tertiary structure by Mg2+ may be observed as has been seen in the case of tRNA (16, 17). The conditions that were established previ- ously to give only primary cleavage of the pre-tRNA were used in these experiments. We assumed that if the pre-tRNA structure was altered we would observe the appearance of new bands, either as primary cleavages or in addition to the primary cleavage in the native structure. This is seen in Fig. 2 for pre-tRNAEA. In the absence of M e (plus 1 mM EDTA), predominant cleavages by RNase T1 were detected at guan- osine residues in the D (G15, GI7, and G18) and T+C (Gg8) loops. In addition, we detected destabilization of certain hel- ices of the secondary structure in the absence of M e at 37 "C. These helices include the D stem (GZ4, GZ5), anticodon stem (G31), variable stem (G", Gs5) and the stem formed by anticodon and intron sequences (G38, G4'). Cleavages at these normally base-paired guanosines could be the result of lower stability of these secondary structures, the loss of tertiary structure, or cleavages in single-stranded regions. The latter alternatives could result in lower stability of these short helices. However, as the M$+ concentration increased from 0 to 10 mM, cleavage at these guanosines seemed to disappear more rapidly than the cleavages in the D and T+C loops which supports the former hypothesis. The primary cleavage at residue 57, seen under the standard conditions, can be ob- served at all M e concentrations tested.

Fig. 3 shows the effect of varying spermidine concentration on the RNase T1 cleavage pattern of pre-tRNAEA in the presence (10 mM) or absence (plus 1 mM EDTA) of M e .

D LOOP [

5"SPLICING CLEAVAGE SITE"

EXTRA x LOOP[

CLEAVAGE SITE+ 3"SPLICING

VARIABLE REGION

TYC LOOP [

SEQUENCE STRUCTURE PROBING

- 66

- 81

85 -

- 98

B

FIG. 2. Effects of Mg2+ on pre-tRNAeA structure. RNase T1 at 1 X units/pg of RNA was used to study the effect of varying MgC12 concentration of pre-tRNAFA structure. Reaction conditions were essentially the same as those in Fig. 1, except that Mg2+ concen- trations were varied as indicated on the figure. Cleavage sites in the absence of Mg2+ (1 mM EDTA) are shown in the right panel. See legend to Fig. 1 and "Experimental Procedures" for other explana- tions.

IO mM M T No Mg" Spd (mM) Spd (mM) CLEAVAGE -_____________ SITES

00.050.1 05 I 3 5 00050.1 05 I 3 5 ( G ) L

D LOOP[

5"SPLICING - CLEAVAGE SITE+ - 31 38

EXTRA " LOOP[

CLEAVAGE SITE"

r

3 " S P L I C I N G

VARIABLE REGION

a,[ B

FIG. 3. Effects of spermidine on pre-tRNA& structure. RNase T1 at 1 X units/pg of RNA was used to study the effects of varying spermidine (Spd) concentrations on pre-tRNMA structure either in the presence (10 mM) or absence (plus 1 mM EDTA) of M$+. The reactions conducted in 10 mM MgC12 were essentially the same as those in Fig. 2 except for the presence of different spermidine concentrations as indicated on this figure. The reactions done in the absence of M$+ were also the same as the RNase T1 reaction in the presence of EDTA in Fig. 2 except for the presence of different spermidine concentrations as indicated. Guanosine positions which were cleaved by RNase T1 are indicated in the right panel. These nucleotides were assigned by comparing to parallel sequencing reac- tions (not shown).

Spermidine has no additional detectable effect on pre- tRNA&A structure in the presence of 10 mM M e . The guanosine residue G57 remains as the sole cleavage site at all spermidine concentrations. However, spermidine does substi- tute effectively for Mg2+ in stabilizing secondary structure. At very low spermidine concentrations cleavage could not be detected at guanosine residues in the double helical regions previously mentioned. But, spermidine cannot substitute as efficiently in the tertiary structure. It required 3 to 5 mM spermidine but only 0.5 to 1 mM M$+ to eliminate cleavage in the D and T+C loops. These data are consistent with NMR studies which suggest that Mg2+ is more efficient than sper- midine at restoring tertiary hydrogen bond resonances in tRNAPhe (16). The RNase T1 primary cleavage site at G57 was seen at all spermidine concentrations tested. These effects of Mg2+ and spermidine on the secondary structure and the probable tertiary interactions between D and T+C loops of pre-tRNAEA further substantiate a structure of pre-tRN&yA which has native tRNA structure in the tRNA portion of the precursor. The cleavage of pre-tRNAFAU and pre-tRNAFZ: by RNases showed similar dependence on M$+ and spermidine concentrations (data not shown).

Consensus Secondary Structure of Yeast tRNA Precursors- When the data gathered from structure-probing of four dif- ferent yeast pre-tRNAs are compiled, it appears that although pre-tRNAs might vary in the size and sequence of their introns, several common structural features exist among these tRNA precursors. Fig. 5 shows a consensus secondary struc- ture, which is compiled from our four structure-probing stud- ies and the predicted secondary structures obtained from

3112

A. pre-tRNAk:: :OH C A

pG C G - C U - A U * AIIO

5G.C

Structure of Yeast Intron-c,

A

G - U G * C

SG . C A . U

pG . ClOO

C. pre- tRNG C ACH

C A

p G * C C * G U * A m C - G

G .n

C ClW

p G * C G * C G . C

C - 0

FIG. 4. Secondary structures of the pre-tRNAs. A . The sec- ondary structure of pre-tRNA& is shown with the nucleotides numbered starting from the 5' end. Sequence variations observed in the two genes that have been sequenced (28, 29) are shown by the double-headed arrows. The three-pronged bracket indicates the anti- codon triplet. P and OH are the 5'-phosphate and 3'-hydroxyl ter- mini, respectively. Base-paired nucleotides are connected by . . Heauy arrows indicate the positions of the 5' and 3' splicing cleavage sites. The primary cleavage sites for the single-strand-specific RNases are indicated by squares and are the following residues: RNase U2 (A4', A"), RNase A (U"'), RNase T1 (G5'), and mung bean nuclease (A4', A4'). The primary cleavage site for duplex-specific RNase V1 (G") is indicated by the diamond. The circles indicate other nucleotides which are secondary sites of cleavage under the standard-probing conditions. Triangles indicate nucleotides which become very susceptible to RNase cleavage upon removal of M F . The inuerted triangles indicate nucleotides of lesser intensity of cleavage under the latter conditions. R. The secondary structure of pre-tRNA& is shown. The symbols used in this figure are as described in A . The primary cleavage sites are as follows: RNase U2 (A"), RNase A (U45), RNase T1 (G41, G4*), mung bean nuclease (U43), and RNase V1 (C51, U5', A53). C. The secondary structure of pre-tRNAgAu is presented with primary cleav- age sites as follows: RNase A (U7?, mung bean nuclease (A8', A"), and RNase V1 (Cs6). Symbols are as described in A . Cleavage

ontaining tRNA Precursors

sequences of the other five pre-tRNAs (pre-tRNAPh', pre- tRNATy', pre-tRNA?&, pre-tRNA&, and pre-tRNATT (11, 29, 36-39)'). In this consensus structure, the acceptor stem, D and T+C hairpin stem-loop structures, and anticodon stem are the same as those found in the tRNA cloverleaf. The intron is always located in the same position between the sixth and seventh bases of the anticodon loop. Sequences complementary to some of the nucleotides of the anticodon loop are always found within the intron, allowing the forma-

C 0

PO 0 0.0 0.0 0 - 0 0 - 0 0.0 0.0 u o o o o c y O A

G x x x C X O G R . . . . . R

G X

.... p:XOoGT " x x x x ~ x o y 0 x x

0.0 0.0 0.0 0.0 0 O# y OO

r O o x x U 0

FIG. 5. Consensus secondary structure for yeast tRNA pre- cursors. The tRNA portion of the precursor retains the conventional cloverleaf structure except in the anticodon loop where the intron is always found inserted between the sixth and seventh bases (arrows) of the loop. A sequence complementary to part of the anticodon loop can always be found within the intron forming a double helical structure. The smallest is three base pairs (found in pre-tRNATYr). The 5' and 3' splice junctions (broad arrow) are located a t either end of this helix and are almost always single-stranded. Nucleotide se- quences which are always conserved are indicated by sequence: G, guanosine; U, uridine; A, adenine; C, cytidine; T, thymidine. Invariant purine and pyrimidine positions are indicated by R and Y, respec- tively. Open circles indicate conserved positions with varied se- quences. X ' s indicate positions with varied length as well as sequence. The three-pronged bracket indicates the anticodon triplet. P and OH indicate the 5'-phosphate and 3'-hydroxyl termini, respectively. Base-pairs are indicated by . by RNase T1 (G17, G", G63, G6') was enhanced a t lower Mg2+ concen- trations; thus, these residues were not considered "primary" sites. D. The secondary structure of pre-tRNAEE2 is shown and presents an intron sequence of another member of the group of precursors for tRNAg& The primary cleavage sites are as follows: RNase U2 (A43, A40, RNase T1 (G3', G35, G36), mung bean nuclease (A43, A", and U"), and RNase V1 (U38 and G3'). Symbols are as described in A.

Structure of Yeast Intron-c

tion of a double helical structure. In two of the four cases all three anticodon bases are included in traditional base pairs in this helix. In the two other cases (pre-tRNA%3‘ and pre-tRNAFAu), it may be possible that nontraditional purine. purine base pairs exist. The 5’ and 3’ splice junctions, located at either end of this helix, are single-stranded except for the 5’ splice junction in the predicted secondary structure of pre-tRNA#”. In this pre-tRNA the intron-anticodon helix may be extended by three additional base pairs involving complementary intron sequences.’ This places the 5‘ splicing cleavage site in the middle of a double helix. Differences among the introns of the various pre-tRNAs are accommo- dated in two intron-specific regions denoted by X’s in Fig. 5. These two regions can be simple single-stranded loops (e.g. pre-tRNA&,J or more complex stem and loop structures. The pre-tRNAs shown in Fig. 4 are representative of the variations that occur.

Model for a Tertiary Structure of Yeast tRNA Precursors- Structure probing of these four pre-tRNAs also provides insight into elements of their probable tertiary structure. The primary cleavage data and the effects on cleavage site avail- ability by variation in Mg’+ and spermidine concentrations support the concept of an organization of the “cloverleaf” secondary structure into a tertiary structure consistent with that directly observed in the yeast tRNAPh‘ crystal structure. Strikingly, the intron region is always the portion of the

T Q C LOOP

3’

:ontaining tRNA Precursors 3113

molecule most sensitive to cleavage by the RNases. This finding strongly indicates that the intron is an exposed region of the molecule and, therefore, is readily accessible for inter- action with the splicing endonuclease. The concerted loss of cleavage of single-stranded nucleotides in the D and TI1.C loops as Mg’+ or spermidine concentration is increased in structure-probing reactions mimics the results obtained for tRNAPhe (Ref. 17 and data not shown). This observation suggests that a similar tertiary structure is found in the yeast pre-tRNAs. Fig. 6 summarizes these observations in a consen- sus tertiary structure. In this model, the tRNA portion of the precursor retains the conventional ‘%“-shape conformation that is stabilized by tertiary interactions between different regions of the molecule, especially between D and T#C loops. These interactions are also stabilized by M e which specifi- cally complexes in the crystal structure in the pocket formed by the D and TI1.C loops (40, 41). The stem formed between anticodon loop and intron sequences extends from the anti- codon stem to form a continuously base-stacked helix on one arm of the L conformation. Additional sequences may be found at the 5’ and/or 3’ ends of the intron in two variable regions (hatched lines in Fig. 6).

DISCUSSION

RNA splicing is ubiquitous in eukaryotes, but we know little about it mechanistically in terms of how splice junctions are uniquely recognized by the enzymes which cleave and ligate. In the various systems under study, consensus se- quences have been observed among only certain classes of spliced RNA precursors. However, even these consensus se- quences may not have a direct role in the recognition of the

( 1- associated splice junction (e.g. certain cryptic sequences un- CCA masked by P-thalassemia mutations (42)). It is becoming End increasingly apparent that RNA secondary and/or tertiary

structure may define the selection of cleavage and ligation positions (8-10). To investigate the significance of RNA struc- ture in tRNA splicing and to eventually understand its mech- anism, we have probed the native structures of four yeast pre- tRNAs containing introns of different sizes and structural complexities.

The limited RNase cleavage technology (15, 22, 27) has been widely used for the study of RNA structure, especially those RNAs which would be difficult to study using physical methods, such as NMR or x-ray crystallography, due to lim- ited availability of substrate. The structure of yeast tRNAPhe obtained using this method has proved to be consistent with the structure obtained by physical methods (15, 22, 27). In using this method, it can still be argued that the specific interactions between probing enzymes and RNA may, in some cases, result in artifacts, e.g. sequence preferences or failure to detect an accessible region because of steric hinderance. To minimize this possibility, we have included in our struc- ture-probing study a battery of enzymes with different speci- ficities and a variety of ionic conditions in which yeast tRNAPhe was known to exhibit dynamic changes in tertiary structure. The standard conditions were chosen because they were known to maintain the native tertiary structure of yeast tRNAPhe (reviewed in Ref. 34) and were close to conditions used in sdicine endonuclease assavs. -

F ~ G . 6. Model for the tertiary structure of yeast tRNA pre- The observation that a single enzymatic activity is respon- cursors. The tRNA portion (thin line) of the precursor retains the sible for the endonucleolytic cleavage of the intron from nine conventional “L” shape conformation with tertiary interaction be- precursors (13) suggests that a feature of these pre- tween different regions of the molecule. The intron (hick line) is tRNAs is being recognized. As we have shown here, the fairly inserted into the anticodon loop (circles indicate anticodon triplet). Intron segment.s t,hat form part of the consensus secondary structure diverse sequences Of the pre-tRNAs produce secondary are shown by the thick black line. Positions of variable structure are and tertiary Structures that maintain certain common features shown by the hatched thick line. (summarized in Figs. 5 and 6). Similar observations of the

3114 Structure of Yeast Intron-containing tRNA Precursors

formation of base pairs between anticodon loop and intron sequences have been made for yeast precursors of tRNAT!'' and tRNAPhe (43). This conserved double helix is flanked by single-stranded regions and by the most variable elements of pre-tRNA secondary structure. In the four structures that we studied, these two regions show diverse structural organiza- tion. Single-stranded loop, single hairpin stem-loop, and mul- tiple hairpin stem-loop structures are found in pre-tRNAs which are all cleaved by the same partially purified splicing endonuclease. These two regions must contribute a minimum number of nucleotides to connect the conserved regions to allow for optimum structural stability of the consensus struc- ture, i.e. particular sizes of hairpin loops and bulge loops may be more thermodynamically favored (25). The single-stranded region which includes the 3' splice junction, while variable in size, never contains less than three nucleotides (e.g. Fig. 4C). Beyond these requirements the splicing endonuclease is ap- parently insensitive to sequence and structure of these vari- able regions. In several cases where multiple genes for a tRNA have been sequenced, it has been shown that introns for identical tRNAs vary in sequence (28, 29, 36, 37).' These variations are always located in the variable portions of the consensus structure.' In addition, in vitro mutagenesis of one cloned gene for pre-tRNA&* has provided altered genes with insertions and deletions at the HpaI site (GTTAAC) located in the extra arm of the intron (44, 45). These alterations had no effect on splicing of the variant precursors except in one case. The exception was a mutant in which all of the extra arm was deleted except for one nucleotide. In this mutant the 3' end of the intron terminates with a cytosine which is not found in any of the nine yeast pre-tRNAs.' This mutant also deviates from the consensus structure in that there are only two single-stranded nucleotides at the 3' cleavage site. All of the other mutants in which splicing was unaffected main- tained our consensus structure.

The finding of a consensus structure, which is an extension of the conventional tRNA structure, may suggest that the splicing endonuclease recognizes some portion of the tRNA structure. Another laboratory has reported that elimination of base pairing of the D stem in a yeast pre-tRNAEA gene resulted in loss of cleavage by the Xenopus tRNA splicing endonuclease (46). It appears that the integrity of the struc- ture of the tRNA portion of the precursor is an essential feature. It has also been demonstrated that Mg2+ and sper- midine are required for splicing (4, 13). It is tempting to speculate that this requirement may be related to the tertiary structure stabilization by these cations. Whether the tertiary structure is necessary for recognition or for the maintenance of stable helical structures more proximal to the cleavage sites remains to be demonstrated.

The 5' splicing cleavage site is shown single-stranded in our model; however, the predicted secondary structure for pre-tRNA&, suggests that it may be double-stranded.' The anticodon-intron helix and single-stranded 3' splicing cleav- age site are the two invariant elements of yeast pre-tRNA structure. This strongly indicates that they have an important role for the binding site and/or active site of the yeast tRNA splicing endonuclease. Some illumination is shed by the cor- relation of other experimental results which utilized heterol- ogous systems. Transfer RNA genes from some other orga- nisms have been sequenced and shown to contain introns which cannot form the anticodon-intron helix (e.g. Refs. 47- 49). In the fission yeast, Schizosaccharomyces pombe, pre- tRNALy" has an eight nucleotide-long intron that contributes no probable secondary structure and can be spliced in S.

cerevisiae extracts although not as efficiently as S. cerevisiae pretRNAPA (47). Conversely, when wild type and SUP4 pre- tRNATy' from s. cerevisiae are compared in Xenopus oocytes the SUP4 pre-tRNA, in which the anticodon-intron helix is compromised by the suppressor mutation, appears to splice more efficiently than the wild type pre-tRNA (50). The Xen- opus pre-tRNATyr sequence cannot form the anticodon-intron helix (48). In both examples there is a correlation between the known (or predicted) structure of homologous pre-tRNAs and the relative efficiency of splicing of heterologous pre- tRNAs with dissimilar details of structure. This suggests that in the finer points of endonuclease-precursor recognition var- ious organisms may differ.

In conclusion, we have probed secondary and tertiary struc- tures of four yeast intron-containing tRNA precursors and have found a consensus pre-tRNA structure. Elements of this structure which are conserved may serve as recognition points for the yeast tRNA splicing endonuclease. In addition, through studying the dynamics of the pre-tRNA structure in response to its ionic environment we have inferred that the tRNA moiety of these pre-tRNAs assumes a tertiary structure similar to that of the mature tRNA.

Acknowledgments-We thank Dr. Donald Lightfoot for kindly supplying RNase V1; Fran Roland for expert rendering of our model; and Carolyn Travis, Stephanie Bragg, Bonnie Pfanenstiel, and Yvette Cook for technical assistance. We also thank Dr. Richard Ogden for useful discussions.

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