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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).