A versatile method for cell-specific profiling of translated mRNAs in Drosophila

Abstract

In Drosophila melanogaster few methods exist to perform rapid cell-type or tissue-specific expression profiling. A translating ribosome affinity purification (TRAP) method to profile actively translated mRNAs has been developed for use in a number of multicellular organisms although it has only been implemented to examine limited sets of cell- or tissue-types in these organisms. We have adapted the TRAP method for use in the versatile GAL4/UAS system of Drosophila allowing profiling of almost any tissue/cell-type with a single genetic cross. We created transgenic strains expressing a GFP-tagged ribosomal protein, RpL10A, under the control of the UAS promoter to perform cell-type specific translatome profiling. The GFP::RpL10A fusion protein incorporates efficiently into ribosomes and polysomes. Polysome affinity purification strongly enriches mRNAs from expected genes in the targeted tissues with sufficient sensitivity to analyze expression in small cell populations. This method can be used to determine the unique translatome profiles in different cell-types under varied physiological, pharmacological and pathological conditions.

Figure 1. Polysome affinity purification strategy using GAL4/UAS-GFP::RpL10A.

(A) Schematic representation of the polysome affinity purification method from adult Drosophila brains expressing GFP-tagged RpL10A in a small population of neurons. Lysates from heads of transgenic animals are incubated with beads (shaded grey) coated with GFP antibodies (red). Ribosomes (light blue) associated along the actively translated mRNA strands (orange) are captured on the beads and washed, followed by an RNA extraction step. RNA can then be used for qRT-PCR or purified for sequencing. (B) Live image of an adult brain from a fly expressing GFP-tagged RpL10A in all neurons (Elav-GAL4>UAS-GFP::RpL10A). The GFP expression pattern is consistent with the GAL4 driver. (C) Enlarged view of a section of the brain in (B), showing that GFP localization is predominantly perinuclear and nucleolar (white arrow).

Figure 2. Polysome incorporation of GFP tagged RpL10A.

(A) Sucrose gradient polysome fractionation from heads of Elav-GAL4>UAS-GFP::RpL10A flies shows the different ribosomal and polysomal fractions. Protein extracts were run on Western blots and probed with GFP and RpS6 antibodies, showing that the GFP-tagged RpL10A displays qualitatively similar incorporation into the polysomes but not into the small 40S ribosomal unit. (B) Immunoprecipitation from lysted made from 50 adult heads of Elav-GAL4>UAS-GFP::RpL10A flies following polysome immunoprecipitation with PAS beads coated with GFP antibodies (IP) or mouse IgG antibodies as a mock control (m). Lysate from the input fraction without the immunoprecipitation step was also loaded on the gel. The GFP-tagged fusion protein is efficiently precipitated from the GFP coated beads but not from the mock coated beads. Actin is only present in the unprecipitated lysate and not in the IP fraction. (C) Western blot of lysates from heads of the Elav-GAL4 (c155) strain and flies expressing GFP or GFP-tagged RpL10A (strain BF14; Table S1) with the Elav-GAL4 driver. All total lysates (16% input) show strong signal for the small ribosomal protein RpS6. After polysome affinity purification, only the immunoprecipitate from Elav-GAL4>UAS-GFP::RpL10A flies (strain BF14) show staining for RpS6, showing that whole ribosomes are precipitated.

Figure 3. Enrichment of 18S rRNA and Gapdh mRNA from neuronal ribosomes and polysomes.

Total RNA was extracted from lysted from 50 adult heads of Elav-GAL4>UAS-GFP::RpL10A compared to Elav-GAL4>GFP flies following immunoprecipitation with PAS beads coated with GFP antibodies. The RNA samples were reverse transcribed and amplified with 18S rRNA primers (A) or Gapdh primers (B). Fold enrichment of RNA was calculated in the Elav-GAL4>UAS-GFP::RpL10A compared to Elav-GAL4>GFP samples, set at 1x. Data are means +/− S.E.M. averaged from three replicates from 2 biological repeats.

Figure 4. Tissue specific expression levels of significantly enriched or depleted genes in the neuronal translatome compared to whole head mRNA.

Comparison of the lists of genes that have transcripts that are significantly and substantially enriched (A; 872 genes) or depleted (B; 1,755 genes) in the polysome-enriched fraction (q<0.05, >2-fold enriched or depleted, respectively) to Flyatlas microarray expression data. For this analysis we generated two lists of genes: those that are either significantly enriched or depleted in the polysome pull-down fraction relative to mRNA derived from whole heads. The two lists were uploaded to Flymine , which generated the graphs presented that shows the number of genes from each list for which the levels of expression are significantly high or low, in several tissues of the fly, according to FlyAtlas microarray data analysis. The genes with transcripts enriched in the polysome-enriched fraction have a greater number of genes with high expression in neuronal tissues as compared to low expression (A; see adult brain, eye, larval CNS, thoracicoabdominal ganglion), and this is not seen in the polyosme-depleted fraction. In other tissues examined by Flyatlas, the polysome-enriched and -depleted fractions do not show this pattern of substantially more genes with high expression in that tissue.

Figure 5. Enrichment of dIlp2 and depletion of NPF from the PI translatome.

qRT-PCR from total RNA extracts from affinity purified polysome fractions from neurons in the pars intercerebralis (from 50Y>UAS-GFP::RpL10A flies) compared to whole head extracts from control flies. (A) dIlp2 is expressed in PI neurons and shows ∼55 fold higher expression in the immunoprecipitate from PI neurons (PI-IP) as compared to whole head extract (Head). (B) NPF is not expressed in PI neurons and shows the opposite pattern: ∼80 fold lower in PI-IP than in whole head extract. Data are means +/− S.E.M. averaged from three replicates from 2 biological repeats.