The DExH box helicase domain of spindle-E is necessary for retrotransposon silencing and axial patterning during Drosophila oogenesis

Abstract

Transposable selfish genetic elements have the potential to cause debilitating mutations as they replicate and reinsert within the genome. Therefore, it is critical to keep the cellular levels of these elements low. This is especially true in the germline where these mutations could affect the viability of the next generation. A class of small noncoding RNAs, the Piwi-associated RNAs, is responsible for silencing transposable elements in the germline of most organisms. Several proteins have been identified as playing essential roles in piRNA generation and transposon silencing. However, for the most part their function in piRNA generation is currently unknown. One of these proteins is the Drosophila melanogaster DExH box/Tudor domain protein Spindle-E, whose activity is necessary for the generation of most germline piRNAs. In this study we molecularly and phenotypically characterized 14 previously identified spindle-E alleles. Of the alleles that express detectable Spindle-E protein, we found that five had mutations in the DExH box domain. Additionally, we found that processes that depend on piRNA function, including Aubergine localization, Dynein motor movement, and retrotransposon silencing, were severely disrupted in alleles with DExH box domain mutations. The phenotype of many of these alleles is as severe as the strongest spindle-E phenotype, whereas alleles with mutations in other regions of Spindle-E did not affect these processes as much. From these data we conclude that the DExH box domain of Spindle-E is necessary for its function in the piRNA pathway and retrotransposon silencing.

Keywords: Drosophila; RNA helicase; embryonic patterning; oogenesis; piRNA; retrotransposon.

Figure 1

Eight of the fourteen spn-E alleles express detectable protein and have point mutations in the SPN-E coding region. (A) Domain structure of Drosophila SPN-E and its human homolog, TDRD9. SPN-E contains a highly conserved DExH box and a Tudor domain as well as a Zinc finger, whereas TDRD9 only has a DExH box and Tudor domain. The position of the two mutations outside of the conserved domains, the five mutations that do not produce detectable protein, and the Zinc finger are shown. (B) The amino acid sequence of the SPN-E DExH box domain compared with its human homolog TDRD9, yeast splicing factor Prp16, and vaccinia virus protein NPH-I. The positions of the five mutations identified in the SPN-E DExH box domain are shown. Amino acid numbering is according to Ensemble Genome Browser release 73. (C) SPN-E protein expression in mutant ovary extracts as measured by Western blotting. Protein was isolated from hemizygous ovaries of the genotype spn-E^mutant/spn-E^∆125. Eight alleles express detectable protein of the correct size for SPN-E. Four alleles do not express detectable protein. Spn-E/Bal = spn-E^∆125/Balancer chromosome. Line 7G2-5 is not shown. Several extraneous bands are found on the Western blots shown above. We did not detect these bands when we used a second antibody developed in the laboratory of Dr. Toshie Kai (Patil and Kai 2010; data not shown); therefore, we think that the extra bands are most likely nonspecific bands recognized by our SPN-E antibody. (D) SPN-E protein levels in the various mutant ovaries relative to spn-E^∆125/Balancer. Error bars represent SD of at least 2 separate protein isolates. SPN-E protein levels were normalized to beta-tubulin. (E) A listing of each spn-E allele name along with its corresponding mutation.

Figure 2

AUB nuage localization is lost in some, but not all, of the spn-E mutant egg chambers. spn-E mutant germline clones are marked by the absence of GFP. All egg chambers were stained with α-GFP (green), α-AUB (red), and DAPI to mark the DNA. In wild-type egg chambers, AUB localizes around the nurse cell nuclei to a structure known as the nuage (A-A′′). spn-E^4-48 (B-B′′) and spn-E^66-21 (not shown) show wild-type localization of AUB to the nuage. spn-E^23-17 (C-C′′) and spn-E^9A2-17 (not shown) show an intermediate phenotype where AUB expression is punctate and only partially localized to the nuage (C′, chamber outlined). In the spn-E^155-55 DExH box mutant allele (D-D′′) as well as most of the other DExH box alleles (not shown), AUB is not localized to the nuage and levels of AUB protein appear to be strongly decreased in mutant egg chambers (D′, outlined). This phenotype is also seen in spn-E^9A9-18 mutant egg chambers (E-E′′) as well as the remainder of the spn-E alleles that do not express detectable protein (not shown). Scale bars = 20 μm.

Figure 3

Dynein motor complex aggregates form in some, but not all, spn-E mutant ovaries. spn-E mutant germline clones are marked by the absence of GFP. All egg chambers were stained with α-GFP (green) to mark clones, α-Egalitarian (EGL) (red), and the DNA dye DAPI. In wild-type egg chambers, EGL is dispersed throughout the nurse cells and localizes to the oocyte (A-A′′). spn-E^4-48 (B-B′′), spn-E^23-17 (C-C′′), as well as spn-E^9A2-17 and spn-E^66-21 (not shown) show wild-type EGL localization. In spn-E^155-55 DExH box mutant egg chambers (D-D′′), EGL forms aggregates throughout the egg chamber. This phenotype is present in spn-E^9A9-18 mutant egg chambers (E-E′′) as well as the DExH box alleles: spn-E^2A9-14, spn-E^7G2-5, spn-E^8D4-11, and the remainder of the spn-E alleles that do not express detectable protein (not shown). Note the small size of the oocyte in spn-E^155-55 and spn-E^9A9-18 egg chambers (arrow in D′ and E′). Scale bars = 20 μm.

Figure 4

Retrotransposon RNA levels are increased to varying degrees in the various spn-E mutant ovaries. (A) Quantitative real-time RT-PCR for the retrotransposons Het-A and Blood using extracts from mutant ovaries (spn-E^mutant/spn-E^∆125). (B) Quantitative real-time RT-PCR for retrotransposons I Factor, TART, and Roo. Relative expression was calculated in comparison to respective RNA levels obtained from heterozygous siblings for each individual allele. All RNA was normalized to Adh. Error bars represent SD of four experiments using two independent RNA isolates.