Mei-P26 is a TRIM–NHL family RNA-binding protein in Drosophila that functions as a post-transcriptional regulator of stem cell fate (Insco et al., 2012; Li et al., 2012; Liu et al., 2009). In the ovary, Mei-P26 acts through interactions with the miRNA pathway, including Ago1- and GW182-containing complexes, to repress target mRNA translation and maintain the balance between stem cell self-renewal and differentiation (Neumüller et al., 2008; Salerno-Kochan et al., 2022). Although this regulatory framework is well established, the contribution of individual miRNAs to Mei-P26 regulation remains poorly defined. In Drosophila , miRNA-mediated silencing is primarily mediated by Ago1-associated RISC complexes (Azzam et al., 2012; Förstemann et al., 2007; Lyu et al., 2014), while a subset of miRNAs is generated through Drosha-independent non-canonical pathways, producing intron-derived miRNAs termed ‘mirtrons’(Okamura et al., 2007).

Recent studies implicate Mei-P26 in neuroblast regulation during brain development (Hu et al., 2025), as well as increased Mei-P26 in proteomic analysis of adult offspring brains following paternal Western diet (Murashov et al., 2023). miRNA enrichment analysis of this proteomic dataset using MIENTURNET identified an evolutionarily conserved miRNA, miR-10-3p, as a potential regulatory candidate ( Figure 1A ) (Licursi et al., 2019; Murashov et al., 2023). Notably, miR-10-3p shares its seed sequence with the mirtron miR-1006-3p (Ruby et al., 2007), raising the possibility that these miRNAs may contribute to the post-transcriptional regulation of mei-P26 expression ( Figure 1D ). To place these candidates in a broader regulatory context, TargetScanFly 7.2 predicted 20 conserved miRNA families with binding sites in the mei-P26 3′ UTR ( Figure 1B ) (Agarwal et al., 2018). Expression-based prioritization using a meta-analysis of testis RNA-seq datasets from the Eric Lai laboratory (Mohammed & Lai, 2016) revealed that miR-10 exhibits the highest testis expression among the top predicted candidates, based on reads per million mapped miRNA reads (RPMM) ( Figure 1C ). Quantitative PCR analysis showed that germline-specific overexpression of mei-P26 resulted in a marked reduction of miR-10-3p levels, whereas miR-1006-3p levels were only modestly affected ( Figure 1E ). Reciprocally, germline-specific overexpression of miR-10 caused a significant reduction in mei-P26 expression ( Figure 1F ), while miR-1006 overexpression produced only a minor decrease ( Figure 1G ).

Consistent with these effects, germline-specific knockdown of miR-10 led to an expansion of Mei-P26–positive spermatocytes relative to controls ( Figure 1I ) compared to control ( Figure 1H ). In contrast, germline-specific knockdown of miR-1006 did not produce a comparable increase in Mei-P26–positive spermatocytes ( Figure 1J ). Together, these results indicate that miR-10-3p, but not miR-1006-3p, plays a predominant role in regulating Mei-P26 expression in the Drosophila male germline. Although miR-1006-3p was expressed at substantially lower levels than miR-10-3p in the testis, it was included as a predicted mei-P26 regulator to experimentally assess whether computationally predicted targeting is sufficient to confer functional regulation. The comparatively weaker effects observed following miR-1006 manipulation support miR-10-3p as the predominant endogenous regulator of mei-P26 in the testis. These findings establish miR-10-3p as the principal regulator of mei-P26 in the Drosophila testis. Nevertheless, the contribution of miR-1006-3p remains to be fully resolved, and future studies will be required to determine whether it plays a context-dependent or developmental stage-specific role in mei-P26 regulation.

Fly husbandry

All flies were raised on standard cornmeal-based food at room temperature. Crosses were raised at room temperature until eclosion and adult males with desired genotypes were shifted to 26°C for 5-7 days to ensure maximum GAL4 activity. Fly strains used in this research were obtained from Bloomington Drosophila Stock Center (BDSC) and KYOTO Stock Center (DGRC). Following fly lines were used in this study: w ¹¹¹⁸ (BDSC# 3605), UAS-mei-P26 (BDSC# 25771), UAS-miR-10 (BDSC# 41169), UAS-miR-1006 (BDSC# 41204), UAS-miR-10-sponge (BDSC# 61377), UAS-miR-1006-sponge (BDSC# 61489), and vasa-GAL4 (DGRC# 109996).

Immunostaining

The testes of aged (5 to 7 days after eclosion) unmated F1s with desired genotype were dissected in ice cold PBSTX (1X PBS with 0.2% Triton X-100, 0.1% Tween-20). The dissected testes were fixed in 4% paraformaldehyde (PFA) for 30 minutes, washed thrice with PBSTX, and blocked in PBSTX + 5% NGS (5% normal goat serum) in PBSTX for an hour. The blocked samples were then nutated with primary antibodies at 4˚C overnight. The testes samples were then washed thrice in PBSTX, followed by blocking for one hour in PBSTX + 5% NGS before incubating with secondary antibodies at room temperature for 2.5 hours. The primary antibodies used in this study were guinea pig anti-Mei-P26 (1:400; a kind gift from Paul Lasko, McGill University) (Liu et al., 2009), mouse anti-α-Spectrin (α-Spec; 1:100; DSHB), and rabbit anti-Cleaved Caspase-3 (1:400; Cell Signaling Technology). The secondary antibodies used in this study were goat anti-rabbit IgG Alexa Fluor 488, goat anti-mouse IgG Alexa Fluor 488, and goat anti-guinea pig IgG Alexa Fluor 594 (Invitrogen). All secondary antibodies were used at dilution of 1:500. Then, the testes samples were washed thrice in PBSTX and mounted on glass slide with 10 μL SlowFade mounting medium with DAPI (Biotium).

RNA Isolation and Quantitative Real-Time PCR

Adult unmated males of 5 to 7 days in age were dissected quickly in filter sterilized ice cold PBSTX. A maximum of thirty males were processed per round. As much of the dissecting medium was removed prior to the addition of 100μl of TRIzol (Invitrogen). Tubes were immediately placed in the -80°C refrigerator for flash freezing. Three batches of testes (30 pairs each batch) were consolidated for a singular RNA extract. Total RNA from tissue samples was extracted with an RNAqueous Micro Kit (Thermo Fisher–Life Technologies) according to the manufacturer's instructions. For miRNA quantification, polyadenylation and reverse transcription were performed using Mir-X miRNA First Strand Synthesis Kit (Takara Bio; Cat. No. 638313) and for gene, first-strand cDNA was synthesized from total RNA using the SuperScript™ VILO™ cDNA Synthesis Kit (Thermo Fisher Scientific; Cat. No. 11754250). Real-time PCR reaction on cDNA was performed using the TB Green Advantage qPCR Premix Kit (Takara Bio; Cat. No. 639676) on qTOWER ³ iris 384 PCR System (Analytik Jena) according to the manufacturer’s instructions. For miRNAs as internal controls, primers for U6 (the noncoding small nuclear RNA) supplied with Mir-X miRNA First Strand Synthesis Kit were used and for gene, RpL32 was used as a reference gene for normalization. For specific miRNAs, miRNA-specific primer (Table 1) and mRQ 3’primer supplied with the Mir-X miRNA First Strand Synthesis Kit according to the instructions provided by the manufacturer. The relative quantifications of miRNA expression were calculated against U6 and of mRNA expression were calculated against RpL32 by the ∆∆ C _t ² method. The experiments were performed three times independently. Following are the sequence of primers used in this study:

Table 1: List of primers used in this study.

Sequences and miRNA Target Prediction

Sequences for miR-10-3p and miR-1006-3p were recovered from miRBase.org for schematic representation. For D. melanogaster mei-P26 miRNA target prediction, we used TargetScanFly 7.2 (Agarwal et al., 2018). An umbrella record for Lai lab miRNA RPMM expression values consolidated from shortRNA-seq assays of various tissues published in FlyBase release (FB2026_01) was used to plot miRNA RPMM expression in testis for top predicted mei-P26 miRNA targets (Öztürk-Çolak et al., 2024).