YEAST

VOL.

6: 345-352 (1990)

An Essential Gene in Saccharomyces cerevisiae Shares an Upstream Regulatory Element with PRP4 SARA PETERSEN-BJIZIRN,T. R. HARRINGTON AND JAMES D. FRIESEN* Hospital for Sick Children and Department of Medical Genetics, University of Toronto, 555 University Av., Toronto, Ontario MSG 1 x 8 , Canada

Received 16 January 1990

URF2 is an essential gene immediately upstream of PRP4 (formerly RNAI), a gene involved in nuclear mRNA processing in Saccharomyces cerevisiae. The two genes are arranged head-to-head. An 8 base-pair conserved sequence element is found upstream of both genes, as well as upstream of certain other genes that are known to be involved in pre-mRNA processing. Through deletion analysis we have found that both of the conserved sequence elements are important for transcription of both genes. We have cloned ORF2 and have isolated temperature-sensitive orf2 mutants. The phenotype of these mutants does not suggest a role for ORF2 in mRNA processing. The deduced amino acid sequence of ORF2 indicates significant similarity to D P R l , a gene encoding a protein that is involved in the carboxyterminal processing of G-protein. KEY WORDS -RNA

processing, divergent transcripts, temperature-sensitive mutants.

INTRODUCTION The PRP4 gene is essential for nuclear mRNA processing in Saccharomyces cerevisiae. PRP4 was cloned on a 2.4kb Hind11 fragment by complementation of a temperature-sensitive prp4 mutant (Soltyk et al., 1984). The PRP4 gene product was subsequently identified as a 52 kilodalton protein that is associated with the U4/U6 snRNP (Banroques and Abelson, 1989; Petersen-Bjerrn et al., 1989). This is consistent with its essential role in mRNA splicing, since the U4/U6 snRNP is one of four snRNPs known to be involved in nuclear mRNA processing both in yeast and higher eukaryotes (Steitz et al., 1988). Sequence analysis of the clone revealed two interesting features in addition to identification of the PRP4 open reading frame (ORF; Petersen-Bjerrn et al., 1989). One was a 187 amino-acid-long O R F upstream of the PRP4 coding region, reading in the opposite direction and remaining open at the Hind11 cloning site; we refer to this gene as ORF2. Genetic analysis showed that ORF2 is an essential gene (Petersen-Bjerrn et al., 1989). Analysis of the sequence of PRP4 and ORF2 identified a short sequence, (C)TAGTAAAG, upstream of both genes; *Corresponding author. 0749%503X/90/04034548$05.00 0 1990 by John Wiley & Sons Ltd

we refer to this sequence as the box. The box is also found upstream of PRP3 (J. Anthony and J. Woolford, personal communication), and a closely related sequence is found upstream of P R P l l (K. Schappert and J. D. Friesen, unpublished observation); both of these genes are involved in, and essential for, mRNA splicing in yeast (Lustig et al., 1986).In all cases the box sequence is approximately 90 base-pairs upstream of the presumed ATG translation start-site of the protein. The major transcription start-site of PRP4 has been mapped; it is 36 bases upstream of the ATG (Petersen-Bjerrn et al., 1989). Thus in the case of PRP4, at least, the conserved box sequence is in the 5’-non-coding region of the gene. A search with the box sequence against yeast sequences in Genbank version 59 did not identify any other genes containing it upstream of protein-coding regions. The fact that the box is found upstream of several genes, some of which are known to be involved in the same cellular function, namely mRNA processing, suggests that it could be important for expression of this group of genes. An example of this is the ‘RPG’-box, which is important for transcription of many genes involved in translation (Huet et al., 1985). To test this we examined the effect on transcription of deleting one or both of the boxes upstream of PRP4 and ORF2.

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S. PETERSEN-BJBRN ETAL.

ORF2

PRP4

lac2

PRP4

ORF2

lacZ

Figure la

347

AN ESSENTIAL GENE IN SACCHAROMYCES CEREVISIAE

PRP4-lac2

ORF2-lac2

I

I

I1

vec wt Aa Ab Aab.wt Aa Ab Aab I

1

I

I

I

I

I

l

l

3.4

fus. 1.8

PRP4 ORF2

ACTlN

Figure 1b. Figure I . Deletion of conserved upstream sequence-elements. (A) Diagram of deletion constructs. Protein-coding regions of IucZ, P R P 4 and ORF2 are indicated by boxes. The P R P 4 - O W 2 intergenic region is indicated by a line. The box sequence upstream of PRP4 is designated ‘a’, the one upstream of ORF2 is designated ‘b’.Arrowheads indicate the presence of a box and its orientation. Triangles indicate deletion of a box. PRP4-ORF2 sequences are a 690 bp BglII-BgnI fragment fused in both orientations to lucZ in a URA3marked integrative vector. (B) RNA-blot analysis of total RNA from strain W303-1A carrying single copies ofvector alone (lane 1) or of the plasmids indicated above (lanes 2-9). Five micrograms of total RNA from all strains were fractionated on an agaroseformaldehyde gel, and transferred to a membrane. The filter was probed with the 690 bp BglII-BglII fragment that hybridizes to the fusion-transcripts (labelled ‘fus’) as well as to endogenous PRP4 and ORF2 transcripts (Soltyk etul., 1984),as indicated on the right. The positions of 3.4 kb and 1.8 kb rRNAs, as indicated by ultraviolet shadowing, are indicated on the left. The membrane was subsequently reprobed with an actin mRNA specific probe (lower panel), as a control for RNA recovery.

MATERIALS AND METHODS

D N A manipulations and analysis

Both strands of the relevant DNA fragment were sequenced by the chain-termination method (Sanger Saccharomyces cerevisiae strain W303-1A has the et al., 1977). Site-directed mutagenesis was carried genotype: MATa, canl-100, his3-11,15, leu 2-3,112, out as described (Zoller and Smith, 1983). Mutatrpl-I, ura3-1, ade2-1 (Petersen-Bjerrn et al., 1989); genesis with N-methyl-N’-nitro-N-nitrosoguanidine YS33 is an isogenic diploid derived from strain has been described (Friesen et al., 1983). Other W303 (provided by R. Rothstein), which in addition DNA and RNA manipulations were essentially as has the LEU2 gene integrated at one of the ORF2 described by Maniatis et al. (1982). loci (Petersen-Bjrarn et al., 1989); YS34 is a haploid derivative of YS33 carrying the ORF2::LEU2 mutation. Plasmids YIP358 (Myers et al., 1986), RESULTS AND DISCUSSION pYF548 (Petersen-Bjsrn et al., 1989), pYS104 We used site-specificmutagenesis (Zoller and Smith, (Petersen-Bjerrn et al., 1989) and pYS128 (Petersen- 1983) to delete precisely the box upstream of PRP4 Bjerrn et al., 1989) have been described previously. (CTAGTAAAG) or upstream of ORF2 (GTAGGrowth media were prepared as described by TAAAG) or both. The 690 bp BglII fragment that Sherman et al. (1986). contains the PRP4-ORF2 intergenic region with Strains. plasmids andgrowth media

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S. PETERSEN-BJQRN ET AL.

Figure 2a. Figure 2. Cloning and sequence of ORF2 (A) Cloning strategy for ORF2. Plasmid pYF548 carries ORF2IPRP4 sequences on a 848 bp HindII-HpaII fragment in a URA3-marked integrative vector. The chromosomal PRP4/ORF2 locus (strain W303-1A) is indicated below the plasmid. The chromosomal configuration following integration of pYF548 at ORF2 is indicated. The SstI sites, which allowed isolation of a clone containing a complete ORF2, are indicated. (B) Nucleotide sequence of ORF2. Overlapping clones o f both strands were sequenced. The ORF2 open reading frame starts at position 260 and terminates at position 1234. The PRP4open reading frame starts at position 6 and reads in the opposite direction. The predicted amino acid sequence in one-letter code is indicated below the middle of codons. The conserved boxes upstream of PRP4 (on the complementary strand) and ORF2 are underlined. A partial ORF2 sequence has been published (Petersen-Bjerrn ef al., 1989); the Hind11 site at position 816 is equal to position 1 in the previously published PRP4 sequence.

the deletion(s), and which extends approximately 250 bp past the ATG of both PRP4 and ORF2, was fused in both orientations to lac2 in YIP358 (Myers et af., 1986), a URA3-marked integrative vector. Yeast transformants that carried a single plasmidcopy integrated at the ura3 locus in the S. cerevisiae strain W303 were isolated (Figure IA). RNA was harvested from exponentially growing cells and was probed for transcription from the PRP4-lacZ and ORF2-EacZ fusions, using the actin mRNA as a control. We chose to analyse the amount of RNA by blot analysis rather than assaying P-galactosidase activity since RNA-blot analysis is a more direct measure of transcript levels; furthermore, the ORF2lacZ fusion is out-of-frame relative to lacZ. The result is shown in Figure 1B. Both fusions gave rise to a transcript of approximately 3 kb (lanes 2 and 6). The relative amount of both transcripts decreased when the box closest to PRP4 was deleted (lanes 3 and 7) or when the box closest to ORF2 was deleted (lanes 4 and 8). When both boxes were deleted both transcripts were barely detectable (lanes 5 and 9), even though the actin control shows that relatively

more RNA was loaded in these lanes. These results suggest that both boxes are important for the level of expression of both PRP4 and ORF2. Since ORF2 is essential and shares a regulatory element with PRP4, and since genes that are arranged 'head-to-head' often have related functions (Bech and Warren, 1988), it is possible that ORF2 might be a gene involved in mRNA processing. To test this we cloned the entire ORF2 gene, as outlined in Figure 2A. A URA3-marked plasmid carrying part of ORF2 and PRP4 (pYF548) was integrated at the ORF2 locus. This partially duplicates this region of the chromosome and vector sequences adjacent to a complete ORF2 gene. The chromosomal arrangement of transformants was confirmed by DNA-blot analysis (data not shown). Total chromosomal DNA was then digested with various restriction enzymes known not to cut inside vector sequences. The cleaved DNA was ligated under dilute conditions to favour circularization, and was then used to transform an Escherichia coli host. Plasmids were recovered and screened for the presence of contiguous ORF2 sequences by DNA-

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AN ESSENTIAL GENE IN SACCHAROMYCES CEREVISIAE

1 ACTCATATTC S M 61 TTACGATTAG 121 CGTACTTAAT 181 AAGAAACTGT 241 CCACTAGAAA

CGTTCAACAA GGTAATAGTA CTAGTCATTT CCCTCAGGCT CGTTGCAAAG TGATCATGTA GACTTCGCTT GTATATATAT ATTGTGAACA

301 TTATATTGAA TCATTGGATA

Y

I

E

S L D

361 TCGTTTGAAT GGGATATATT

R L N

G I Y

421 ATTTGTGAAA GAAGAGGTTA

F V K

E

E

V

481 TTTCGCACCA TTTCCAAGAC

F A P

F P R

541 CTTGGCCACT TATGATGCTT

L A T

Y D A

601 CTTCATCCGT GGGAATCAAT

F I R

G N Q

661 AGATACAAGG TTTGTTTATA

D T R

F V Y

721 TGAAGTTGTT GACCCTGCTG

E V V

D P A

781 TGGATTATGT CCTAATGCTG

G L C

P N A

841 GGCAATTGCT AACAAATTGG

A I A

N K L

901 GCTTTGCGAA CGACAATTAC

L C E

R Q L

961 TGTTTGCTAT AGTTGGTGGG

V C Y 1021 AAATTATGAG N Y E 1081 AAGCGACAGG S D R 1141 AAGTTTAATG S L M 1201 CGTTACATCA V T S 1261 TGCTTGTTTA 1321 TTTTCTTTGA 1381 CAGGGTTAAT 1441 AAGAACCATT 1501 TGGGCGCAAG 1561 TTTTCTTGTT 1621 TGGTACAATA 1681 CTCGTTAATG 1741 TGACTATTTC

S W W AAGCTAACTG K L T CCTGAAAATG P E N GGATACGACA G Y D AAATTCAAAA K F K AGTGTTCTTA GGATGGAACG TACTTTACAA GTCGATGGTA GAATCAAGAA TCTTGAACCT TCGGTGTCTT GAATTCCAAA TTGGGATAAG

ACAGCCTTTC CTTTACTAGC GTAATAGCGA TAGTAATACC ATTTCAGATA CCACTACTGT TGTCAGGATC TCTTACGCTA L T L M S G S CAAACAAGCA TAATTTTGAA T N K H N F E GGGGACTTAC TGCCTTGTGT W G L T A L C TATCATTTGT TCTGAGCTGT I S F V L S C ATGATGCACA TTTATTAACT H D A H L L T TGGATGTTCT T G G W G A T L D V L G K D TGGAAGATGG TTCGTTCCAG L E D G S F Q CAGCCTTGAG TGCACTATCA T A L S A L S TAGACTTTGT ACTCAAGTGT V D F V L K C AATCCCATGC AGCTCAAGCT E S H A A Q A ATATGCTTAG TGACGACCAA D M L S D D Q CAGAAGGTGG ACTAAACGGT P E G G L N G TTTTATCTTC ATTAGCTATT V L S S L A 1 AGTTTATACT CAAATGTCAA E F I L K C Q AAGTGGATGT CTTCCATACA E V D V F H T ATTTAGTCCC CATAGATCCC N L V P I D P AGTATCCATA CAAATAGATG K Y P Y K TTTTGATGCA TTATTTAGAT TACTCCATAT ACCTAGCACG CCGTTGTATG TAAATCAAAT TTGGGCAAAG TGTCAAAGTG ATGGCGATAA ATGACCCTGG TTTGTGGTAA CTGGTCTGGT TAGACATCAT GTTATCGTTT CGGTGTCTTT TGAATCTTCC TATGCTATTC TAGTTGGATT Figure 2b.

TCTCTACGTT AGTAGTAAAG TACTAATGAT CTTAAGGAAA L K E TACTGGCTTA Y W L GTGCTTGATT V L D TGGGACGACA W D D ACATTGTCCG T L S CGTAAGGTTC R K V GGTGACAGGT G D R ATTTTGGGTG I L G TATAATTTTG Y N F TTTACCTGTC F T C CTAGAAGAAA L E E AGGCCAAGTA R P S ATTGGTAGAC I G R GATGAGAAGA D E K GTTTTTGGTG V F G ATATATTGTA I Y C GCAGAGTACC

TAAACATTTC ACAACAATTT TCTGTTTTCT AGCATATACG K H I R CCGAGCATCT T E H L CTCCAGAGAC S P E T AATACGGCGC K Y G A CCGTACAGAT A V Q I GCTTGATTTC R L I S TTGGGGAGGT F G E V AATTAACGTC E L T S ATGGTGGCTT D G G F TTGGCGCTTT L G A L TTGGGTGGTG I G W W AACTGCCCGA K L P D TAGATTGGAT L D W I AGGGTGGAAT K G G I TTGCTGGTTT V A G L TGCCGAAATC M P K S AACACTACCG

ATATATCTCT TCTATTATGA TGTGCAGAAG TGGTCCAAAT TCTAGTATTG TCTTTAACTA TCAGTATAAA GGATAAATCG TGAGCTC

TCTTGTATAG ATGAAACTTC TAGTGTCGCA CCCATGAAAT TTTTAACGAG ATTCATCGAT CATCTTCGCT TCCCTGAAAA

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wt I

ts- 1

a b cRT a

ts-2 r

l l

b cRT a

PrP2 - 1

b cRTRT37

P M

Figure 3. Analysis of or$?-ts mutants. RNA-blot analysis of total RNA from strain YS34 (ORF2::LEU2) carrying wildtype pYS104 (ORFZ), from two pYS104 derivatives (ts-1, ts-2) carrying temperature-sensitive or@ mutations, and from a temperature-sensitiveprp2 strain. Total RNA was harvested from cells growing at 23°C (RT) or from cells shifted to 37°Cfor 1,2 or 3 h (a, b and c respectively). For the prp2-ts strain, RNA was harvested 1 h following temperature shift. Five micrograms of total RNA were fractionated on an agarose-formaldehyde gel, transferred to a membrane, and probed with the introncontaining ribosomal protein gene CRY. ' P and 'M' indicate the positions of pre-mRNA and mature RNA.

and RNA-blot analysis (data not shown). The presence of a complete ORF2 gene was assayed genetically by testing whether a plasmid could rescue an ORF2::LEU2 null allele. Strain YS33 (ala, ade2lade2, his3,his3, ura3/ura3,ORF2/ORF2:: LEU2) was transformed with potential ORFZ plasmids that had been linearized within URA3 in order to direct integration to the chromosomal ura3 locus. URA' clones were selected, confirmed by DNAblot analysis, sporulated and dissected. The smallest clone that could rescue the ORF2::LEU2 allele (i.e. gave rise to Leu+Ura+ haploid progeny) was an Sstl clone that contained approximately 1 kb of additional sequence beyond the original Hind11 site (Figure 2A). Sequence analysis extended the 187 amino acid ORF2 ORF that was originally identified on the HindIII-Hind11 clone (Petersen-Bjsrn ef al., 1989) for an additional 138 amino acids, predicting the ORFZ gene product to be a 325 amino acid, 36.5 kilodalton protein (Figure 2B). This size agrees well with the size of the 1.3 kb mRNA. The ORF2

sequence shows an interesting similarity to D P R l (Tamanoi et al., 1988), a protein that is thought to be involved in the C-terminal processing of G-proteins (Goebl, M., Tamanoi, F. and Friesen, J. D. (unpublished results). To assess the possible role of ORF2 in mRNA processing, we isolated temperature-sensitive (ts) mutants of ORF2;the idea was to determine whether the mutants accumulate unprocessed pre-mRNA following shift to the non-permissive temperature, as do the prp2-11 ts-mutants (Teem and Rosbash, 1983; Lustig et al., 1986).We used the 'plasmid shuffling' method (Budd and Campbell, 1987) to obtain ORF2 mutants. A replicating plasmid, pYS104 (ORF2, URA3, CEN3) was mutagenized with Nmethyl-N'-nitro-N-nitrosoguanidinein E. coli and was used to transform into strain YS34, a haploid yeast strain (derived from YS33) carrying an ORFZ:: LEU2null-allele on the chromosome but with ORF2 function provided from a replicating plasmid, pYS128 (ORF2, TRPI). Approximately 1000 Leu' Ura+Trp+ transformants were selected at 23°C and

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AN ESSENTIAL GENE IN SACCHAROMYCES CEREVISIAE

screened for temperature-sensitivity at 37°C to detect dominant ts-mutants. None were found. The clones were then replicated serially at 23°C on Leu' Ura' selective medium until clones had become Trp-, i.e. they had lost pYS128, the plasmid that carries wild-type ORF2. Leu'Ura'Trpclones were then screened for temperature-sensitivity at 37"C, and two ts clones were recovered; we do not know if these are independent mutations. The temperature-sensitivity was mapped to the plasmid by retransformation and subsequently to ORF2 by recloning the 1.7 kb MstII-SstI fragment containing ORF2. Both mutants rapidly ceased to proliferate following shift from 23°C to 37°C; the cells stopped proliferating within 1-2 h and arrested randomly with the cell cycle as determined by microscopic examination. RNA was harvested from both ts ORF2 mutants grown at permissive and non-permissive temperature. The RNA was analysed by RNA blots, using DNA of the intron-containing genes CRY (Himmelfarb et al., 1984) and ACTZN(Gal1witz and Sures, 1980) as probes. The result for the CRY probe is shown in Figure 3; a similar result was obtained using the ACTZN probe. Neither or$? mutant accumulates unprocessed pre-mRNA following shift to the non-permissive temperature (lanes 5-1 2), although a prp2-ts mutant does (lanes 13-14). The or$?-ts mutants also do not accumulate intronexon2 intermediate or free intron, since the probe used would have detected these species. This could mean: (i) the ORF2 gene product is not involved in mRNA processing, even if it shares a regulatory element with PRP4. An example of two genes that have unrelated functions despite sharing a regulatory element are HZS3 and pet56 (Struhl, 1985), although in this case the sequence element is poly(dAdT) rather than a more specific sequence, or (ii) the ORF2 gene product might be involved in processing of a protein that is itself involved in mRNA processing, possibly a G-like protein that could be membrane-associated (see above). ACKNOWLEDGEMENTS We thank our colleagues for helpful suggestions and comments, M. Drebot for help with microscopic observations, and H. Willard for first calling our attention to the 'ORF2-box'. This work was supported by The National Sciences and Engineering Council of Canada (grant A8060). S.P.B. held Connaught and Medical Research scholarships.

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352 Struhl, K. (1985). Naturally occuring poly(dA-dT) sequences are upstream promoter elements for constitutive transcription in yeast. Proc. Nail. Acad. Sci. USA 82,8419-8423. Tamanoi, F., Hsueh, E. C., Goodman, L. E., Cobitz, A. R., Detrick, R. J., Brown, W. R. and Fujiyama, A. (1988). Post-translational modification of ras proteins. Detection of a modification prior to fatty acid acylation and cloning of a gene responsible for the modification. J. Cell Biochern. 36,261-273.

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Teem, J. L. and Rosbash, M. (1983). Expression of a pgalactosidase gene containing the ribosomal protein 51 intron is sensitive to the rna2 mutation of yeast. Proc. Nail. Acad. Sci. USA 80,4403-4407. Zoller, M. J. and Smith, M. (1983). Oligonucleotidedirected mutagenesis of DNA fragments cloned in M 13 vectors. Methods Enzymol. 100,468-500.