The conserved modification N6-methyladenosine (m6A) modulates mRNA processing and activity. Here, we establish the Drosophila system to study the m6A pathway. We first apply miCLIP to map m6A across embryogenesis, characterize its m6A ‘writer’ complex, validate its YTH ‘readers’ CG6422 and YT521-B, and generate mutants in five m6A factors. While m6A factors with additional roles in splicing are lethal, m6A-specific mutants are viable but present certain developmental and behavioural defects. Notably, m6A facilitates the master female determinant Sxl, since multiple m6A components enhance female lethality in Sxl sensitized backgrounds. The m6A pathway regulates Sxl processing directly, since miCLIP data reveal Sxl as a major intronic m6A target, and female-specific Sxl splicing is compromised in multiple m6A pathway mutants. YT521-B is a dominant m6A effector for Sxl regulation, and YT521-B overexpression can induce female-specific Sxl splicing. Overall, our transcriptomic and genetic toolkit reveals in vivo biologic function for the Drosophila m6A pathway.

In the Drosophila ovary, germline stem cells (GSCs) are maintained primarily by bone morphogenetic protein (BMP) ligands produced by the stromal cells of the niche. This signaling represses GSC differentiation by blocking the transcription of the differentiation factor Bam. Remarkably, bam transcription begins only one cell diameter away from the GSC in the daughter cystoblasts (CBs). How this steep gradient of response to BMP signaling is formed has been unclear. Here, we show that Fused (Fu), a serine/threonine kinase that regulates Hedgehog, functions in concert with the E3 ligase Smurf to regulate ubiquitination and proteolysis of the BMP receptor Thickveins in CBs. This regulation generates a steep gradient of BMP activity between GSCs and CBs, allowing for bam expression on CBs and concomitant differentiation. We observed similar roles for Fu during embryonic development in zebrafish and in human cell culture, implying broad conservation of this mechanism.

Increasing evidence supports the idea that the regulation of stem cells requires both extrinsic and intrinsic mechanisms. However, much less is known about how intrinsic signals regulate the fate of stem cells. Studies on germline stem cells (GSCs) in the Drosophila ovary have provided novel insights into the regulatory mechanisms of stem cell maintenance. In this study, we demonstrate that a ubiquitin-dependent pathway mediated by the Drosophila eff gene, which encodes the E2 ubiquitinconjugating enzyme Effete (Eff), plays an essential role in GSC maintenance. We show that Eff both physically and genetically interacts with dAPC2, a key component of the anaphase-promoting complex (APC), which acts as a multisubunit E3 ligase and plays an essential role in targeting mitotic regulators for degradation during exit from mitosis. This interaction indicates that Eff regulates the APC/C-mediated proteolysis pathway in GSCs. Moreover, we show that expression of a stable form of Cyclin A, but not full-length Cyclin A, results in GSC loss. Finally we show that, in common with APC2, Eff is required for the ubiquitylation of Cyclin A, and overexpression of full-length Cyclin A accelerates the loss of GSCs in the eff mutant background. Collectively, our data support the idea that Effete/APC-mediated degradation of Cyclin A is essential for the maintenance of germline stem cells in Drosophila. Given that the regulation of mitotic Cyclins is evolutionarily conserved between flies and mammals, our study also implies that a similar mechanism may be conserved in mammals.

Relatively little is known about the in vivo functions of newly emerging genes, especially in metazoans. Although prior RNAi studies reported prevalent lethality among young gene knockdowns, our phylogenomic analyses reveal that young genes are frequently restricted to the nonessential male reproductive system. We performed large-scale CRISPR/Cas9 mutagenesis of "conserved, essential" and "young, RNAi-lethal" genes and broadly confirmed the lethality of the former but the viability of the latter. Nevertheless, certain young gene mutants exhibit defective spermatogenesis and/or male sterility. Moreover, we detected widespread signatures of positive selection on young male-biased genes. Thus, young genes have a preferential impact on male reproductive system function.

Epitranscriptomic modifications can impact behavior. Here, we used Drosophila melanogaster to study N6-methyladenosine (m6A), the most abundant modification of mRNA. Proteomic and functional analyses confirm its nuclear (Ythdc1) and cytoplasmic (Ythdf) YTH domain proteins as major m6A binders. Assays of short term memory in m6A mutants reveal neural-autonomous requirements of m6A writers working via Ythdf, but not Ythdc1. Furthermore, m6A/Ythdf operate specifically via the mushroom body, the center for associative learning. We map m6A from wild-type and Mettl3 mutant heads, allowing robust discrimination of Mettl3-dependent m6A sites that are highly enriched in 5’ UTRs. Genomic analyses indicate that Drosophila m6A is preferentially deposited on genes with low translational efficiency and that m6A does not affect RNA stability. Nevertheless, functional tests indicate a role for m6A/Ythdf in translational activation. Altogether, our molecular genetic analyses and tissue-specific m6A maps reveal selective behavioral and regulatory defects for the Drosophila Mettl3/Ythdf pathway.

Post-transcriptional regulatory strategies that involve coupling between terminal uridyltransferase (TUTase) and exoribonuclease enzymes have recently been characterized in diverse species. Of note, the 3' exoribonuclease Dis3L2 has received substantial attention as a factor that metabolizes uridylated substrates in contexts such as general mRNA degradation, turnover of specific miRNAs, and quality control of noncoding RNAs. To date, most studies of Dis3L2 have focused on fungi and mammalian cells. Here, we introduce as a system that permits analysis of molecular mechanisms as well as the ability to interrogate organismal phenotypes. We started with a structure-function analysis of the TUTase Tailor, which we recently identified to inhibit biogenesis of splicing-derived miRNA hairpins. Next, we show that Tailor/Dis3L2 form a complex via N-terminal domains in the respective proteins that are distinct from their catalytic domains. In vitro, Dis3L2 has nuclease activity, but substrate oligouridylation by Tailor stimulates their degradation by Dis3L2, especially for structured substrates. We analyzed mutants of and, which are viable and lack overt morphological defects. Instead, these mutants exhibit defects in female and male fertility, implying specific requirements in the germline. defects are more severe than, and their requirements appear stronger in males than in females. In particular, loss of Dis3L2 completely blocks productive spermatogenesis, causing male sterility. RNA-seq analysis from single- and double-mutant testes reveals aberrant gene expression programs and suggests that noncoding RNAs may be preferentially affected by Dis3L2. Overall, our studies of a new tailing/trimming complex reveal unexpectedly specific requirements during gametogenesis.

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BackgroundRecent clinical trials using c-kit+ human cardiac stem cells (CSCs) demonstrated promising results in increasing cardiac function and improving quality of life. However, CSC efficiency is low, likely due to limited cell survival and engraftment after transplantation. The Rho-associated protein kinase (ROCK) inhibitor, Y-27632, significantly increased cell survival rate, adhesion, and migration in numerous types of cells, including stem cells, suggesting a common feature of the ROCK-mediated apoptotic pathway that may also exist in human CSCs. In this study, we examine the hypothesis that pretreatment of human CSCs with Y-27632 protects cells from Doxorubicin (Dox) induced apoptosis.Methods and Resultsc-kit+ CSCs were cultured in CSC medium for 3–5 days followed by 48hr treatment with 0 to 10μM Y-27632 alone, 0 to 1.0μM Dox alone, or Y-27632 followed by Dox (48hrs). Cell viability, toxicity, proliferation, morphology, migration, Caspase-3 activity, expression levels of apoptotic-related key proteins and c-kit+ were examined. Results showed that 48hr treatment with Y-27632 alone did not result in great changes in c-kit+ expression, proliferation, Caspase-3 activity, or apoptosis; however cell viability was significantly increased and cell migration was promoted. These effects likely involve the ROCK/Actin pathways. In contrast, 48hr treatment with Dox alone dramatically increased Caspase-3 activity, resulting in cell death. Although Y-27632 alone did not affect the expression levels of apoptotic-related key factors (p-Akt, Akt, Bcl-2, Bcl-xl, Bax, cleaved Caspase-3, and Caspase-3) under basal conditions, it significantly inhibited the Dox-induced increase in cleaved Caspase-3 and reduced cell death under Dox treatment.ConclusionsWe conclude that preconditioning human CSCs with Y-27632 significantly reduces Dox-induced cell death and possibly involves the cleaved Caspase-3 and ROCK/Actin pathways. The beneficial effects of Y-27632 may be applied to stem cell-based therapy to increase cell survival rates after transplantation or to act as a cardiac protective agent for Dox-treated cancer patients.