Copyright 0 1995 by the Genetics Society of America

Two Distinct Temperature-Sensitive Alleles at the elav Locus of Drosophila Are Suppressed Nonsense Mutations of the Same Tryptophan Codon

MarieLaureMichael J. Lisbint andKalpana Whitet

* Waksman Institute, Rutgers, The State University, Piscataway, New Jersey 08855, and tDepartment of Biology and Volen National Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02254 Manuscript received May 30, 1995 Accepted for publication August 14, 1995

ABSTRACT The Drosophila gene ehv encodes a 483-amine-acid-long nuclear RNA binding protein required for normal neuronal differentiation and maintenance. We molecularly analyzed the three known viable alleles of the gene, namely ela.c/"', elaflJ1,and elaflJ2, which manifest temperature-sensitive phenotypes. The modification of the ehfly' allele corresponds to the change of glycinelz6 (GGA) into a glutamic acid (GAA). Surprisingly,el&' and ehflyz were both found to have tryptophanlle (TGG) changed into two different stop codons, TAG and TGA, respectively. Unexpectedly, protein analysis from ehv"' and ehdTqZreveals not only the predicted 45-kD truncated ELAV protein due to translational truncation, but also a predominant full-size 50-kD ELAV protein, bothat permissive and nonpermissive temperatures. The full-length protein present inelav"' and ehfly2canapim. be explained by one of severalmechanisms leading to functional suppression of the nonsense mutation or by detection of a previously unrecognized ELAV isoform of similar size resulting from alternative splicing and unaffected by the stop codon. Experiments described in this article support the functional suppression of the nonsense mutation as the mechanism responsible for the full-length protein.

ANY ts mutations have been reported to cause been reported. First, ts alleles of the Drosophila genes M amino acid substitutions in open reading frames white and Ubx are caused by the insertion of transpos- (.g., BAER et al. 1989; TH'NG et al. 1990; MASAI and able elements within intronic regions (BINGHAMand HOTTA 1991;MULLINS and RUBIN1991; JOHNSON et al. CHAPMAN1986; ASHBURNER 1989), presumably affect- 1993; SAVILLEand BELOTE1993), and many have suc- ing the production of mature transcripts in a tempera- cessfully been engineered in vitro, leading to amino acid turedependent fashion. Second, a nonsense mutation substitutions or, occasionally, to the insertion of one or in the Drosophila gene sevenless leads to a ts mutation two amino acids (see, e.g., ENGELMANand ROSENBERG that has been suggested as being due to reinitiation of 1987; KIPREOS et al. 1987). This is consistent with the translation downstream of the stop codon, resulting in fact that ts mutations are mostly caused by mutagenic a temperature-sensitive N-terminally truncated protein agents generating missense mutations rather than by (MULLINSand RUBIN1991). In this study, we analyzed mutagens causing frame shifts or larger molecular re- three elad mutations and found thatwhile one of them arrangements (reviewed in SUZUKI1970). Theresulting (eZadTq2)corresponds to a missense mutation, the other thermolability of the mutated proteins is only one of two (ela4' and elafly') are surprisingly due to nonsense the reasons these alterations can be associated with ts mutations in the codon for tryptophan,,, of the 483 phenotypes. Modified forms of protein whose activity, amino-acid-long ELAV. binding, folding, etc., is affected by temperature consti- The elav gene provides a function necessary for nor- tute additional possibilities. For instance, both the de- mal differentiation and maintenance of the Drosophila creased affinity (&) of N-myristoyltransferasefor myri- nervous system (for a review, see YAO et al. 1993). Most stoyl-Coa (JOHNSON et al. 1993) andthe defective mutations at the locus are embryonic recessive lethal, assembly of the catalytic RNA M1 and its protein cofac- and homozygous mutant embryos show abnormal neu- tor C5 into a functional RNAse P holoenzyme (BAERet rites, except for three alleles (elavt"', elafly', and elas2"J2), al. 1989) are due to ts missense mutations. which allow development up to the adult stage. Each of Two notable cases of ts mutations that do not corre- the viable alleles exhibits temperature-sensitive pheno- spond to minor nucleotide substitution/additionshave types. Two of them, elafly' and were induced in a genetic screen for behavioral mutants (HOMYKet aZ. Corresponding author: Marie-Laure Samson, Department of Bio- 1980) and show clear temperaturesensitivity of their chemistry and Molecular Biology, University of Nebraska Medical pleiotropic phenotype. These mutants were reported to Center, 600 S. 42nd St., Omaha, NE 68198-4525. E-mail: [email protected] have good flying and hoppingabilities at permissive tem- ' Present address: Department of Biochemistry and Molecular Biol- perature (22"), but to be flightless and uncoordinated ogy, University of Nebraska Medical Center, Omaha, NE 68198. when raised at the restrictive temperature (29") ( HOMYK

Genetics 141: 1101-1111 (November, 1995) 1102 M.-L. Samson, M. J. Lisbin, and K. White et al. 1980; HOMYKand GRIGLIATTI1983). The allele The possible presence of two forms of ELAV protein in eladTq' was described as more severe than ela?y2. The the elab"' and eladZq2extracts is intriguing, as previous eladZi1' mutants have abnormal electroretinograms and studies had never suggested two ELAV isoforms. An no optomotor response whether raised at 22" or 29", alternative possibility is that the wild-type gene encodes while mostof the flies carryingthe elad'zJzmutation have only one protein and that the full-size ELAV found in a normal ERG and optomotor response at 22". In addi- elad'' and e1azfLq2results from the functional suppres- tion, the morphology of photoreceptor cells and optic sion of the nonsense codon-for instance, by RNA edit- lobes of e,?adlg'flies is abnormal in flies raised at 29" but ing, translational readthrough, translational hopping, not at 20" (HOMYKet al. 1985). The mutation elav"' was or frameshifting (for reviews, seeMURGOLA 1985; VAILE independently recovered from an EMS screen for lethal and MORCH 1988; CATTANEO1991). We use the term elav alleles (CAMPOS et al. 1985) and shows an accentu- suppression to refer to these possible mechanisms. Exper- ated ts phenotype. At 25", the emerging adults are unco- iments to differentiate between these possibilities are ordinated and die within a few days, and the stock sur- described. vives only when raised at 18". The morphology of the visual system is abnormal at 18" and, to a greater extent, MATERIALS AND METHODS at 25" (CMPOS et al. 1985). The structure of elav protein (ELAV)was deduced Constructions: We refer to the location of nucleotides in from the study of two cDNAs: cDNA-1 from an embry- the eluv clones according to theirposition in the 9285 nucleo- onic library and cDNA-2 from an adult head library tide published sequence (ROBINOWet ul. 1988) and to the location of restriction sites by the position of the first nucleo- (ROBINOWet al. 1988).Both cDNAs encode a 483-amine tide of the recognition site. The 8.5-kb fragment included acid open reading frame (ORF1) corresponding to a between nucleotides 757 and 9285 (fused to termination of polypeptide with an A/Q-rich N-terminus, adjacent to transcription and polyadenylation signals) is sufficient for three domains approximately 80 amino acids long con- transformation rescue (Figure 1; YAO and WHITE1991). The taining ribonucleoprotein consensus sequences (RNP- 483-amino-acid-long open reading frame (nucleotides 7099- 8546) found in cDNA-1 is contained in the third exon,except CS; BANDZIULISet al. 1989) that are characteristic of for the A of the ATG initiation codon (nucleotide 4890). proteins that bind RNA. This class of RNA binding do- Isolation and sequencin of genomic fragments containing main is referred to as either an RNA binding domain elav ORF from elad', elazh', and elau@@flies: A 2.9-kb Hin- (RBD; for a review, see BANDZIULISet al. 1989) or RNA dII-EcoRI (6378-9280) genomicfragment, containingthe eluv ORF, was cloned for each of the three eluv alleles by recognition motif KENAN et al. 1991; MATTAJ (RRM; creating a "mini" library of 2.9-kb HindIII-EcoRI fragments 1993). Members of this family of RNA binding proteins into pBluescriptKS+ for each genotype and screening with have diverse binding specificities and affinities and play the wild-type eluu cDNA. Several clones containing the 2.9-kb roles ina variety of processesrelated to RNA metabolism eluv ORF fragment were identified for each mutant and are (for reviews,see BANDZIULISet al. 1989; KENAN et al. referred to as p2.9HEts1, p2.9HEJ1, and p2.9HEJ2. For each 1991; MATTAJ 1993). Theelav gene is the first identified eluv mutation, the entireORF was sequenced with Sequenase DNA polymerase (U.S. Biochemicals) in both directions for member of a neuron-specific RNA binding multigene at least two individual clones. family that exists also in vertebrates, including humans Plasmid p2.6SE containing the 2.6kb XmuI-EcoRI (6639- (SZABOet al. 1991; KIM and BAKER1993; KING et a[. 1994; 9280) elnu genomic fragment (YAO andWHITE 1991) was SAKA~et al. 1994; GOOD 1995; PERRONet al. 1995; G. modified by filling in the BumHI site of the vector backbone, yielding p2.6SEhst. The wild-type1.8-kb Xmd-BumHI frag- MANLEY and H. FURNEAUX,personal communication). ment of p2.6SEhst was replaced with that from each of the In this study, we analyzed molecular alterations in constructs p2.9HEts1, p2.9HEFliJ1, and p2.9HEFliJ2, generat- three elnu ts mutations and found that one of them ing p2.6SEtslhst, p2.6SEJlhst, and p2.6SEJ2hst. A 3.4kb (elazf';'') corresponds to anamino acid substitution, XmuI-XbuI fragment (containing the mutated eluu ORF and consistent with the traditional view of ts mutations. In the al-tubulintrailer sequences) was purified from these plas- contrast, we found that in two independant mutations, mids and subjected to a three-fragment ligation together with the 5.9-kb PstI-XmuI eluv genomic fragment and transforma- elad" and eladTc12,tryptophan4lo TGG is changed into tion vector pCaSpeR (detailed in YAO and WHITE1991). two different stop codons, TAG and TGA, respectively. Construction of elav transformation vectors carrying new Furthermore, analysis of protein extracts from flies of mutations: Plasmid pe336 differs from the wild-type elud'""w" the genotypes elavL" and elaJ'1q2revealed the presence construct (YAO and WHITE1991) by the insertion of oligonu- of ELAV forms. One of these forms has an apparent cleotide 5"CCGAATTCGGS' between nucleotides 8357 and two 8358, inducing a frame shift mutation in the elm ORF. In size of 45-kD, consistent with the presence of the prema- order to build pe336, a pBluescript derivative (pe325) con- ture stop codonmutations, and the other has an appar- taining the Suu3AI-BumHI (8102-8402) eluu fragment was ent size of 50-kD, identical to the size of normal ELAV. partially digested with PvuII and religated in the presence of The full-size ELAV protein in elab"' and elaJ'1i'2 flies the oligonucleotide, generating a setof recombinant plasmids might be explained by alternative splicing, where the among which pe330 was identified as carrying the linker be- tween nucleotides 8357 and 8358. Plasmid pe333 was gener- mutant stop codonis absent in one of the spliced forms. ated by using the BslXI-RnmHI (8269-8402) fragment from Under this interpretation, the wild-type gene encodes pe330 to replace the wild-type BstXI-BumHI fragment in two distinct proteins that comigrate in a denaturinggel. pe332, a pUC18 derivative that contains a SmuI-XbuI insert Suppressed Nonsense Mutations 1103 distal proximal

B H HPB P E HPEE PRPPPPPSHH E X R PR I I Ill I I I I I I II II Ill Ill I I Ill man of elavmRNA -lkb 3' , ,;...GU&ORFl 5' cDNAl

TRP/STOP 8.5 kb transformation rescue

FIGLXII I.-Stnlcture of the c/nu locus. A restriction map of the elnu locus is shown as a continuous line. The 9.3 kh BnmHI- IhRI at the 5' end of the locus (first 5' BnmHI site and second 3' LmRI site on the figure) has been entirely sequenced (Ronlso\v e/ nl. 1988).Transcripts extend mwh furthcr downstream, as demonstrated by Northern ancl cDNA analysis. The structure and splicing pattern of cDNA-I is shown below, as well as the 8.5 kb I!d-lcut with Pd and XhnI yielded pc336. on 12% SDSpolvaclylamide (37.5:l) minigels (9 x 3 x 03 Plasmid petl3I) differs from the wild-type e/n~/""~""'constrrlct cm) and transferred to nitrocellulose in a scmidn blotting (YAO and M'lrrn: 1991) by the insertion of oligonucleotide apparatus for30 min at 1.50 mA. EIAV immunotlctection was 5"CTAGCTAGCTAGJ' between nucleotides 8650 and 8657. performed using either colorimetric or chemiluminescence The mutation affects ORF located downstream of P/nv ORFl. (Amersham ECI.) methods according to manufacturers' in- It shortens a 74-amino-acid-long ORF (nucleotides 8449- structions. A rat anti-ELAV polyclonal antibody (Ronlso\v 8670; Figure 6R, frame a) by 6 amino acids. It also modifies and Mrl-IITF. 1991) or a mouse anti-ELAV monoclonal antibody and splits a 131-amino-acid-long ORF (nucleotides 8462- (1:.5000 dilution, a gift from G. RLws) were used for primary 8854; Figure 6R, frame b) into a series of short ORFs (from incubation. The secondary antibodies were either alkaline .5' to 3'):6.5, 3, 1,27, 15, and 9 amino acids long, respectively. phospllataseconjugated anti-rat IgG or anti-mouse IgG The Camino-acid-long ORF in reading frame c that is modi- (Sigma or Amersham) or peroxidaseconjrlgated anti-mouse fied by the mutation is unlikely to be relevant (Figure 6R). IgG (Amersham NNA 931). Comparisons between the The mutagenizcd plasmid was a Rluescript derivative, p3.4SX, amounts of protein loaded in each lane were performed by containing the same 3.4-kb Smnl-Xl,nI fragment as its dcriva- detectingfhubulin immunoreactivity with chemilumines- tive, pe333 (see above; Y;\oand WIIITII 1991). The oligonucle- cence, via a 1:10,000 mouse primary antibody (Sigma) and otide was inserted at the ,Vwl site (8649) of the dm Smnl- 1:10,000 peroxidaseconjrlgated antimouse secondary anti- EmR1 (6639-9280) fragment by cutting p3.4SXwith Nwl, body (Amersllam NA 932). treatingwith T4 DNA polymerase, and ligatingwith the linker, Approximate quantificationof the amountof ELAV protein generating pe337. Plasmid pes37 was partially cut with XhnI in a given test sample \vas performed by preparing an immu- and cut tocompletion with SrnnI, antl the4.2-kb X/~nl-.SrnnIe/nv nohlot with twofold serial dilutions of the test and the refer- fragment (2518-6639) was inserted, Icading to a Bluescript ence extracts. The ratio between the dilution factors of the derivative, pc338, containing nucleotides 2.518-9280 of e/m refercncc/test extract lanes that gavebest matching signal with the in ni/rogeneratcd mntation, andfused to the termina- intensities were considered to reflect the ratio of ELAV pro- tion of transcription and polyadenylation signals of the a1 tein abundance between reference and test extracts. tubulin gene. The final step for the construction of pe339 Germline transformations: Emblyos from the stock y was to replace the 7.Ckb XlmI fragment of pe336 with the were coinjected with 'LOO ng/ml pCaSpcR derivatives and .50 analogous Xl>nI fragment 01' pe338. ng/ml px25.7 (KARESS and RL.RIN 1984; PIRRO'ITA 1988: Thc sequence of the mrltations was verified by didcoxy- TIILWMIII. P/ nl. 1988).Standard procedures were used to iden- scquencing with Tnq DNA polymerase. As expected, themuta- tify the transformants ancl homozygous or balanced stocks tion in pe336 changes the wild-type 5". . . GGCAGCTGTT generated (Table 1). . . .-3' into .5'-. . . G(;<;AGCCGAATTCGG(;TG~.. .-3', where Geneticanalysis: All crosses were maintained on stan- the inserted nucleotides arc underlined. As expected, pc339 dard cornmeal mediwn. The t.. mutants P/~TI"', ~lnd"",and contains the oligonucleotide with a stop codon in all three c/n~t"~'were previously described (HOMYKe/ nl. 1980; Hosnl; possible open reading frames. However, the sequence of the and ~KI<~I.ISI-1'11983; C..\vrose/ d. 1985; Hosnx P/ nl. 1985). mutation is 5". . . AAATCTAGCTAGCTAGGGTAT. . .-SI, and c/nd5is an amorphic allele and ~/nr/~'""''is the I). T1idf.s mini- differs from the predicted 5". . . AAATACTAGCTAGCT- gene, capable of providing dnlno function (YAOand M'IIITF. A(;TGGTAT . . .-.?I, where the insertedoligonucleotide is 1991; YAO e/ nl. 1993). In order to test for n no""" rescue by underlined and the missing nucleotides are bold. one copy of an P/~TItransgene ( 7y),females P/nrfs/eCnzfr; e/ndh- Protein preparation and immunoblot analysis: Protein ex- ""/c,~o, were mated toy TU'"''^; ~y/~yorp TI?"'?; 7~/~?0maIes tracts were. made by homogenizing adult heads (10-30) or carrying the transgene (7Y) to be tested, at 18" and, for some cmhlyos (50-100) at 4" in 100-.500 p1 1 X PRS, 0.5% NP40, of the transformant lines, at 25" (see Table 1). The males 1 rnxl EGTA pH 8, PMSF0.2 mM, pepstatin 1 pg/ml, leupeptin cclosing from this cross must carry a transgene that provides 0.5 pg/ml. Extracts were spun for 10min in a microcentrifuge rlmT function; that is, cirhcr they have straight wings and 1104 M.-L. Samson, M. J. Lisbin, and JS. White

TABLE 1 transposon, the following crosses were established: females FM7a/FM7a; CyO/Sco were mated to males elazP5/Y;FliJl-7/ Characteristics of the transformant lines FliJl-7. Female el~v'~/FM7a;FliJll-7/CyO progenyfrom this cross were mated to males y w67'23/Y; FliJ1-7/FliJ1-7. Rescue Line Elav immunoreactivity (kD) Rescue of elaunUN by one copy of FliJl-7 was never obtained. Thepercentage of rescue by two copies of FliJl-7 was calculated as the ratio FliJl-2 50 NT between the number of males elauP5/Y;FliJ1-7/FliJ1-7 and the FliJl -5 50 No* number of females elaue5/FM7a;FltJlI-7/HiJl-7. FliJl-6 50 Not Analysis of elm RNA using reverse transcription PCR To- FliJl - 7 50 No: tal RNA from adult heads was isolated by the guanidine-HCl FliJl -1 3 50 No method (COX 1968), followed by chloroform extraction and niJ2-2 45 and 50 No ethanol precipitation. Six independent annealing reactions FliJ2-3 45 and 50 No of 500 ng elad1,/2RNA to 60 ng of oligonucleotide E6 5'- TSl-12 45 and 50 No TGCACTGATTCGTTGTGGS' (complementary to nucleo- TSl-13 45 and 50 No tides 8424-8407 of elav) were performed as in LO et al. (1994). TSl-271 NT No* The annealing mixes were suplemented with MgC12, dNTPs TSl-2 72 45 and 50 Not and RNasin as in LO et al. (1994), and their volume brought 336.10 47 No up to 20 pl. For each reaction, 1 unit of avian myeloblastosis 336-11 47 No reverse transcriptase (Promega) was added to 10 pl of the N T No 336-14 NT sample, and the remaining 10 pl were used as a control. The 336-1 9 NT No 12 tubes were incubated at42" for 40 min. The reactions were 336-26 NT No brought up to 25 pl 20 mM Tris pH 8.4, 25 mM KCl, 1.5 mM 336.30 NT No MgCle, 0.05 mM dNTPs, and 0.4 ng of oligonucleotide E7 5'- 339-1 7 50 loo%* CACACGCCAGTTAACAAG-3' (complementary to nucleo- 50 92% 50 tides 8169-8187 of elav) was added to each tube. The reac- 339-Spl7 tions were overlaid with 14 pl liquid wax, incubated 10 min 50 87% 339-sp9 at 95", and therafter maintained 10 min at 80" in a thermal The size of the protein produced from the transgenes was cycler, during which 0.5 unit of Taq polymerase (Promega) determined by immunoblot analysis of Drosophila head pro- was added. Cycling was performed for 60 cycles at 95" for 1 tein extracts as detailed in MATERIALS AND METHODS. Rescue min, 53" for 1 min, 72" for 2 min. The products of the reac- experiments with one copy of transgene were performed at tions were analyzed on an agarose gel and by Southern blot 18" as described in MATERIALS AND METHODS. In the case of hybridization with a probe corresponding to elau genomic lines FliJl-7, TSl-12, TSl-13, TSI-272,and 339-17, crosses were DNA. None of the reactions where reverse transcriptase was also performed at25" and yielded the same results. Two copies omitted gave any detectable product, while, as expected, the of FliJl-7 were necessary to rescue the elme5 mutation (see reverse transcription PCRs all yielded a product of approxima- text). NT, not tested. Several minigene insertions were reces- tively 240 bp, which hybridized to the elav probe. These PCR sive-lethal, presumably because their insertion generated a products were pooled and purified on Promega Wizard PCR mutation in a vital gene. prep DNA purification column. The ends of the products * Homozygous lethal. were polished with T4 DNA polymerase before cloning into No rescue with two copies. the pCR-Script SK(+) vector, according to the instruction of Rescue with two copies. the manufacturer (Stratagene). The sequence of the inser- tions contained in five independent clones was determined by dideoxysequencing using Taq DNA polymerase. carry ekeom,or they have curly wings and are rescued by the tested transgene. The percent rescue as listed in Table 1 is number of males carrying the transgene/number of males RESULTS carrying elaPom. Rescue with two copies of transgenes was tested for the transformed lines FliJl-6, FliJl-7, and TS1-272, in which the Temperatar-ensitive elm mutations areclustered transposons are inserted on the second chromosome. The within the third RNA binding domain of the coding following crosses were performed at18" to test for theviability sequence: The open reading frame encoding ELAV is of males ela7Y5/K Tf/TJ Females FM7a/FM7a; CyO/Sco were contained in a single exon, except forthe first nucleo- mated with y w"""/Y; Tf/Tfmales. F1 males FM7a/Y;Tf/Sco tide of its ATG initiating codon (ROBINOWet al. 1988). were then mated toelaIf5/elad5; ela~""/CyO females. elaIf5/ Sincetemperature-sensitive mutations often corre- FM7a; Tf/ Cy0female progeny were mated to males from the stock y w~~"~/Y;Tf/Tf; and the progenyscored for males spond to DNAalterations leading to minor changes elad5/Y; Tf/TJ When they survived, such males were mated in the structure of the protein, we cloned the 2.9-kb to females el~v"~pS/FM61.The progeny of this cross yielded only restriction fragment HindIII-EcoRI (Figure 1, and MATE- Bar females, confirming that two doses of this transposon are RIALS AND METHODS) from the stockselad", eZur/'l"J', and necessary for rescue of elaZP5. Females homozygous for the ela#'J2 and determined their nucleotide sequence. In allele and for the FliJl-7 transposon were obtained by backcrossing these Bar females (elabS/FM61; FliJ1-7/+) to each case, we founda different point mutation presum- elaIf5/Y;FliJ1-7/FliJll-7. The raresurviving females elaue5/elazP5; ably altering the protein structure (Figure2). The allele FliJ1-7/F1iJ117were sterile. Therefore, males elag5/Y; FliJ1-7/ eZad"J' has glycine (G) codon 426 (GGA) changed into FliJl-7 were mated to their sisters elaIf5/FM6L,F1~1-7-/~~1~-7. a glutamic acid (E) codon (GAA) . The alleles elad" and After a few generations, afew fertile females elav" /elav" , FhJ- eZu7f'q2have tryptophan (W) codon 419 (TGG)changed 7/Flij-7were found andwere used to establish a stock dad5/ elav" /Y; RiJ1-7/FlzJ1-7. into stop codons (TAG and TGA,respectively). This Inorder to quantify the rates of rescue by the FliJl-7 translational truncation is expected to result in a mu- Suppressed Nonsense Suppressed1105 Mutations

RRM RRM RRM RRM RRM 1234567 50 kD, ""- 45 kD-45 " --

tubulin -' i. .... Leu Trp Gln Leu Phe Gly Pro Phe Gly Ala ...... - - - - ....CTG T G CAG CTG TIT GGT CCA TIT GGA GCT... VI I FIGURE4.-Analysis of elm expression in embryonic pro- tein extracts of the temperature sensitive mutants raised at ehTS' .... 25", using the affinity purified rat anti-ELAV antibody. Tu- bulin was used as an internal loading control.ELAV migrates elavFW2 ....Leu END as a 50-kD protein, and the truncatedELAV migrates as a 45- elav FIN kD protein. Degradation products are seen below the 45-kD .... Leu Trp Gln Leu Phe Gly ProPhe ...... bands in some of the lanes. The age of the embryos is indi- cated for each sample: (1) elnu', 12-15 hr; (2) eluv'", 6-9 hr; FIC;URE2.-The elnv" mutations. The wild-type ELAV pro- (3) eluv"', 12-15 hr; (4) elnu"', 18-21 hr; (5) ~lud"~~,6-9 hr; tein is shown as a box. The N-terminus of ELAV contains a (6) elad''/', 12-15 hr; (7) elnd1'12, 18-21 hr. Glutamine-Alanine (QA) rich region (amino acids 24-126). The protein contains three RRh4s (RNA recognition motif). result is consistent with the single amino acid substitu- Each RRM is characterized by two conserved motifs about 30 amino acids apart, a hexapeptide (RNP2, most N-terminal), tion predicted for the elad';'' allele. The patterns of andan octapeptide (RNPl, most Gterminal),represented expression are identical at 18" and 25" (Figure 3A for here as solid vertical bars. The nucleotide sequence (nucleo- the adult head extracts). tides8349-8378) andthe corresponding amino acid se- In contrast, eld' and elad'"' do not show the pre- quence (aminoacids 418-427) in the regionof the mutations dicted pattern. In adult head protein extracts of eluv"' are shown. T tophan (W) 419 and Glycine (G) 426 are and eLad"i12, an unexpected full-size 50-kD protein and altered in elad'T /e/d ,142 and in elad"", respectively. a less abundant 45kD protein,consistent with the pre- dicted truncated ELAV protein, are observed (Figure tant ELAV polypeptide that is 65 amino acids shorter 3A). The 50-kD protein contributes >90% of the total than the wild-type polypeptide. ELAV immunoreactivity, while the 45-kD protein ac- Immunoblot analysis reveals two forms of proteins counts for

A elav[+] 8340 G~C~~~~~~-~TAAA~~~~C~~C~~~A~~.. . .AAAGCCAAGTAG 8549 415EAALWQLPGPPGAVQSVKIVKDPTTNQCKGYGFVSM....KAK* 483

FRAME SHIFT INDUCED STOP B elav[+] eIav3361 8632 ACMCAUCATATATATAAATA"AT~~~~~ a tthiykyawyngn9ap ? b phtyinmhgitvtkrn E nthi*icmv*r*lsat

FIGURE6.-Stmcture and predicted effect on elav expression of the mutant minigenes. A: Nucleic acid sequence of 3' end of elav ORFl (starting at nucleotide 8340) and conceptualtranslation of the sequences (starting at aminoacid 415) in wild-type elav, and in the mutant transgenes flml", lab"^', and ehv'". The RNPl of the third RRM is underlined. €3: Nucleic acid sequence of wild-type elau and mutant transgenes. The conceptual translation in three frames is indicated below the sequence and is specifically indicated in lower case to emphasize the hypothetical use of these ORFs. C: Predicted effects of dazt mutations on the RNA corresponding to ELAV and on the hypothetical mRNA encoding ELAV* (see text). The structure of elav mRNA as deduced from the structure of cDNA-1is represented at the top of the figure,where ORFl is shown as a gray box. The hypothetical alternatively spliced mRNA and ORF specific for the hypothetical ELAV* protein are diagrammedbelow. The solid lines indicate the location of the genomic DNA fragments contained in the minigenes that were tested in our experiments, relative to the structure of the ORFs. Four of the transgenes (eluv+,elub", elad"/2,elnu"", and elav"', respectively), the mutations that they carry (arrows), the effect of the mutations on theORFs of both ehv mRNA, and the presumptive alternatively spliced elav mRNA are also shown. this structure, the alteration of the ts alleles is inferred ELAV protein are thermolabile, nor is the process lead- to lie within the region encoding the first alpha helix ing to the generation of two forms of ELAV, in the case of the RRM, a region that does not directly contact of elav'"' and el~d"'~,temperature-sensitive. Rather, this RNA. G42fjis a preferred consensus amino acid in loop finding suggests that the mutant proteins themselves 2 leading into the 02, as suggested by NMR studies of are functionally impaired. For instance, ELAV affinity the Sxlprotein RRh42 and the hnRNPC RRM (BANDZIU- for its targets may be affected by the mutations at non- LIS et a[. 1989; COUCH et a/.1992; LEEet dl. 1994). On permissive temperature. the other hand, W419in that position of a helix turn is As opposed to nonmutated elav transgenes, none very rare, being specific to the elmsubfamily. Wd19is in of the mutant transgenes were able to rescue elav""" a position that corresponds to position RZ5in RRM of mutations when present as a single copy. However, we U2B" which is involved in protein-protein interaction were able to obtain rescue of e1uvna" mutations with (SCHERLYel dl. 1990; BENTLEYand KEENE 1991). A dif- two copies of FliJl-7. These flies show very clear tem- ferent amino acid in this position could compromise perature sensitivity: 70% rescue at 18" and 12% rescue the function of the protein. at 25" (Table 1) and abnormal eye morphology at 18" Biological activity of the mutant ELAV proteins: and to a greater extent at 25". Consistent with their Both the genomic ehv mutations and the mutant elm extreme phenotype, these flies have a lower level of minigenes show profiles of ELAV protein(s) that are ELAV expression (about twofold) compared to flies not affected by temperature (Figure 3, and data not carrying an elaJ'"' mutation. Our attempts to rescue shown), indicating that neither the mutant forms of elav"""with two copies of FliJl-6 and TSI-272 failed, Suppressed Nonsense Suppressed Mutations 1109 but the number of flies that we examined may not ment from eZav sufficient to rescue an eZav null muta- have been sufficient. tion. The mutant transgenes generated the same two We attribute the difficulty of the elad minigenes to forms of ELAV as the genomic mutant genes, indicating rescue eZavnuLLto the lower than normal level of ELAV that the 8.5-kb genomic fragment contains all the cod- expression from the minigenes and the impaired func- ing information for the two forms of ELAV protein to tional activity of the protein products themselves. This be produced. Second,we analyzed a mutanttransgene, insufficient expression is consistent with finding fewer ela~?~~,with a frameshift mutation in ORF-1 mapping females than males carrying two copies of FZiJ-7 between the mutations in elavl"1/elavn"72(codon 419) transgenes as their only source of elav function, re- and eladZq1(codon 426). This frame-shift mutation in- flecting dosage compensation in males. Dependence of troduces a stop codon at position 457 (wild-type stop, the elav phenotype on the copy number of hypomor- 483) and thus should result in a truncated protein.The phic alleles has previously been reported ( CAILIPOSet al. putative ELAV* should remain unaffected, assuming 1985; YAO et al. 1993). that the presumed alternatively spliced intron includes Nonsense mutations at position419 of ELAV appear theframe shift. Transgene elad3' expresses a single to be suppressedSurprisingly, analysis ofthe nonsense truncated protein of about 47 kD whose level of expres- mutants elav"l and elavF""/'shows that both the predicted sion is very low,probably as a consequence of the insta- 45-kD protein and an unexpected 50-kD normal size bility of the mutated RNA or protein. Since only a trun- ELAV protein are produced. cated ELAV product is detected, we conclude that We considered the idea of alternative splicing as a unless the frameshift mutation alters sequences essen- mechanism producing these two forms ofELAV be- tial for normal splicing of the presumed alternative in- cause (1) alternative splicing occurs within the 9 kb tron, asingle form of ELAV isencoded by the elav gene. downstream of ORFl thatbelong to the elav locus To determine whether the frame shift mutation of (CAILIPOSet al. 1987); (2) alternative splicing is widely elad3' might alter splicing signals, we searched the se- used as a mechanism to generate multiple protein iso- quence in the vicinity of thismutation for the presence forms (for reviews, see BREITBARTet al. 1987; R~o1993); of a Drosophila consensus 3'splice sites (MOUNT et and (3) alternatively spliced protein forms have been al. 1992). A sequence matching 10/15nucleotides was reported for othermembers of the ELAV protein family found, 8344CTGCCCTGTGGCAGI CT8359, which is modi- (Szmo et al. 1991; KIM and BAKER1993; GAOet al. 1994; fied into CTGCCCTGTGGCAGICC in the ela~?~~ LIU et al. 1995). transgene (Figure 6A). However, this is an unlikely We also considered the possibility that the full-length splicing site for the following reasons: ELAV in the elav"" and ela#@ mutants is generated by 1. As opposed to the 3' splicing sites found in the suppression of the stop codon because: (1) both elav'"' database, which are consistently Gpoor, it is buried in and ela#@alter W419,suggesting that that these muta- a Grich region (MOUNT 1993). Moreover, if this site tions might have been selected because they can be were used, none of thethree possible ORFs that it suppressed by an inherent mechanism able to identify would introduce in the ELAV* sequence would be long the sequence surrounding the mutations (for reviews, enough to generate a protein of size similar to the size see MURCOLA1985; VALLE and MORCH 1988; CATTANEO of ELAV*. 1991); and(2) the specificity ofthe phenotype of elad'?'' 2. The D. virilis elav gene encodes a proteinidentical us. elav'"' (CAILIPOSet al. 1985; HOMYKet ~1.1985) and to D. melanogaster ELAV in the region corresponding to the similarity of the ELAV pattern of expression in the the RNA binding domains (YAO and WHITE1991). We two mutants suggest that they encode distinct impaired searched D. virilis sequences for the potential ORFs proteins (see above). Thus, different amino acids might identified in D. melanogaster eZav, but did not find simi- be substituted for the conserved W419(see above) in larities, even under low stringency comparisons. the mutant elav"' and elau@lz proteins. This could result 3. We searched for experimental evidence for splic- from suppression of the stop codons, either by editing ing. Several elau cDNA clones from an embryonic library or readthrough of the nonsense codon to yield inser- and a headcDNA library were characterized and found tion of different aminoacids. Although both ribosomal to match the structureof the genomic DNA (M.-L. SA" hopping and frameshifting are formal possibilities for SON, unpublished results). We identified a minimum nonsense suppression, we think that they are unlikely of seven different head and seven different embryonic to be occurring, because they would give rise to forms cDNA clones in which the region corresponding to nu- of ELAV whose third RRM is significantly altered, most cleotides 8090-8781 is intact. Consistently, RNAse pro- likely totallydisrupting the protein function,and, in the tection assays of adult head and pupal RNA between case offrameshifting, whose size wouldbe incompatible nucleotides 8324 and 8472 reveals only an unspliced with our data. protected RNA (data not shown). To distinguish between the possibilities of alternative Thus, taken together, our data suggest that elav en- splicing us. suppression, we first analyzed TSl and HiJ2 codes a single protein and that, in the mutants elav" mutant transgenes that span the 8.5-kb genomic frag- and eZaS1"J2, translation of the mutated RNA generates 1110 M.-L. Samson, M. J. Lisbin, and K. White high levels of a truncated and unstable 45-kDELAV proteinand through interactions withU2 snRNP-A' protein. Mol. Cell. Biol. 11: 1829-1839. and, at a lower level, a stable 50-kD ELAV arising from BINGHAM,P. M., and C. H. CHAPMAN, 1986Evidence that white-6lood the suppression of the stop codons. Presumably, the is a novel type of temperature-sensitive mutation resulting from original W419is substituted by a different aminoacid in temperature-dependent effects of a transposon insertion on for- mation of white transcripts. EMBO J. 5 3343-3351. each mutant, since the mutant phenotypes are distinct. BREITBART,R.E.,A.ANDREADI~~~~B.NADAL-GUINARD,1987 Alterna- We found that five independent head mRNA elaJ""J2 tive splicing: a ubiquitous mechanism for thegeneration of multi- mRNAs were all complementary to the genomic elaS'1EJ2 ple protein isoforms from single genes. Annu. Rev. Biochem. 56: 467-495. sequence. Thus, editing of the stop codon seems to WPOS,A. R., D. GROSMANand K WHITE,1985 Mutant alleles at the be unlikely to be the mechanism responsible for the locus elav of Drosophila melanogasterleadto nervous system defects: a suppression. However, we cannot completely rule out developmentalgenetic analysis. J. Neurogenet. 2: 197-218. C~POS,A. R., D. R. ROSEN,S. N. ROBINOM!and K. WHITE, 1987 thispossibility, because it might be thatthe edited Molecular analysis of the locus elav in Drosophila melanagaster: a mRNA would be a minor species in the bulk of elau gene whose embryonic expression is neural specific. EMBO J. 6: mRNAs. Suppression by translational readthrough or 425-431. CATT.~KEO,R. 1991 Different types of messenger RNA editing. editing are the most likely mechanisms to account for Annu. Rev. Genet. 25: 71-88. our observations. Cox, R. A,, 1968 The use of guanidium chloride in the isolation of The suppression mechanism is able to act similarly nucleic acids. Methods Enzymol. 12: 120-129. ENGELMAN, A,, andN. ROSENBERG,1987 Isolation of temperature- on the two different stop codons (TAG and TGA, re- sensitive Abelson virus mutants by site-directed mutagenesis. spectively, for elau"' and elafl!''). Such a case of sup Proc. Natl. Acad. Sci. USA 84 8021-8025. pressed nonsense mutationshas been reported fornina- GAOF.-B., C. C. CARSON,T. LEVINEand J. D. KEENE, 1994 Selection of a subset of mRNAs from combinatorial 3' untranslated region E, which encodes Drosophila's major rhodopsin (WASH- libraries usingneuronal RNA-binding protein Hel-N1. Proc. Natl. BURN and O'TOUSA1992). All three nonsense codons Acad. Sci. USA 91: 11207-11211. at position 309 of the nina-EORF are suppressed,restor- GooD, P. J., 1995 A conserved family of elav-like genes in verte- brates. Proc. Natl. Acad. Sci. USA 92: 4557-4561. ing partial or total function. GORIACH,M., M. WIITEKIND,R. A. BECKMAN,L. MUEI.I.ERand G. A variety of previous examples of suppression have DREWUSS,1992 Interaction of the RNA-binding domain of the been reported that proved to function withlow effi- hnRNPC proteins with RNA. EMBO J. 11: 3289-3295. HOFFMAN,D. W., C. C. QUERY,B. L. GOLDEN, S. W. WIIITE andJ. D. ciencies (see, e.g., XUEand COOLEY1993). Thekinetics KEENE, 1991 RNA-binding domain of the A protein component of ELAV embryonic expression also support low levels of the U1 small nuclear ribonucleoprotein analyzed byNMR of suppression. When ELAV protein is synthesized at a spectroscopy is structuraly similar to ribosomal proteins. Proc. Natl. Acad. Sci. USA 88: 2495-2499. high level, as in early stages of neuronal differentiation, HOMYK,T., J. J. SLIDONYA~~~D. T. SUZUKI,1980 Behavioral mutants the 45-kD form predominates over the 50-kD form. A of Drosophila melanogaster. 111.Isolation and mappingof mutations similar pattern would probably be observed during the by direct visual observations of behavioral phenotypes. Mol. Gen. Genet. 177: 553-565. second waveof neuronaldifferentiation that occurs HOMYK,T. J,, andT. A. GRIGLIATTI,1983 Behavioral mutants of during metamorphosis. As development proceeds, the Drosophila melanogaster. rV. Analysis of developmentally tempera- stable 50-kD ELAV form progressively accumulates, and ture-sensitive mutations affecting flight. Dev. Genet. 4: 77-97. HOMYK,T. J., K. ISONOand W. L. Pm, 1985Developmental and its levels reach and eventually surpass that of the unsta- physiological analysis of a conditional mutation affecting photo- ble 45-kDELAV, generating the protein patterns ob- receptor andlobe development in Drosophila melanogaster.J. Neu- served in late embryogenesis and in adults. rogenet. 2: 309-324. JOHNSON, R. D., R. J. DURONIO,C. A. LANGNER, D. A. RUDNICK, and We thank S. MOLINTfor sharing his knowledge on splicing, J. I. GORDON,1993 Genetic and biological studies of a mutant L. RABINow for his constructive comments on the manuscript, and Saccharomycescermisiae myristoyl-CoA. protein N-myristoyltrans- ferase, nmt72p' r'i'''i~pr'', that produces temperature sensitive myris- P. PAKMENTER forhelp with the manuscript. We are grateful to D. tic acid auxotrophy. J. Biol. Chem. 268 483-494. BORDNE forthe tine technical support and S. KOUSHIKAfor data on MESS,R., and G. M. RUBIN,1984 Analysis of P-transposable ele- 339Sp transformant lines. This work was supported by grant 693- ment functions in Droso$hila. Cell 38: 135-146. 028 from the the New Jersey Commission on Cancer Research to M.- KENAN, D. J., C. C. QUERY and J. D. KEEN^.:, 1991 RNA recognition: L.S. and by grant GM22350 from the National Institute of Health towards identifying determinants of specificity. Trends Biochem. (NIH) to K.W. M.-L.S. was supported by NIH Training Grant T32NS Sci. 16: 214-220. 07292 and NRSA 1F32 NSO8948. M.J.L. was supported by NIH Train- KIM, Y.:J., and B. S. 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