SMG6 localizes to the chromatoid body and shapes the male germ cell transcriptome to drive spermatogenesis.

Nonsense-mediated RNA decay (NMD) is a highly conserved and selective RNA turnover pathway that depends on the endonuclease SMG6. Here, we show that SMG6 is essential for male germ cell differentiation in mice. Germ-cell conditional knockout (cKO) of Smg6 induces extensive transcriptome misregulation, including a failure to eliminate meiotically expressed transcripts in early haploid cells, and accumulation of NMD target mRNAs with long 3' untranslated regions (UTRs). Loss of SMG6 in the male germline results in complete arrest of spermatogenesis at the early haploid cell stage. We find that SMG6 is strikingly enriched in the chromatoid body (CB), a specialized cytoplasmic granule in male germ cells also harboring PIWI-interacting RNAs (piRNAs) and the piRNA-binding protein PIWIL1. This raises the possibility that SMG6 and the piRNA pathway function together, which is supported by several findings, including that Piwil1-KO mice phenocopy Smg6-cKO mice and that SMG6 and PIWIL1 co-regulate many genes in round spermatids. Together, our results demonstrate that SMG6 is an essential regulator of the male germline transcriptome, and highlight the CB as a molecular platform coordinating RNA regulatory pathways to control sperm production and fertility.

The Neonatal and Adult Human Testis Defined at the Single-Cell Level.

Spermatogenesis has been intensely studied in rodents but remains poorly understood in humans. Here, we used single-cell RNA sequencing to analyze human testes. Clustering analysis of neonatal testes reveals several cell subsets, including cell populations with characteristics of primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). In adult testes, we identify four undifferentiated spermatogonia (SPG) clusters, each of which expresses specific marker genes. We identify protein markers for the most primitive SPG state, allowing us to purify this likely SSC-enriched cell subset. We map the timeline of male germ cell development from PGCs through fetal germ cells to differentiating adult SPG stages. We also define somatic cell subsets in both neonatal and adult testes and trace their developmental trajectories. Our data provide a blueprint of the developing human male germline and supporting somatic cells. The PGC-like and SSC markers are candidates to be used for SSC therapy to treat infertility.

Attenuated Codon Optimality Contributes to Neural-Specific mRNA Decay in Drosophila.

Tissue-specific mRNA stability is important for cell fate and physiology, but the mechanisms involved are not fully understood. We found that zygotic mRNA stability in Drosophila correlates with codon content: optimal codons are enriched in stable transcripts associated with metabolic functions like translation, while non-optimal codons are enriched in unstable transcripts, including those associated with neural development. Bioinformatic analyses and reporter assays revealed that similar codons stabilize or destabilize mRNAs in the nervous system and other tissues, but the link between codon content and stability is attenuated in the nervous system. We confirmed that optimal codons are decoded by abundant tRNAs while non-optimal codons are decoded by less abundant tRNAs in embryos and in the nervous system. We conclude that codon optimality is a general determinant of zygotic mRNA stability, and attenuation of codon optimality allows trans-acting factors to exert greater influence over mRNA decay in the nervous system.

EC-tagging allows cell type-specific RNA analysis.

Purification of cell type-specific RNAs remains a significant challenge. One solution involves biosynthetic tagging of target RNAs. RNA tagging via incorporation of 4-thiouracil (TU) in cells expressing transgenic uracil phosphoribosyltransferase (UPRT), a method known as TU-tagging, has been used in multiple systems but can have limited specificity due to endogenous pathways of TU incorporation. Here, we describe an alternative method that requires the activity of two enzymes: cytosine deaminase (CD) and UPRT. We found that the sequential activity of these enzymes converts 5-ethynylcytosine (EC) to 5-ethynyluridine monophosphate that is subsequently incorporated into nascent RNAs. The ethynyl group allows efficient detection and purification of tagged RNAs. We show that 'EC-tagging' occurs in tissue culture cells and Drosophila engineered to express CD and UPRT. Additional control can be achieved through a split-CD approach in which functional CD is reconstituted from independently expressed fragments. We demonstrate the sensitivity and specificity of EC-tagging by obtaining cell type-specific gene expression data from intact Drosophila larvae, including transcriptome measurements from a small population of central brain neurons. EC-tagging provides several advantages over existing techniques and should be broadly useful for investigating the role of differential RNA expression in cell identity, physiology and pathology.

Dynamic regulation of mRNA decay during neural development.

BACKGROUND: Gene expression patterns are determined by rates of mRNA transcription and decay. While transcription is known to regulate many developmental processes, the role of mRNA decay is less extensively defined. A critical step toward defining the role of mRNA decay in neural development is to measure genome-wide mRNA decay rates in neural tissue. Such information should reveal the degree to which mRNA decay contributes to differential gene expression and provide a foundation for identifying regulatory mechanisms that affect neural mRNA decay. RESULTS: We developed a technique that allows genome-wide mRNA decay measurements in intact Drosophila embryos, across all tissues and specifically in the nervous system. Our approach revealed neural-specific decay kinetics, including stabilization of transcripts encoding regulators of axonogenesis and destabilization of transcripts encoding ribosomal proteins and histones. We also identified correlations between mRNA stability and physiologic properties of mRNAs; mRNAs that are predicted to be translated within axon growth cones or dendrites have long half-lives while mRNAs encoding transcription factors that regulate neurogenesis have short half-lives. A search for candidate cis-regulatory elements identified enrichment of the Pumilio recognition element (PRE) in mRNAs encoding regulators of neurogenesis. We found that decreased expression of the RNA-binding protein Pumilio stabilized predicted neural mRNA targets and that a PRE is necessary to trigger reporter-transcript decay in the nervous system. CONCLUSIONS: We found that differential mRNA decay contributes to the relative abundance of transcripts involved in cell-fate decisions, axonogenesis, and other critical events during Drosophila neural development. Neural-specific decay kinetics and the functional specificity of mRNA decay suggest the existence of a dynamic neurodevelopmental mRNA decay network. We found that Pumilio is one component of this network, revealing a novel function for this RNA-binding protein.

Miz-1 activates gene expression via a novel consensus DNA binding motif.

The transcription factor Miz-1 can either activate or repress gene expression in concert with binding partners including the Myc oncoprotein. The genomic binding of Miz-1 includes both core promoters and more distal sites, but the preferred DNA binding motif of Miz-1 has been unclear. We used a high-throughput in vitro technique, Bind-n-Seq, to identify two Miz-1 consensus DNA binding motif sequences--ATCGGTAATC and ATCGAT (Mizm1 and Mizm2)--bound by full-length Miz-1 and its zinc finger domain, respectively. We validated these sequences directly as high affinity Miz-1 binding motifs. Competition assays using mutant probes indicated that the binding affinity of Miz-1 for Mizm1 and Mizm2 is highly sequence-specific. Miz-1 strongly activates gene expression through the motifs in a Myc-independent manner. MEME-ChIP analysis of Miz-1 ChIP-seq data in two different cell types reveals a long motif with a central core sequence highly similar to the Mizm1 motif identified by Bind-n-Seq, validating the in vivo relevance of the findings. Miz-1 ChIP-seq peaks containing the long motif are predominantly located outside of proximal promoter regions, in contrast to peaks without the motif, which are highly concentrated within 1.5 kb of the nearest transcription start site. Overall, our results indicate that Miz-1 may be directed in vivo to the novel motif sequences we have identified, where it can recruit its specific binding partners to control gene expression and ultimately regulate cell fate.