Drosophila Polypyrimidine Tract-Binding Protein (DmPTB) Regulates Dorso-Ventral Patterning Genes in Embryos

Abstract

The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during embryogenesis. A loss of function mutation, heph03429, results in varied defects in embryonic developmental processes, leading to embryonic lethality. However, the suite of molecular functions that are disrupted in the mutant remains unknown. We have used an unbiased high throughput sequencing approach to identify transcripts that are misregulated in this mutant. Misregulated transcripts show evidence of significantly altered patterns of splicing (exon skipping, 5′ and 3′ splice site switching), alternative 5′ ends, and mRNA level changes (up and down regulation). These findings are independently supported by reverse-transcription-polymerase chain reaction (RT-PCR) analysis and in situ hybridization. We show that a group of genes, such as Zerknüllt, z600 and screw are among the most upregulated in the mutant and have been functionally linked to dorso-ventral patterning and/or dorsal closure processes. Thus, loss of dmPTB function results in specific misregulated transcripts, including those that provide the missing link between the loss of dmPTB function and observed developmental defects in embryogenesis. This study provides the first comprehensive repertoire of genes affected in vivo in the heph mutant in Drosophila and offers insight into the role of dmPTB during embryonic development.

Cell Cycle Kinase Polo Is Controlled by a Widespread 3′ Untranslated Region Regulatory Sequence in Drosophila melanogaster

“…Such an example is human polypyrimidine tract-binding protein (PTB/ PTBP1/hnRNPI) (reviewed in references 47 to 50), which is a multifunctional RBP involved in alternative splicing regulation (50), polyadenylation (44,(51)(52)(53)(54), mRNA localization in Xenopus (55), and internal ribosome entry site-driven translation (56). In Drosophila melanogaster, Hephaestus (Heph) (the PTBP1 homologue) is involved in alternative splicing (57,58) and regulates oskar mRNA translation and mRNA levels (57), Gurken protein location (59), embryo dorsoventral patterning, and germ line-soma signaling (59)(60)(61), as well as spermatogenesis (58,(62)(63)(64). However, a function for Heph in APA had not been identified yet.…”

High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis

“…However, for the heph 03429 mutant, showing embryonic lethality, the P element is inserted into an intron downstream of the proximal 5’ transcript start site. This transcript start site is relevant for the non-sex-specific, including embryonic, function of dmPTB ; no reads were found that spanned the other end of the P element insertion into the heph gene [ 26 ].…”

“…In addition to RNA-Seq [ 26 ] and the phylogenetic sequence analysis used here, several other approaches have been employed in other studies of RNA-protein interaction: yeast-three-hybrid assay [ 45 ]; CLIP [ 46 ]; Fast-FIND ( Fast - F ully I ndexed N ucleotide D atabase) [ 47 ]; PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) [ 48 ]; and RIP-Seq (or RIP-ChIP). As noted above, the short, degenerate (pyrimidine-rich sequence) PTB binding site appears frequently in the genome and makes identification of RNA-protein interactions that are functionally relevant even more difficult.…”

“…Traces were generated using the UCSC genome browser/tools. For additional methods details on RNA-Seq expression and isoform analysis, see [ 26 ].…”

Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals

“…Hence, we checked whether the main repressor of Srrm4-dependent exons in mammals (Ptbp1) 38,40 has a similar role in Drosophila. We analysed two RNA-seq datasets upon knockdown of Hephaestus (heph), the D. melanogaster Ptbp1/2/3 ortholog, in fly embryos 40 and SL2 cells 53 , and found no evidence for a role in repressing the inclusion of eMIC targets, unlike for equivalent experiments in mammalian cells 54,55 (Figure 5D and Supp. Figure 6A).…”