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.

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

“…We show that Heph binds to the USE RNA but not to a mutated USE. We demonstrate that the heph 2 /TM6B mutant, which has a phenotype in testis (62) and defects in spermatogenesis (58,63) that ultimately lead to male sterility (64), presents abnormal polo mRNA 3=-end formation with selection of the cryptic pA2, which is also chosen by gfp-poloΔUSE; polo 9Ϫ/Ϫ flies. We also observed that gfp-poloΔUSE; polo 9Ϫ/Ϫ flies use another cryptic pA signal upstream of pA1 (cryptic pA1) which is not used by heph 2 /TM6B mutants, implying that the in vivo deletion of the polo USE is more severe in pA signal selection than the lack of Heph.…”

“…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.…”

Abundant small RNAs in the reproductive tissues and eggs of the honey bee, Apis mellifera

“…4 D). Of tRFs that were significantly upregulated at 72 h (red-eyed pupae) relative to other pupal stages, many multi-mapped to eleven genes, two of which are important gametogenesis genes in D. melanogaster ; Hephaestus (heph) [ 78 , 79 ] and CUGBP Elav-like family member 2 (bru-2) [ 80 ]. 72 h post-fertilisation (red-eyed pupae) coincides with the end of meiosis and the start of spermiogenesis [ 77 ]: the striking changes in small RNA expression during this window strongly suggest that small RNAs play an important role in spermatogenesis.…”

Abundant small RNAs in the reproductive tissues of the honey bee, Apis mellifera, are a plausible mechanism for epigenetic inheritance and parental manipulation of gene expression

“…In contrast, miRNAs that are upregulated at 72 hr testes relative to 24 hr testes tend to remain upregulated in 72 hr testes and 144 hr testes, representing a trend towards increased miRNA production as the testis develop (Fig 4D). Of tRFs that were significantly upregulated at 72 hours (red-eyed pupae) relative to other pupal stages, many multi-mapped to eleven genes, two of which are important gametogenesis genes in D. melanogaster; Hephaestus (heph) (Robida et al 2010;Sridharan et al 2016) and CUGBP Elavlike family member 2 (bru-2) (Dasgupta and Ladd 2012). 72 hours post-fertilisation (red-eyed pupae) coincides with the end of meiosis and the start of spermiogenesis (Lago et al 2020): the striking changes in small RNA expression during this window strongly suggest that small RNAs play an important role in spermatogenesis.…”