Acute myeloid leukemia (AML) is an aggressive clonal disorder of hematopoietic stem cells (HSCs) and primitive progenitors that blocks their myeloid differentiation, generating self-renewing leukemic stem cells (LSCs). Here, we show that the mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human AML and is required for disease initiation as well as propagation in mouse and human AML. YTHDF2 decreases the half-life of diverse m6A transcripts that contribute to the overall integrity of LSC function, including the tumor necrosis factor receptor Tnfrsf2, whose upregulation in Ythdf2-deficient LSCs primes cells for apoptosis. Intriguingly, YTHDF2 is not essential for normal HSC function, with YTHDF2 deficiency actually enhancing HSC activity. Thus, we identify YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion.

YTHDF2 binds and destabilizes N6-methyladenosine (m6A)-modified mRNA. The extent to which this branch of m6A RNA-regulatory pathway functions in vivo and contributes to mammalian development remains unknown. Here we find that YTHDF2 deficiency is partially permissive in mice and results in female-specific infertility. Using conditional mutagenesis, we demonstrate that YTHDF2 is autonomously required within the germline to produce MII oocytes that are competent to sustain early zygotic development. Oocyte maturation is associated with a wave of maternal RNA degradation, and the resulting relative changes to the MII transcriptome are integral to oocyte quality. The loss of YTHDF2 results in the failure to regulate transcript dosage of a cohort of genes during oocyte maturation, with enrichment observed for the YTHDF2-binding consensus and evidence of m6A in these upregulated genes. In summary, the m6A-reader YTHDF2 is an intrinsic determinant of mammalian oocyte competence and early zygotic development.

TUT4 and TUT7 mediate 3′ uridylation of mRNA transcripts, preferentially those with short poly(A) tails; in the absence of TUT4 and TUT7, oocytes cannot mature and female mice are infertile. After fertilization, early embryonic events are dictated by the maternal messenger RNAs stored in the oocyte. Dónal O'Carroll and colleagues show that the appropriate maternal transcriptome is achieved via an essential uridylation process that eliminates unwanted transcripts. The terminal 3′ uridylation is mediated by TUT4/7 complex, and this modification ensures the correct polyadenylation (poly(A)) tail length. In the absence of TUT4/7, oocytes cannot mature, and fertilization is prevented. This study reveals the specific and essential function of poly(A) tail length in sculpting and activating the functional maternal transcriptome. A fundamental principle in biology is that the program for early development is established during oogenesis in the form of the maternal transcriptome1,2. How the maternal transcriptome acquires the appropriate content and dosage of transcripts is not fully understood. Here we show that 3′ terminal uridylation of mRNA mediated by TUT4 and TUT7 sculpts the mouse maternal transcriptome by eliminating transcripts during oocyte growth. Uridylation mediated by TUT4 and TUT7 is essential for both oocyte maturation and fertility. In comparison to somatic cells, the oocyte transcriptome has a shorter poly(A) tail and a higher relative proportion of terminal oligo-uridylation. Deletion of TUT4 and TUT7 leads to the accumulation of a cohort of transcripts with a high frequency of very short poly(A) tails, and a loss of 3′ oligo-uridylation. By contrast, deficiency of TUT4 and TUT7 does not alter gene expression in a variety of somatic cells. In summary, we show that poly(A) tail length and 3′ terminal uridylation have essential and specific functions in shaping a functional maternal transcriptome.

Male fertility requires the continuous production of high quality motile spermatozoa in abundance. Alterations in all three metrics cause oligoasthenoteratozoospermia, the leading cause of human sub/infertility. Post-mitotic spermatogenesis inclusive of several meiotic stages and spermiogenesis (terminal spermatozoa differentiation) are transcriptionally inert, indicating the potential importance for the post-transcriptional microRNA (miRNA) gene-silencing pathway therein. We found the expression of miRNA generating enzyme Dicer within spermatogenesis peaks in meiosis with critical functions in spermatogenesis. In an expression screen we identified two miRNA loci of the miR-34 family (miR-34b/c and miR-449) that are specifically and highly expressed in post-mitotic male germ cells. A reduction in several miRNAs inclusive of miR-34b/c in spermatozoa has been causally associated with reduced fertility in humans. We found that deletion of both miR34b/c and miR-449 loci resulted in oligoasthenoteratozoospermia in mice. MiR-34bc/449-deficiency impairs both meiosis and the final stages of spermatozoa maturation. Analysis of miR-34bc^−/−;449^−/− pachytene spermatocytes revealed a small cohort of genes deregulated that were highly enriched for miR-34 family target genes. Our results identify the miR-34 family as the first functionally important miRNAs for spermatogenesis whose deregulation is causal to oligoasthenoteratozoospermia and infertility. The sustained production of functional motile sperm is critical for male fertility. In recent years, a dramatic increase of cases of male infertility were reported, with the most common cause represented by the production of morphologically abnormal spermatozoa with low motility. Several genetic and environmental factors have been proven to impact on sperm development. In particular, preliminary studies on samples from fertile and sterile individuals suggested that the deregulation of a class of small noncoding RNAs, called microRNAs, might be detrimental for sperm formation. To this end, we investigated the expression of Dicer, a core microRNA pathway component, in male germ cells and observed a peak of expression during meiosis. We performed a microRNA-expression screening and identified 5 members of the miR-34 family (miR-34bc and miR-449abc) as highly expressed from late meiosis to the sperm stage. Deletion of miR-34bc and miR-449 leads to sterility due to the production of abnormal spermatozoa with reduced motility. Thus our work proves for the first time the importance of a microRNA family in sperm formation and male fertility.

The mRNA N⁶-methyladenosine (m⁶A) modification has emerged as an essential regulator of normal and malignant hematopoiesis. Inactivation of the m⁶A mRNA reader YTHDF2, which recognizes m⁶A-modified transcripts to promote m⁶A-mRNA degradation, results in hematopoietic stem cell (HSC) expansion and compromises acute myeloid leukemia. Here we investigate the long-term impact of YTHDF2 deletion on HSC maintenance and multilineage hematopoiesis. We demonstrate that Ythdf2-deficient HSCs from young mice fail upon serial transplantation, display increased abundance of multiple m⁶A-modified inflammation-related transcripts, and chronically activate proinflammatory pathways. Consistent with the detrimental consequences of chronic activation of inflammatory pathways in HSCs, hematopoiesis-specific Ythdf2 deficiency results in a progressive myeloid bias, loss of lymphoid potential, HSC expansion, and failure of aged Ythdf2-deficient HSCs to reconstitute multilineage hematopoiesis. Experimentally induced inflammation increases YTHDF2 expression, and YTHDF2 is required to protect HSCs from this insult. Thus, our study positions YTHDF2 as a repressor of inflammatory pathways in HSCs and highlights the significance of m⁶A in long-term HSC maintenance.