Proteomic analysis reveals CCT is a target of Fragile X mental retardation protein regulation in Drosophila

Abstract

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that is required for the translational regulation of specific target mRNAs. Loss of FMRP causes Fragile X syndrome (FXS), the most common form of inherited mental retardation in humans. Understanding the basis for FXS has been limited because few in vivo targets of FMRP have been identified and mechanisms for how FMRP regulates physiological targets are unclear. We have previously demonstrated that Drosophila FMRP (dFMRP) is required in early embryos for cleavage furrow formation. In an effort to identify new targets of dFMRP-dependent regulation and new effectors of cleavage furrow formation, we used two-dimensional difference gel electrophoresis and mass spectrometry to identify proteins that are misexpressed in dfmr1 mutant embryos. Of the 28 proteins identified, we have identified three subunits of the Chaperonin containing TCP-1 (CCT) complex as new direct targets of dFMRP-dependent regulation. Furthermore, we found that the septin Peanut, a known effector of cleavage, is a likely conserved substrate of fly CCT and is mislocalized in both cct and in dfmr1 mutant embryos. Based on these results we propose that dFMRP-dependent regulation of CCT subunits is required for cleavage furrow formation and that at least one of its substrates is affected in dfmr1- embryos suggesting that dFMRP-dependent regulation of CCT contributes to the cleavage furrow formation phenotype.

Figure 1. Comparative proteomic analysis of control and dfmr1- cleavage stage embryos

(A) Schematic showing general procedure for 2D DIGE analysis. Differential interference contrast (DIC) images of control and dfmr1- embryos show the morphological stage of sorted embryos. (B) A master gel is pseudocolored with control extracts labeled with CY3 (red) and dfmr1- extracts labeled with CY5 (green). The approximate isoelectric point (pI) is indicated at the top of the gel and the approximate molecular weight (MW) is indicated to the left. Difference spots are indicated with white arrow and numbered. The labeled difference spots were observed in at least four gel replicates. (C) High magnification examples of difference spots are shown with red arrows, and corresponding spot number in (C) is indicated to left. (D) The 28 difference proteins identified by mass spectrometry are categorized by proposed gene ontology.

Figure 2. Immunoprecipitation of candidate target mRNAs with dFMRP

(A) Immnuoblots showing input and supernatant (S) and pellet (P) fractions of IPs performed using an anti-dFMRP antibody with WT and dfmr1- extracts probed with dFMRP and Tubulin antibodies. Although no Tubulin co-IPd with dFMRP, IgG heavy chain (HC) was present in equal volumes in the P fraction of each IP as indicated with asterisks. (B) RNA was extracted from P fractions shown in (A) and subjected to qRT-PCR. Histogram shows the fold enrichment of each mRNA in WT vs. dfmr1- IPs normalized to RpL32. tral mRNA is a know target of dFMRP and its enrichment was tested as a positive control in these experiments. Error bars indicate standard deviations (SD). Significance was assessed using the Student's t test (*, P≤0.05 and **, P≤0.005). Red box highlights those mRNAs that are at least 2-fold in enriched.

Figure 3. CCT subunits in Drosophila melanogaster

(A) Table shows known genetic and molecular information for each of the eight CCT subunits in fly. Percentages of identity and similarity in protein sequence for each of the subunits was compared between fly and budding yeast and fly and human. The protein difference detected in dfmr1- embryos the 2D DIGE gels is indicated for those proteins identified in the screen. (B) A Clustal-W alignment of the protein sequences of the fly (dm) and budding yeast (sc) CCT subunits affected in dfmr1- embryos shows regions of high conservation.

Figure 4. CCT is required for proper cleavage furrow formation and loss of cct enhances dfmr1- phenotype

(A) Frames from representative DIC movies of WT and cct- cleavage stage embryos. Time (t) is in minutes from the start of interphase of nuclear cycle 14. Percentage and number of embryos examined (n) with shown phenotype is indicated at top. Arrowheads and brackets indicate the furrow front position and nuclear elongation, respectively. Scale bar indicates 10 μm. (B) Bar graph shows percentage of embryos with severe cleavage furrow phenotype. Genotypes are indicated at the bottom with the total number of embryos observed (n). The inset is a representative DIC image of an embryo with a severe furrowing phenotype. Arrow indicates where furrow is completely disrupted.

Figure 5. CCT holocomplex assembly is disrupted in dfmr1- embryos

(A) Quantitative immunoblot (IB) of WT and dfmr1- cleavage stage embryo extracts probed with antibodies against CCT1, dFMRP, and Myosin II (MYOII, loading control) indicated to right. MW in kDa is indicated to the left. (B) IB of fractions collected from 1.3 mg of WT and dfmr1- cleavage stage embryo extracts separated using a Superose 6 column and probed with anti-CCT1 antibody. 10 mg of protein loaded onto column is indicated at far left of blots. 660kDa complex was present in fraction 11 of 20. Cartoon representation of possible state of CCT complex formation indicated below blots. (C) Immunofluorescence (IF) analysis of fixed cleavage stage WT and dfmr1- embryos shows CCT1 localization in surface (top) and sagital (bottom) views. Arrows indicate abnormal accumulation of CCT1. Scale bar indicates 10 μm.

Figure 6. PNUT localization is dependent on CCT and is a likely substrate of CCT

(A) IF analysis of fixed cleavage stage WT and cct- embryos shows PNUT and F-actin (Phalloidin) localization. (B) IF analysis of fixed WT and dfmr1- embryos shows PNUT and Anillin localization. Arrow heads indicate normal localization of PNUT and Anillin to furrow fronts, and arrows indicate abnormal accumulation of PNUT along lateral membrane. Scale bar indicates 10 μm. (C) Immunoblots showing PNUT IPs from WT and dfmr1- extracts. 10 μg starting extract was loaded in input lanes (4% of total input). 10 μg of supernatants (S) and 50% of pellet (P) from mock and anti-PNUT IPs was loaded in indicated lanes. Proteins probed for indicated to left.