Substitution of critical isoleucines in the KH domains of Drosophila fragile X protein results in partial loss-of-function phenotypes

Abstract

Fragile X mental retardation proteins (FMRP) are RNA-binding proteins that interact with a subset of cellular RNAs. Several RNA-binding domains have been identified in FMRP, but the contribution of these individual domains to FMRP function in an animal model is not well understood. In this study, we have generated flies with point mutations in the KH domains of the Drosophila melanogaster fragile X gene (dfmr1) in the context of a genomic rescue fragment. The substitutions of conserved isoleucine residues within the KH domains with asparagine are thought to impair binding of RNA substrates and perhaps the ability of FMRP to assemble into mRNP complexes. The mutants were analyzed for defects in development and behavior that are associated with deletion null alleles of dfmr1. We find that these KH domain mutations result in partial loss of function or no significant loss of function for the phenotypes assayed. The phenotypes resulting from these KH domain mutants imply that the capacities of the mutant proteins to bind RNA and form functional mRNP complexes are not wholly disrupted and are consistent with biochemical models suggesting that RNA-binding domains of FMRP can function independently.

Figure 1.—

Schematic of fragile X protein and expression analysis of dfmr1 KH domain alleles. (A) RNA-binding domains of FMRP. Two KH domains, an RGG box, and two tandem copies of a Tudor/Agenet-related domain have all been demonstrated to bind RNA. KH domains with conserved isoleucine residues mutated to asparagine for this study are depicted, (B) Western blot of total fly extracts from wild-type, dfmr1³ heterozygote, and flies expressing one or two (2X) copies of a transgene harboring a dfmr1 genomic rescue fragment coding for either an I244N or an I307N substitution in the KH domains. Extracts were prepared from males aged 2–3 days. Signals were scanned and quantified by ImageQuant software (Molecular Dynamics, Sunnyvale, CA), and average expression levels compared to a w¹¹¹⁸ control from six independent blots are given.

Figure 2.—

Numbers of larval NMJ boutons are not increased in flies expressing KH domain I244N or I307N substitutions as a sole source of dFMR1 protein. Third instar larvae were dissected and probed with antibodies against horseradish peroxidase to assess numbers of type I NMJ boutons, which are increased in flies homozygous for strong or null alleles of dfmr1 (Zhang et al. 2001; Jin et al. 2004b). Analyses of several muscle types from segment A3 show that type I boutons are significantly increased in all muscle types examined from animals homozygous for a null allele of dfmr1 compared to all wild-type and KH domain alleles examined (P < 0.001 for muscle 4, Kruskal–Wallis test and Dunn post-test; P < 0.01 for muscle 6/7, one-way ANOVA, followed by a Tukey–Kramer post-test; P < 0.001 for muscle 12, Kruskal–Wallis test and Dunn post-test). There are no significant changes in type I bouton numbers when wild-type controls are compared with either of the two KH domain mutants. The allele designations denote the sole source of dFMR1 protein. Results are from analysis of at least 20 hemi-segments for each genotype.

Figure 3.—

MB β-lobe phenotypes of flies with dfmr1 KH domain alleles. (A) Representation of the frequency with which a midline crossing of β-lobe neurons was observed. Anti-FasII staining of MBs from 2-day-old flies shows that the I244N and I307N substitutions result in a frequency of midline crossing phenotypes intermediate to flies with a wild-type allele of dfmr1 and to those homozygous for a dfmr1 null allele. The genotypes denote the allele of dfmr1 being expressed, while the presence of a wild-type allele to test for dominant effects of the mutant allele is denoted by a “+.” The frequency with which a no-crossing phenotype occurred did not change upon an increase in dosage of either mutant KH domain protein. Expression of either mutant KH domain transgene in a background with a wild-type copy of dfmr1 present has no effect on midline crossing frequency, indicating that the transgene insertions and mutant proteins do not elicit a detectable dominant effect. Genotypes grouped under a common numerical designation do not differ from each other in percentage of brains observed with a midline crossing of β-lobe axons, while those under different numerical designations differ from each other as judged by chi-square tests of homogeneity. KH domain alleles differ from the null allele in frequency of midline crossing (P < 0.0001) and from flies with a wild-type allele of dfmr1 (P = 0.0152). (B–E) Representative examples of MB morphology illustrating the variety of midline crossing phenotypes observed. α- and β-Lobes are noted, while arrows point to the midline where crossovers of the β-lobe neurons may occur. NC, no crossover.

Figure 4.—

Analysis of circadian locomotion activity of flies expressing dFMR1 with mutant KH domains in constant darkness. An assignment of rhythmic vs. arrhythmic activity for individual flies was determined using ClockLab software as described in materials and methods. The percentage of flies from each genotype judged to be rhythmic was compared by a chi-square test for homogeneity. Genotypes that are grouped by a common number do not have any significant difference between them in the percentage of animals displaying a rhythmic locomotion phenotype, while separate groups differ to a confidence level of <0.0001. Increasing the dose of mutant dFMR1 protein had no significant effect on the percentage of animals judged to have maintained rhythmic locomotion activity, indicating that other RNA-binding domains of the mutant proteins are unable to compensate for the defects in the KH domains. The genotypes denote the allele of dfmr1 that is the sole source of dFMR1 protein, while the presence of a wild-type allele to test for dominant effects of the mutant allele is denoted by a “+.” Flies that express both a mutant and wild-type allele of dfmr1 resemble wild-type flies in the percentage of animals judged to be rhythmic, demonstrating that the mutant allele and transgene insertion do not have a detectable dominant effect.

Figure 5.—

Analysis of naive courtship activity of flies expressing dFMR1 KH domain mutations. Naive courtship was analyzed as described in materials and methods. At least 25 flies of each genotype were tested. For each mutant KH domain transgene, expression in a background with a wild-type allele of dfmr1 does not result in a phenotype differing from wild type, indicating that the transgene insertion and mutant protein do not induce a detectable dominant effect. (A) Flies expressing dFMR1 with the I244N substitution as the sole source of dFMR1 protein have a significant decrease in naive courtship activity compared to flies with a wild-type allele of dfmr1, but the decrease in courtship activity is not as strong as is observed in flies homozygous for a dfmr1 null allele. Increasing the dosage of the I244N allele does not result in a significant increase in courtship activity. Courtship indexes were arcsin transformed and the data were analyzed by a one-way ANOVA, followed by a Tukey–Kramer post-test. The P-value for the ANOVA is <0.0001. Genotypes under the same numerical heading do not vary from each other to a significant extent, while genotypes under different numerical headings are significantly different from each other (P < 0.05). (B) The I307N substitution results in a significant decrease in naive courtship activity compared to flies expressing wild-type dFMR1, but not to the degree observed with flies homozygous for a null allele of dfmr1. As was seen with the I244N allele, an increase in dosage of the I307N allele does not produce a significant increase in naive courtship activity. The data for these genotypes were processed in the same manner as the I244N flies, and the P-value for the ANOVA is <0.0001. Genotypes under the same numerical grouping do not differ from each other in courtship activity to a significant extent, while genotypes under different numerical groupings have a significant variation in the courtship index (P < 0.01).