Intron Sequence and Structure Requirements for tRNA Splicing in ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Val. 263, No. 27, Isaue of September 25, pp. 13839-13847,1988 Printed in U.S.A.

0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Intron Sequence and Structure Requirements for tRNA Splicing in Saccharomyces cerevisiae” (Received for publication, March 24, 1988)

Elizabeth SzekelyS, HeatherG . Belford, and ChrisL. GreerB From the Department of Biological Chemistu, California College of Medicine, University of California, Irvine, California, 92717

Predicted single-stranded structure at the 3’ splice sors from Schizosaccharomycespombe (5,6) and theability of siteis a conservedfeatureamong intervening se- amphibian (7) and mammalian cell (8) extracts to process tRNA precursors. yeast pre-tRNAs indicate features required for splicing are quences (IVSs)in eukaryotic nuclear Theroleof 3‘ splice site structurein splicing was conserved across species boundaries. examined through hexanucleotide insertions at a cenThe sequences of 33 eukaryotic nuclear tRNA genes with tral intron position in the Saccharomyces cerevisiae introns have been obtained to date (9-16). Allcontain asingle tRNA gene. These insertionswere designed toalter the structure at the splice site without changing its se- small intron (8-60 nucleotides) which interrupts the coding quence. Endonucleasecleavage of pre-tRNA substrates sequence in the same relative position, one nucleotide 3’ to was then measured in vitro, and suppressor activity the anticodon. As a result, the pre-tRNA transcripts of these was examined in vivo. A precursor with fully double- genes share certain general structural features. Both lowest stranded structureat the 3’ splice site was not cleaved free energy calculations (11)and theresults of solution structure probing (17, 18) suggest these precursors contain a mabyendonuclease.Theintroductionofoneunpaired nucleotide at the3’ splice site was sufficient to restore ture tRNA-like domain which retains the conserved cloverleaf cleavage, althoughat a reducedrate. We have also secondary structure and L-form tertiary structure of their observed that guanosine at the antepenultimate posi- spliced counterparts. The identification of numerous exon tion provides a second consensus feature among IVSs mutations which affect splicing (19-24) provides good eviin tRNA precursors. Point mutations at this position dence that thesplicing enzymes, like many other tRNA procwere found to affect splicing although there was no essing enzymes (cf. Ref. 25), recognize conserved sequence specific requirementforguanosine.Theseandpreand/or structuralelements within the mature domain. Among vious results suggest that elements of secondary andl these mutationsare several which affect the accuracy of or tertiary structure at the3’end ofIVSs are primary determinants in pre-tRNA splice site utilization cleavage by endonuclease. An insertion mutation (Ai26) in whereas specific sequence requirements are limited. the S. pombe sup3-e gene creates a potential 6th base pair in the conserved 5-base pair anticodon stem and results in new 5’ and 3’ cleavage sites, each effectively one nucleotide closer to the base of the anticodon stem (23). Alterations in the The activities required for splicing of tRNA precursors (pre- length of the anticodon stem in pre-tRNAPh”have also been shown to result in relocation of the cleavage sites.3 These tRNAs) in extracts of Saccharomyces cerevkiae are a sitespecific endonuclease and anATP-dependent RNA ligase (1- observations suggest splice sites may be identified in part by 3). Theseactivities can be separated and arecapable of acting their distance and position relative to a primary binding site independently (2). Theendonuclease cleaves precisely at the within the tRNA-like domain. A comparison of the sequences at the 5’ and 3’ ends of boundaries of the intervening sequence (IVS),’ producing paired tRNA “halves” and the linear IVS (4). In this work, IVSs in tRNA precursors is shown in Fig. 1. In contrast to we have examined features of the substratewhich affect splice the mature domain, the IVS contains few conserved elements. This lack of conservation and the ability of the splicing site utilization by the tRNA endonuclease. The profiles of endonuclease activity determined with a apparatus to tolerate large intron insertions, deletions, and variety of pre-tRNAs are coincident throughout extensive rearrangements (26-28) suggest much of the IVS plays no purification (2).’ This result suggests that there is a single role in splicing. However, limited elements of IVS sequence enzyme which recognizes features common to all of these or structure mustbe essential since 1)endonuclease does not substrates. The ability of the yeast enzyme to process precur- cleave “mature-sequence” tRNA (28); and 2) certain intron mutations and deletions have been shown to affect splicing * This work was supported by Grant GM-35955 from the National (23, 27-29). Institutes of Health and Grant DMB-8614092 from the National Most of the splicing-defective intron mutations have been Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must found to affect the 3’ splice site where two consensus elements therefore be hereby marked “advertisement” in accordance with 18 can be identified. The firstis the predicted structure of the 3‘ splice site region. Single-stranded structare at the 3’ splice U.S.C. Section 1734 solely to indicate this fact. $ Supported by Fellowship GM-09483 from the National Institutes site is predicted by lowest free energy calculations for all of of Health and Fellowship S-52-86 from the California Division of the precursors (( 11)data not shown) and has been confirmed The American Cancer Society. 5 Established Investigator of the American Heart Association. To for several by solution structure probing (17, 18). Deletions within a yeast tRNAP’” gene and a tRNkh” which alter both whom correspondence should be sent. The abbreviations used are: IVS, intervening sequence; SSI, sin- the sequence and predicted structure at the3‘ splice site have gle-strand insert, DSI, double-strand insert. P. Green and J. Abelson, personal communication.

V. Reyes and J. Abelson, manuscript in preparation.

13839

Affecting Mutations Intron

13840

tRNA Splicing secondary structure of the IVS-containing pre-tRNAencoded by this gene is shown in Fig. 2. The numbering convention used throughout is that based on tRNAPhe(33) which allows constant numbering of conserved positions. Nucleotides within the IVS (which begins after position 37) are numbered 321, 37:2, etc., as is done for other regions in which length varies. Role of Conserued Intron Sequences-In 27 of the 33 pretRNA sequences currently available, guanosine is found at the antepenultimate position of the IVS. The SUP53precur-

L

$

40 20 0 1

3

2

4

5

-2

-1

Position 1001

I

-3

Position FIG. 1. Intron sequences at splice sites intRNA precursors. A comparison of the sequences at the 5' (A) and 3' ends ( E )of IVSs in pre-tRNAs is shown. The position indicated is the distance in nucleotides (with the 5' to 3' direction positive) from either the 5' splice site (panel A ) or the 3' splice site (panel B ) . For each position, the percent of the totalrepresented by each of the 4 bases is indicated. Thirty-three sequences were compared including 16 from S. cereuisiae (11, 17), 7 from other fungi (12, 13, 47-50), 2 from plants (14, 51),1 from Dictyosteldum (52), 1from Drosophila (53), and 6 from vertebrate sources (15, 16, 54,55).

been shown to affect splicing (30).4Although the sequence at other IVS positions is not highly conserved, the presence of guanosine at the antepenultimate position (-3 from the 3' splice site)in 80% of the precursors compared in Fig. 1 provides a second consensus feature. A guanosine to adenosine change at this position in the sup3-e precursor from S. pombe has been shown to affect splicing (24). Thus thisposition is a candidate for a sequence-specific recognition site. This observation andprevious analyses of IVS mutations suggested that both sequence and structure at the 3' splice site may affect tRNA splicing, a situation which would be similar to thatfor other classes of splicing reactions. To examine whether these conserved features are required for splicing, a series of directed mutations within the intron of the SUP53 (tRNA?) gene from S. cereuisiae was constructed. Splicing of pre-tRNA transcripts was measured in uitro, and suppressor activity was examined i n uiuo. The results indicate that while structure at the 3' end of IVSs is a critical factor insplice site utilization, specific IVS sequence requirements are limited. EXPERIMENTAL PROCEDURES' RESULTS

The S. cerevisiae SUP53 gene encodes an amber suppressor derivative of tRNA? (31, 32). The sequence and predicted M.-C. Lee, J. Milligan, and G. Knapp, personal communication. "Experimental Procedures" are presented in miniprint at theend of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal thatis available from Waverly Press.

I

A

A A

II

A

U

B. SSI

A

A

C. D S I

A

n A

u u

U

FIG. 2. Primary sequence and secondary structure of SUP53 and mutant pre-tRNAs. A , the sequence and structure of pre-tRNASUPS3 is shown, Numbering of the mature domain is by the convention established for tRNAPhe (33) and predicted secondary structure is as described by Lee and Knapp (18). The broad filled-in arrows indicate the positions of splice sites. The open arrow indicates the position of the SSI and DSI hexanucleotide insert sequence. Z indicates the intron loop region of the precursor referred to in the text. In panels B and C , only the anticodon stem-intron regions of these precursors (corresponding to the boxed region in panel A) are shown. The SSI and DSI 6-nucleotide insert sequence is shown in boldface. The star in panel C indicates the nucleotide that is altered in DSI pre-tRNA in order to obtain the mutant DSI-1C precursor.

Intron Mutations Affe tcting tRNA Splicing sor is an exception with adenosine at this position. To determine whether sequence is essential for optimal splicing, directed point mutations which placed a consensus guanosine or anonconsensus uridine at this position were made. Labeled pre-tRNA substrates were prepared by transcription, and endonuclease cleavage was measuredin vitro. The results are shown in Fig. 3. Both the uridine and guanosine replacements reduced the rateof cleavage. This result is not consistent with a specific requirement for guanosine.Thus thepredominance of guanosine at the antepenultimate position may reflect a structural requirement such as pairing with another conserved position or may be essential for a process other than splicing. Structure of the 3' Splice Site-Deletions and rearrangements which include the 3' end of the IVS have been shown to affect splicing (27-30): While this suggests some aspect of this region is essential, the natureof these mutations makes it difficult to attribute splicing defects to effects on structure as opposed to changes in sequence. To overcome this limitation, a series of hexanucleotide insertions was made at a central intronposition in the SUP53gene. The insertion site was chosen so as to allow potential interaction between the IVS insert in the pre-tRNA and the 3' splice site loop. The sequences of two inserts are shown in Fig. 2. The first insert is not complementary to the splice site loop, and this SUP53 variantwas designated SI for singlestrand insert. The second is fully complementary to the splice site loop and creates the potential for an extended helix which would include the 3' splice site. This variant was designated DSI for double strand insert. Limited ribonuclease digestion was used to probe the structures of the SUP53, DSI, and SSI precursors. Two enzymes whichcleave preferentially within single-stranded regions were used. These were RNase T1, which is specific forguanosine, and Neurosporacrassa endonuclease, which has no known base specificity (34). A third enzyme, cobra venom ribonuclease VI, which cleaves preferentially within segments involved in secondary or tertiary interactions (35), was used to probe structured regions. Low specific radioactivity transcripts were prepared and labeled to high specificactivity at 5' ends with T4 polynucleotide kinase and [y3*P]ATPor at 3' ends with T4 RNA ligase and cytidine 3',5'-[3'-32P]bisphosphate. Labeled precursors were purified by gel electrophoresis, subjected to limited nuclease digestion, and the products were analyzed by electrophoresis on thin denaturing gels (these methods are described in the Miniprint Supplement). An example of one

13841

such experiment using the N. crassa nuclease is shown in Fig. 4. A summary of the structure probing experiments is presented in Fig. 5. Within the D, T, and acceptor arms the patterns of cleavage by each of the enzymes were identical among the SUP53, DSI, and SSIprecursors (for simplicity, only the T1 cleavage sites in this region are shown in Fig. 5). Thus, the mature tRNA-like domain of the precursor is not affected by the IVS insertions. The pattern of T1 and N. crassa nuclease cleavage around the 3' splice site was identical for the SUP53 and SSIprecursors. The IVS insert in SSI was also a site for cleavage by both of these enzymes. Thus, the 3' splice sites in the SSI and SUP53 precursors have similar single-stranded character. In contrast, both the 3' splice site and the IVS insert in the DSI precursor were resistant to the single-strand-specific nucleases. This result is consistent with the formation of an extended IVS helix which includes the 3' splice site in DSI RNA. The appearance of three new cobra venom ribonuclease VI sites and the loss of both T1 and N. crassa nuclease sites

A. 1

2

3

4

5

6

7

8

GI 5G237 G24 G30G37-

and DSI - SSI 5' SliceSite

-

SUP53 5' Slice Site DSI

s,",'S:d

G37:17-

d l n t r o n LOOD

G3724G37:26, G3721G37:28-

3' Splice Site Loop

B. 1

2

3

4

5

6

7

8

"

c 3

60 -

G37:24G37:26G37277 G37:28

0

3 Intron LOOP 3' Splice Site Loop

A

FIG.4. Limited digestion by N. C m 8 8 a endonculease of SUP53 and mutant pre-tRNAs. A, the SUP53 (lanes 3 and 0

5

10

Time

15

20

(min)

FIG. 3. Endonuclease cleavage of mutant transcripts. Labeled SUP53 (O), G37:30 (0)and U37:30 (A) precursors were prepared by in vitro transcription and incubated with tRNA splicing endonuclease for the times indicated (see the Miniprint Supplement for methods). The reaction products were resolved byelectrophoresis, visualized by autoradiography, and then quantitated by measuring Cerenkov radiation in gel slices. The percent precursor cut was calculated as in Ref. 23.

4),

SSI (lanes 5 and 6),and DSI precursors (lanes 7 and 8)labeled at 3' ends and incubated with (lanes 4,6, and 8)or without (lanes 3,5,and 7) N. crmsa endonuclease were resolved by electrophoresis on thin denaturing gels as described in the Miniprint Supplement. Endlabeled SUP53 was also digested with RNase T1 under denaturing conditions (lane I ) and with NaOH (lane 2 ) to provide sequence markers. The positions of T1 cleavage sites are indicated at the left edge. Regions of the precursor corresponding to N . crassa nuclease cleavage sites are indicated at the right edge. The autoradiograph in panel A was exposed for 1 day. Panel B shows a &day exposure of the region of the gel corresponding to thelarge bracket in panel A.

13842

Intron Mutations AffectingtRNA Splicing

A. SUP 53

AOH

C C

.

A

PG C G . C

u

U

DG

u

' A70

. A

.C .

GO

C.DSI

U

U l0i

u. u. c. u. c.

...

G G C

c

usoA

n

C C G

AA

AQH

A

G aa

C

G bC b C

bU

-@

a-

FIG. 5. Summary of structure probing experiments. A diagrammatic summary of the results of limited nuclease digestions of end-labeled SUP53 (A), SSI ( B ) ,and DSI (C) precursors is shown. For simplicity, only the RNase T1 cleavage sites are shown for the whole precursor with other nuclease sites shown only for the anticodon stem-intron region (positions 27-43 inclusive). In all cases cleavage was at the 3' side of the base indicated. RNase T1 products readily visible on short exposures (typically 16 h when lo' cpm of substrate was used) were designated major cleavage sites and are indicated by double open triangles. Products readily visible on intermediate exposures (typically 2 days) are indicated by a single open triangle. Within the anticodon stem-IVS region, products only faintly visible after extended exposure (7 days) are indicated by parentheses. N. crmsa nuclease cleavage sites are indicated by circles with major sites as defined above shown with bold circles. Cobra venom endonuclease VI sites are shown with filled triangles.

within the predicted stem provide additional evidence for the comparable efficiencies producing the end-mature IVS-conformation of a stable extendedhelix. taining forms (data not shown). Thus the IVS inserts have Differences in cleavage patterns were also observed near no significant effect on these other transcriptionand processthe 5' end of the IVS in theregion predicted to pair with the ing steps. The rate of cleavage of these labeled substrates was then anticodon segment. For the SSI precursor, two novel VI sites (U37:8 and U37:9) and enhanced T1 cleavage at G37:lO were measured using a partially purified yeast splicing endonucleobserved. Increased cleavage by both single- and double- ase fraction. Reactions containing constant amounts of substrand-specific nucleases in thissmall segment may be due to strate and endonuclease were incubated at 30 "C for varying increased nuclease accessibility reflecting a more open or intervals, stopped with the addition of a sodium dodecyl flexible structure in the SSIIVS domain. For the DSI precur- sulfate-proteinase K mixture (see the Miniprint Supplement), sor, reduced cleavage (relative to the SUP53 and SSI precur- and productswere resolved by gelelectrophoresis and visualsors) by both the T1 and N. crassa nucleases was observed in ized by autoradiography. Examples for the SUP53, SSI, and the 5' IVS region. This might reflect either decreased nuclease DSI precursors are shown in Fig. 6, A-C. For each of these accessibility or an increase in the stability of this structure. substrates, long incubations in the absence of added enzyme Two additional N. crassa nuclease cleavage sites were ob- resulted in the appearance of a small amount of breakdown served for the DSI precursor at positions 31 and 32 at the products. In the presence of added enzyme, the SUP53 and base of the anticodon stem.Note that the patterns of cleavage SSI precursors were converted to 3' and 5' halves and the at sites immediately adjacent to the5' splice site areidentical linear IVS (note that due to theIVS insert, the SSI IVS and 5' half nearly comigrate). The identities of these reaction for all of the precursors. IVS Excision by the tRNA Endonuclease-Labeled pre- products were confirmed by one-dimensional T1 oligonucletRNA substrateswere prepared by in uitro transcription using otide analysis (data not shown). In contrast to the SUP53 a yeast nuclear extract containing RNA polymerase I11 and a and SSI precursors, no correct cleavage products were devariety of tRNA processing enzymes ((36) see the Miniprint tected in incubationswith the DSI precursor. In addition to the expected cleavage products, two smaller Supplement). The SUP53, SSI, and DSI templates were transcribed at similar levels, and transcripts were processed with products accumulated in incubations with the SSI precursor

tRNA Affecting Mutations Intron

Splicing

13843

e. SSI

A. SUP53 Endonuclease: Time (min)

-

- 0 60 .".. ~

t

+ + + +

0 2

5

",=.-."

" + + + + + t 0 60 0 2 5 IO 20 60 - ". ."rr

i-

IO 20 60 a

~"

"

I

- pre-tRNA

-pre-tRNA

FIG.6. Kinetics of cleavage by the tRNA splicing endonuclease.Labeled SUP53 ( A ) , SSI ( B ) ,and DSI ( C ) precursors were prepared by in uitro transcription and incubated with (+) or without (-) yeast tRNA endonuclease as described in the Miniprint Supplement. The reaction products were resolved by polyacrylamide gel electrophoresis and visualized by autoradiography (panels A-C). Incubation times are indicated a t the top of these panels. Note that in panel C, the same bandsthat accumulate in the plus endonuclease lane after 60 min are also present in the no enzyme control, although they are of lesser intensity. The amounts of these endonuclease-independent products were found to vary in our experiments. Reaction products were quantitated by measuring Cerenkov radiation in gel slices, and the percent precursor cut by endonuclease was calculated as described by Greer et al. (23). The results shown in panel D are for two independent experiments distinguished by open versus filledsymbols. The data for SUP53 are indicated by boxes, for SSI by triangles, and for DSI by circles.

-3'half -3'half

/5'half + IVS

-5' half -1VS

,b

C. DSI

-

Endonuclease: t Time(min) +0 60-0 j__

t t

"-

D. t t

2 5 IO 20 60

I

pre-tRNA-

+

I ~

-

O O

20

40

60

Time (min)

(labeled a and b in Fig. 6 B ) . These were shownto be derived from the IVS by T1 oligonucleotide analysis. These products were also found to accumulate when the isolated IVS was incubated in the absence of endonuclease (data not shown). The nature of this endonuclease-independent cleavage reaction is being examined. Precursors and reaction products in these incubations were quantitated by measuring the radioactivity in gel slices. The results are shown in Fig. 6D. Although the initial rate and extent of cutting varied slightly from one substrate preparation to the next, cleavage of the SSI and SUP53 precursors was essentially identical. In no case wereany significant levels of correct cleavage products detected for the DSI precursor. Thus an IVS insertion which cannot pair with the 3' splice site had no effect on splicing. An insertion which is complementary to the3' splicesite completely blockedendonuclease cleavage at both the 3' and 5' splice sites. A series of endonucleasecompetition assays was carried out to examine the basis for the complete splicing defect in the DSI precursor. In these experiments, increasing amounts of unlabeled RNA competitor were addedto reactions containing constant amounts of labeled SUP53 precursor and limiting endonuclease. Following incubation at 30 "C, reactions were stopped and products resolved and quantitated as described above. The results are summarized in Fig. 7. The DSI precur-

sor was found to compete as effectively as SUP53 pre-tRNA and considerably more effectively than a nonspecific RNA, poly(A-C). These results indicate that the splicing defect in the DSI precursor isprobably not due to an inability to interact with the endonuclease. Attempts at measuring binding to endonuclease are under way and should providea means for directly testing thisassertion. Variants Which Restore DSI Cleavage-Minimal requirements for single-stranded structure at the 3' splice site were examined by introducing changes in the DSI insert which reduce its complementarity to the splice site loop in onenucleotide increments. In the first variant, designated DSIIC, guanosine at the first position in the DSI insert (see Fig. 2C) is replaced by cytidine. This replacement was designed to provide oneunpaired nucleotide (C38)at the 3' splicejunction in the pre-tRNA. Thestructure of the DSI-1C precursor was probedby limited nuclease digestion of end-labeled substrates as described above (data not shown). The patterns of cleavage by T1 and N. crassa nucleases were identical for the DSI and DSI-1C precursors. Cobra venom ribonuclease VI cleavage patterns were also similar with the following exception.Positions G37:32 and G37:33 in the extended 3' splice site helix were preferred cleavage sites in the DSI precursor but were not cleaved in DSI-1C pre-tRNA. This loss of cobra venom

13844

tRNA Splicing

Affecting Mutations Intron 100 1

A. D S I 1C - - + + + + + +

I

Endonuclease 0 60 0 Time (rnin) c

-

a

0

c

0

IO 20 30 60

.pre-tRNA

t 20

5

I

4

8 12 18 Cornpetttor/Substrate

20

FIG. 7. Splicing endonuclease competition experiment. Reactions (10 pl) contained constant amounts of endonuclease (2 X units) and high specific radioactivity SUP53 precursor (6 fmol, 5 X lo3 dpm/fmol) with increasing amounts of poly(A-C) RNA (0)or low specific activity (50 dpm/fmol) DSI (A) or SUP53 (0)precursor. Reaction conditions and analysis of products following gel electrophoresis (including calculations for percent precursor cut) were as described by Greer et al. (23). Competitor/substrate is calculated as the ratio of the concentrations of low specific activity precursor or poly(A-C) to high specific activity SUP53 RNA.

- 3‘ half - 5‘ half -IVS

ribonuclease VI sites was not accompanied by restoration of the T1 and N . crass& nuclease sites observed in this region for the SSI and SUP53precursors. Thus, this result may be due to either reducednucleaseaccessibility or to reduced stability of the extended IVS helix. Overall, the resultssuggest too I differences between the DSI-1C and DSI structures arelimited to theregion around the 3‘ splice site. Labeled pre-tRNA substrates were prepared by in vitro transcription, and tRNA endonuclease cleavage assays were carried out as described above. An autoradiograph from an assay with the DSI-1C precursor is shown inFig. 8A. Three cleavage products were detected in these endonuclease incubations. These products were identified as the authentic 3’ and 5’ halves and the IVS by T1 oligonucleotide analysis 5’ half from the (data not shown). Note that the IVS and DSI-1C precursor are the same length but do not comigrate. This is in contrast to the pattern obtained with the SSI precursor in which these two products nearly comigrate (see Fig. 6B). This is presumably due to a difference in conformation between the SSI and DSI-1C IVSs and suggests these Time (min) gels are not fully denaturing. A comparison of the rates of FIG. 8. Kinetics of DSI-1C pre-tRNA cleavage by the spliccleavage of the DSI-1C and SSI precursors is shown in Fig. ing endonuclease. Labeled DSI-1C precursor-preparedin vitro tran8B.Although cleavage of the DSI-1C precursorwas accurate, scription was incubated with (+) or without (-) tRNA endonuclease the reactionproceeded a t a much slower rate than for the SSIas described under “Experimental Procedures.” Reaction products substrate. Additional variants of the DSI insert designed to were resolved by gel electrophoresis and visualized by autoradiograintroduce additional unpaired sitesat thesplice site were also phy (panel A). Incubation times are indicated at the top of the panel. analyzed (data not shown). Variants with two or more mis- The identities of species indicated at theright edge was confirmed by matches to the splice site loop sequence were cleaved by the T1 oligonucleotide analysis. In a separate experiment (panel B ) , the kinetics of cleavage of the DSI-1C (A) and SSI (0)precursors were tRNA endonuclease a t rates similar to that obtained for the compared. Products were quantitated by measuring Cerenkov radiaSSI precursor. These resultssuggest that thefull 6-nucleotide- tion in gel slices, and percent cut was calculated as described in the long single-stranded segment found in the SUP53 precursor legend to Fig. 6. is not requiredfor optimal rates of endonuclease cleavage. Nonsense Suppressor Activity in Vivo-Correlation of splic- colonies (37). Mutations in ade3 block an earlier step in the ing defects measured in vitro with tRNA biosynthetic defects pathway (cf. Ref. 38) and are epistatic toade2, i.e. ade2 ade3 in vivo was examined by analyzingsuppression of amber double mutants produce white colonies (39). Thus, suppres(UAG) nonsense mutations. For this purpose, the SUP53 sion of the ade3-26(Am) mutation in our strain is detected by gene and the insertion variants were subcloned in a stable the appearance of the d e 2 color phenotype with thelevel of low copy yeast transformation vector and introduced into a color reflecting the level of suppressor activity (40).As sumyeast strain carrying multiple nonsense mutations. marized in Table I,cells transformed with the vector alone or Two tests for suppressor activitywere used. The first was with the DSI geneproduced white colonies indicating the a colony color assay which relies on a combination of the absence of suppressor activity. Transformants bearing either ade3-26(Am) and ade2-l(0c) mutations. Strains carrying d e 2 SUP53orSSI producedredcolonies indicating efficient red suppression of the ade3 mutation. Transformants with the mutations accumulatea pigmented substance and produce

B.

tRNA Splicing

Affecting Mutations Intron TABLE I In vivo assays of suuuressor function In

tRNA

SUP53 SSI DSI DSI-1C a

uiuo suppressor function*

I n vitro

Growth

splicing"

Colony color'

Yes Yes No Reduced

R R W DP

37,30,23"C

18 "C

+ +

+ +-

-

+

-

Compilation of data shown in Figs. 6 and 8.

* Suppressor function was assayed by colony color and growth of yeast strain ES24 transformed with plasmids containing the tRNA genes indicated (see the Miniprint for a description of methods). R, red W, white; DP, dark pink.

DSI-1C gene produced pink colonies consistent with intermediate levels of suppressor activity. The second assay for suppressor activity was based on the lys2-801(Am) and met8-1(Am) mutations with suppression measured by the ability to grow on defined media lacking lysine and methionine. Growth of transformants was assessed at 18, 23, 30, and 37 "C, and the results are summarized in or SSI genes grew Table I. Transformants bearing the SUP53 at all of these temperatures whereas those with the DSI gene or vector alone failed to grow at any temperature. The DSI1C transformants failed to grow at 18 "C but produced colonies at higher temperatures. The results of these nonsense suppressor assays are consistent with the splicing defects measured in vitro. In both assays SUP53 andthe SSI variant were indistinguishable, the DSI variant was completely inactive, and DSI-1C showed intermediate activity. DISCUSSION

The primary sequences and predicted secondary and tertiary structures of a number of yeast pre-tRNAs has been described and aconsensus structure hasbeen proposed which includes a single-stranded loop at the 3' splice site (11, 17, 18). The role of this consensus structure was addressed through a series of central intron insertions producing segments which canpair with the 3' splice site loop in the precursor. The effect of IVS insertions on splicing was correlated with the degree of complementarity to the 3' splice site and not with the site or lengthof the insert. Thissuggests that the ability of a fully complementary insert to prevent cleavage by endonuclease is due to a change in structure rather thana change in sequence. This effect might be directly due to theformation of an extended IVS helix at the 3'splice site or might be indirect via induced changes in structure in other regions. Evidence that there was no substantial change in the structureof the mature tRNA-like portion of the DSI precursor includes: 1)similar patternsof nuclease cleavage in the mature domain regions of DSI, SSI, and SUP53 RNAs were observed in structure probing experiments; 2) end maturation of DSI RNA by the nuclear extract fraction inin vitro transcription reactions occurred with efficiencies similar to those observed for the other RNAs; and 3) theDSI precursor could compete effectively for endonuclease in competition assays. The results of enzymatic structure probing suggested that within the IVS domain there were two differences between the SSI and DSI precursors. These were the formation of an extended helix at the3' splice site and decreased accessibility to nucleases of a segment within the 5' half of the IVS (U37:4G37:lO). We attribute defective 3' splice site cleavage to the formation of an extended3' IVS helix for the following

13845

reasons. First, the structures of the DSI and DSI-1C precursors were found to differ only in the region of the proposed 3' splice site helix while other regions of the IVS domain were identical. This limited structural difference was sufficient to restore cleavage by the splicing endonuclease. Second, these results are consistent with previous work by others in which the alteration of both sequence and structure at the 3' splice site has been found to affect cleavage (27, 28, 30).4Using the tRN@" precursor, Strobe1 and Abelson (28, 30) analyzed a series of extensive IVS rearrangements. These workers found that cleavage at the 3' splice site was consistently correlated with predicted single-stranded structure in this region. Finally, deletion of the 5' half of the IVS in pre-tRNLk" (positions A37:1-A37:18; Ref. 28) has no effect on splicing indicating there are no essential elements in this segment. The inability of endonuclease to act at the5' splice site in the DSI precursor was unanticipated. This effect might be due to a requirement for ordered cleavage at the two splice sites in this precursor or an indirect effect of the DSI insert on the structureor accessibility of the 5' splice junction. The first possibility seems unlikely since mutations which result in the accumulation of products derived by cleavage at only the 5' splice junction have been identified for the yeast tRNGL"" and S. pombe tRNAS"' precursors (23, 30). With regard to thesecond possibility, the specific requirements for sequence and/or structure at the 5' splice site are not well defined. Common features in this region include generally conserved tRNA sequence in the anticodon loop (pyrimidine 32, uridine 33, and purine 37; see Ref. 41) and the potential for the formation of an anticodon-IVS (AC-IVS) helix (11, 18). Mutations which alter anticodon-IVS complementarity in the yeast t R N A y gene and theireffects on splicing by the Xenopus endonuclease have been examined (29). Triplet mutations in the anticodon or its IVS complement were found to have noeffect on cleavage indicating that complementarity in this region isnot required. Incontrast, compensatory anticodon and IVS triplet mutations which restore complementarity butchange the sequence composition and predicted stability of the anticodon-IVS helix prevent splicing, potentially by including the 5' splice site in a double-stranded segment. Similar indirect stabilization of the anticodon-IVS helix by the DSI insert in our experiments is unlikely since we observed preferential cleavage by RNase T1 at position G37 within this region. The remaining, and perhaps most likely, possibility is that the extended IVS helix in the DSI precursor produces a change in the overall configuration of the IVS domain so that the 5' splice site is effectively out of register with the active site in the bound enzyme. Minor changes in nuclease cleavage patterns at several sites within the IVS are consistent with this possibility. Insertion and point mutations which increase accessibility within the DSI IVS and which alter the structure of the 5' splice site in the SUP53 precursor are being used to examine these possibilities. The introduction of a consensus guanosine at the antepenultimate position of the SUP53 IVS did not enhance endonuclease cleavage suggesting that guanosine at this position is not required for optimal splicing. These experimentsdo not distinguish between no requirement for a conserved sequence and a limited requirement for a purine at this position. However, this latter possibility is difficult to reconcile with the observed inhibitory effect of a guanosine to adenosine change at this position in the sup3-e precursor (mutation A37:13, Ref. 23). For the former possibility, sequence bias at this site might represent a structural requirementfor interaction with a conserved base. Although nucleotide 32 within the 5' half is an apparent candidatefor such interactions with conserved

Intron Mutations Affecting tRNA Splicing

13846

pyrimidine character (most often cytidine) and positioning opposite the 3‘ splice site, we found that a compensatory second mutation (C32) in the G37:30 variant did not restore efficient cleavage (data not shown). It remains possible that sequence conservation at this position is required for some process other than splicing. In addition to the results presented here, evidence for the absence of a specific IVS sequence requirement intRNA splicing includes: 1) the absence of conserved sequence immediately adjacent to the splice sites; 2) the rare occurrence of point mutations within introns which affect splicing (20); 3) the ability to tolerate extensive IVS insertions and deletions with no effect on splicing (26-28, 30); and 4) the ability of exon mutations to produce new cleavage sites with little sequence similarity to the original sites (23).6 Thus IVS requirements in tRNA splicing may be limited to elements of secondary or tertiary structure rather than primary sequence. Similarities in reaction mechanisms and intermediates as well as analogies drawn among conserved splice siteand intronfeatures have been cited as evidence supportinga common origin for nuclear mRNA splicing and the Group I and Group I1 self-splicing reactions (42,43).Our findings that sequence at the3‘ ends of IVSs may not be essential in tRNA splicing serves to emphasize the difference between tRNA splicing and all other classes. Additional fundamental differences are found in the reaction mechanisms. tRNA splicing is characterized by the apparentabsence of ordered splice site selection (23) and the presence of 3”terminal phosphates in cleavage products (2, 3) whereas ordered cleavage and 5‘phosphates arecommon to all otherclasses. Also, limited size (although this constraintmay be imposed in partby requirements for transcription by RNA polymerase 111) and conserved position within exons are characteristics unique to tRNA gene introns. These extensive differences imply that tRNA splicing either diverged rather early from the progenitor to the other classes or that these two groups of splicing reactions evolved separately. Further analysis of the tRNA splicing reaction andits relationship to other RNA rearrangement reactions such as viroid and virusoid self-cleavage and ligation (44-46) should provide insightsinto the origins andfunctions of intronsintRNA genes andthe evolution of this unique splicing class. Acknowledgments-We thank HorstDomdey for providing several of the oligonucleotides used in this work, Scott Glaser for assistance with certain of the initial experiments, Gayle Knapp, Phil Green, Vicente Reyes, and John Abelson for communicating their results prior to publication, and Eric Phizicky and Craig Peebles for comments on this manuscript.

REFERENCES 1. Peebles, C. L., Ogden, R. C., Knapp, G., and Abelson, J. (1979) Cell 1 8 , 27-35 2. Peebles, C. L., Gegenheimer, P., and Abelson, J. (1983) Cell 32, 525-536 3. Greer, C.L., Peebles, C.L., Gegenheimer, P., and Abelson, J. (1983) Cell 3 2 , 537-546 4. Knapp, G., Ogden, R. C., Peebles, C. L., and Abelson, J. (1979) Cell 18,37-45 5. Pearson, D., Willis, I., Hottinger, H., Bell, J., Kumar, A., Leupold, U., and Soll, D. (1985) Mol. Cell. Biol. 5 , 808-815 6. Gamulin, V., Mao, J., Appel, B., Sumner-Smith, M., Hamao, F., and Soll, D. (1983) Nucleic Acids Res. 11, 8537-8546 7. Ogden, R. C., Beckman, J. S., Abelson, J., Kang, H. S., Soll, D., and Schmidt, 0.(1979) Cell 17,399-406 8. Standring, D. N., Venegas, A., and Rutter, W. J. (1981) Proc. Natl. Acad. Sci. U. S. A . 78,5963-5967 9. Sprinzl, M., Hartmann, T., Meissner, F., Moll, J., and Vorder-

V. Reyes and J. Abelson, personal communication.

wulbecke, T. (1987) Nucleic Acids Res. 15, r53-rl88 10. Abelson, J., Knapp, G., Peebles, C. L., Ogden, R. C., Johnson, P. F., and Johnson, J. D. (1981) in Molecular and Cellular Aspects of Microbial Euolution, Society for General Microbiology Symposium 32 (Carlile, M. J., Collins, J. F., and Moseley, M., eds) pp. 151-173, Cambridge University Press, New York 11. Ogden, R. C., Lee M-C., and Knapp, G. (1984) Nucleic Acids Res. 12,9367-9382 12. Sumner-Smith. M.. Hottineer. H.. Willis I.. Koch. T. L.. Arentzen, R., and’Sol1, D. (1984) Moi. Gen. Genet. 1 9 7 , 4471452 13. Debuchy, R., and Brygoo, Y. (1985) EMBO J. 4,3553-3556 14. Waldron, C., Wills, M., and Gesteland, R. F. (1985) J. Mol. Appl. Genet. 3,7-17 15. Gouilloud, E., and Clarkson, S. G . (1986) J. Bwl. Chern. 2 6 1 , 486-494 16. van Tol, H., Stange, N., Gross, H. J., and Beier, H. (1987) EMBO J. 6,35-41 17. Swerdlow, H., and Guthrie, C. (1984) J. Biol. Chem. 259,51975207 18. Lee, M.-C., and Knapp, G. (1985) J. Biol. Chem. 260,3108-3115 19. Colby, D., Leboy, P. C., and Guthrie, C. (1981) Proc. Natl. Acad. Sci. U. S. A . 78,415-419 20. Nishikura, K., Kurjan, J., Hall, B.D., and DeRobertis, E. M. (1982) EMBO J. 1,263-268 21. Willis, I., Hottinger, H., Pearson, D., Chrisolm, V., Leupold, U., and Soll, D.(1984) EMBO J. 3,1573-1580 22. Pearson, D., Willis, I., Hottinger, H., Bell, J., Kumar,A., Leupold, U.,and Soll, D. (1985) Mol. Cell. Biol. 5 , 808-815 23. Greer, C., Soll, D., and Willis, I. (1987) Mol. Cell. B i d . 7 , 76-84 24. Baldi, M., Mattoccia, E., and Tocchini-Valentini, G . P. (1983) Cell 35,109-115 25. Deutscher, M. (1984) Crit. Reu. Biochem. 17,45-71 26. Johnson, J., Ogden, R., Johnson, P., Abelson, J., Pembeck, P., and Itakura,K. (1981) Proc. Natl. Acad. Sci.U. S. A . 77,25642569 27. Raymond, G., and Johnson, J. D. (1983) Nucleic Acids. Res. 11, 5969-5988 28. Strobel, M.C., and Abelson, J. (1986) Mol. Cell. Biol. 6 , 26632673 29. Baldi, M. I., Mattocia, E., Ciafre, S., Attardi, D. G., and TocchiniValentini, G. P. (1986) Cell 47,965-971 30. Strobel, M.C., and Abelson, J. (1986) Mol. Cell. Biol. 6 , 26742683 31. Reed, C. R., and Liebman, S. W. (1979) Genetics 91,5102-5109 32. Andreadis, A., Hsu, Y.-P., Kohlhaw, G.B., and Schimmel, P. (1982) Cell 31,319-325 33. Schimmel, P. R., Soll, D., and Abelson, J. (1979) in Transfer RNA: Structure, Properties and Recognition (Schimmel, P. R., Soll, D., and Abelson, J., eds) pp. 518-519, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 34. Linn, S., and Lehman, I. R. (1965) J. Biol. Chem. 2 4 0 , 12871293 35. Lockard, R. E., and Kumar, A (1981) Nucleic Acids Res.9,51255140 36. Engelke, D. R., Gegenheimer, P., and Abelson, J. (1985) J . Bwl. Chem. 260,1271-1279 Biol. 2 1 , 37. Roman, H. (1956) Cold Spring Harbor Symp. Quant. 175-185 38. Jones, E. W., and Fink, J. R. (1982) in The Molecular Biology of the Yeast Saccharomyces (Strathern, J. N., Jones, E. W., and Broach, J. R., eds) pp. 181-299, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 39. Jones, E. W. (1972) Genetics 7 1 , 217-232 40. Koshland, D., Kent, J. C., and Hartwell, L. H. (1985) Cell 40, 393-403 41. Cedergren, R. J., Sankoff, J. D., LaRue, B., and Grosjean, H. (1981) Crit. Reo. Biockrn. 11,35-104 42. Cech, T. (1986) Cell 44,207-210 43. Sharp, P. A. (1987) Science 2 3 5 , 766-771 44. Kiberstis, P. A., Haseloff, J., and Zimmern, D. (1985) EMBO J. 4,817-827 45. Hutchins, C. J., Rathjen, P. D., Forster, A. C., and Symons, R. H. (1986) Nucleic Acids Res. 14,3627-3640 46. Forster, A. C., and Symons, R. H. (1987) Cell 49, 211-220 47. Mao, J., Schmidt, O., and Soll, D. (1980) Cell 21,509-516 48. Selker, E., and Yanofsky, C. (1980) Nucleic Acids Res. 8 , 10331042 49. Gamulin, V., Mao, J., Appel, B., Sumner-Smith, M., Yamao, F., ”

Mutations Intron

Affecting tRNA Splicing

13847

andSoll, D. (1983) NucleicAcids Res. 11,853757. Sherman, F., Fink, G. R.,and Hicks, J. B. (1983) in Methodsin 50. Huiet, L., Tyler, B.M., and Giles, N. H. (1984) NucleicAcids.YeastGenetics:LaboratoryManual, Cold Spring Harbor LaboRes. 12,5757-5765 ratory, Cold Spring Harbor, NY 51. Stange, N., and Beier, H. (1986) NucleicAcids Res. 14,8691 58. Zoller, M. J., andSmith, M. (1982) NucleicAcids Res. 10, 648752. Peffley, D. M., andSogin,M. L. (1981) Biochemistry 20, 4015-6506 Natl.Proc. 4021 59. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) 53. Robinson, R., and Davidson, N. (1981) Cell 23, 251-259 Acad. Sci. U. S. A . 74,5463-5467 54. Muller, F.,and Clarkson, S. G. (1980) Cell 19,345-353 60. Donis-Keller, H., Maxam, A. M., and Gilbert, W. (1977) Nucleic 55. McPherson, J. M., and Roy, K. L. (1986) Gene (Amst.) 42, 101Acids Res. 4,2527-2538 106 61. Ito, H., F'ukada, Y., Murata, K., and Kimura, A. (1983) J. Bacte56. Hill, A., and Bloom, K. (1987) Mol. Cell. Biol. 7, 2397-2405 riol. 163, 163-168

S u p p l e m e n t a rHya t e r l a 1 E l l z d b e t hS r r k e l y .H r d t h r r

to

' ' I n t r o n Sequence and S t r u c t ~ r e Requlremenfr G. B e l f o r d .a n dC h r i s

L. G r e w

EXPERIIENTAL PRDCEOURES

for

tRNA S p l i c i n g "