Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster

Abstract

Genome-wide identification of RNAs associated with RNA-binding proteins is crucial for deciphering posttranscriptional regulatory systems. PUMILIO is a member of the evolutionary conserved Puf-family of RNA-binding proteins that repress gene expression posttranscriptionally. We generated transgenic flies expressing affinity-tagged PUMILIO under the control of an ovary-specific promoter, and we purified PUMILIO from whole adult flies and embryos and analyzed associated mRNAs by using DNA microarrays. Distinct sets comprising hundreds of mRNAs were associated with PUMILIO at the two developmental stages. Many of these mRNAs encode functionally related proteins, supporting a model for coordinated regulation of posttranscriptional modules by specific RNA-binding proteins. We identified a characteristic sequence motif in the 3'-untranslated regions of mRNAs associated with PUMILIO, and the sufficiency of this motif for interaction with PUMILIO was confirmed by RNA pull-down experiments with biotinylated synthetic RNAs. The RNA motif strikingly resembles the one previously identified for Puf3p, one of five Saccharomyces cerevisiae Puf proteins; however, proteins encoded by the associated mRNAs in yeast and Drosophila do not appear to be related. The results suggest extensive posttranscriptional regulation by PUMILIO and uncover evolutionary features of this conserved family of RNA-binding proteins.

Fig. 1.

Specific RNAs selectively associated with Pum in Drosophila adults and embryos. Rows represent genes (unique DNA elements) and columns represent individual experiments. The color code indicates the degree of enrichment (green–red ratio scale). (A) Relative enrichments of mRNA targets of Pum expressed in adult ovaries. Four experiments with affinity-tagged Pum proteins and three mock experiments are shown. Genes were ordered from top to bottom according to increasing FDRs determined by significance analysis of microarrays (SAM) analysis. Arrowheads depict enrichment of previously known Pum targets. (B) Pum RNA targets selected in embryos. A total of 174 transcripts representing 165 genes were clustered based on Euclidean distances with smd (50). (C) Diagram showing overlap of adult and embryo Pum associated mRNAs.

Fig. 2.

mRNAs associated with Pum encode proteins of specific macromolecular complexes and regulatory pathways. (A) Subunits of the vacuolar ATPase. Red, subunits whose mRNAs associated with Pum; gray, subunits whose mRNAs were not enriched with a FDR of <1%. Subunits of the V1 domain are labeled with capital letters (34): A, Vha68; B, Vha55; C, Vha44; D, Vha36; E, Vha26; F, Vha14; G, Vha13; H, VhaSFD. Subunits of the V0 domain are indicated by small letters: a, Vha100; c, Vha16; c″, PPA1; e, VhaM9.7; d, VhaAC39. (B) Components of anterior–posterior pattering systems associated with Pum in embryos and/or adults. mRNAs associated with Pum are shown in red and their protein products in blue. Proteins whose mRNAs were not found to be associated are in black.

Fig. 3.

RNA consensus motif in 3′ UTR sequences associated with Pum and yeast Puf3, Puf4 and Puf5 proteins (5). Height of the letter indicates the probability of appearing at the position in the motif. Nucleotides with <10% appearance were omitted.

Fig. 4.

Validation of Pum mRNA targets. RNA–protein complexes formed between biotin-labeled 3′ UTRs and extracts of adult Drosophila expressing TAP-PumHD were purified on streptavidin magnetic beads and monitored for the presence of TAP-PumHD by immunoblot analysis with Peroxidase-Anti-Peroxidase Soluble Complex (Sigma). (A) Biotin-labeled 3′ UTR sequences for indicated genes (lanes 2 to 8) were incubated with Drosophila extract (input, lane 1). Rps26 3′ UTR was used as a negative control probe RNA (lane 8). (B) Validation of the core sequence motif identified in 3′ UTRs sequences of Pum target mRNAs. Lane 1, input (Drosophila extract); lanes 2–4, biotin-labeled RNA corresponding to the Vha16 3′ UTR was combined with Drosophila extract (lane 2) and 100-fold excess of competitor RNA (R1; AUUGUAAAUA; lane 3) or control RNA where the core motif is mutated (R2; AUACAAAAUA; lane 4). The conserved trinucleotide motif UGU in the 3′ UTR of CG8414 was left intact (lane 6) or was mutated to ACA (lane 7). Rps26 3′ UTR is the negative control probe RNA (lane 5).