title: High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis.
abstract: The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. Using an unbiased high throughput sequencing approach, we have identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5' ends. We independently verified these findings by reverse-transcription and polymerase chain reaction (RT-PCR) analysis. Our analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. We show, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB and that the site is conserved in many Drosophila species. We discuss that Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado, United States of America.
authors.forename: Vinod
authors.initials: V
authors.lastname: Sridharan
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado, United States of America.
authors.forename: Joseph
authors.initials: J
authors.lastname: Heimiller
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado, United States of America.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Robida
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado, United States of America.
authors.forename: Ravinder
authors.initials: R
authors.lastname: Singh
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D063990:Gene Ontology; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010802:Phylogeny; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013091:Spermatogenesis / Q000235:genetics*; D024363:Transcription Initiation Site; D059467:Transcriptome / Q000235:genetics
chemical_list: D029721:Drosophila Proteins; C588105:Heph protein, Drosophila; D020033:Protein Isoforms; D012333:RNA, Messenger; D038941:Polypyrimidine Tract-Binding Protein