Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate.

Nonsense-mediated RNA decay (NMD) is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs) demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages

A microRNA cluster in the Fragile-X region expressed during spermatogenesis targets FMR1.

Testis-expressed X-linked genes typically evolve rapidly. Here, we report on a testis-expressed X-linked microRNA (miRNA) cluster that despite rapid alterations in sequence has retained its position in the Fragile-X region of the X chromosome in placental mammals. Surprisingly, the miRNAs encoded by this cluster (Fx-mir) have a predilection for targeting the immediately adjacent gene, Fmr1, an unexpected finding given that miRNAs usually act in trans, not in cis Robust repression of Fmr1 is conferred by combinations of Fx-mir miRNAs induced in Sertoli cells (SCs) during postnatal development when they terminate proliferation. Physiological significance is suggested by the finding that FMRP, the protein product of Fmr1, is downregulated when Fx-mir miRNAs are induced, and that FMRP loss causes SC hyperproliferation and spermatogenic defects. Fx-mir miRNAs not only regulate the expression of FMRP, but also regulate the expression of eIF4E and CYFIP1, which together with FMRP form a translational regulatory complex. Our results support a model in which Fx-mir family members act cooperatively to regulate the translation of batteries of mRNAs in a developmentally regulated manner in SCs

Molecular Determinants of Oocyte and Embryo Developmental Competence

The earliest stages of life, including the transition from the fully differentiated oocyte to the totipotent zygote, the first days of embryo cleavage and cell differentiation to form a blastocyst, and implantation of that blastocyst in the wall of the uterus, are somehow beautifully simple and strikingly complex at the same time. These stages, which represent the beginning of life for us and other vertebrates, are difficult to study, owing to a number of experimental and ethical considerations. However, advances in our understanding of nuclear reprogramming, transcriptional quiescence and activation, the maintenance of pluripotency, and what it takes for an embryo to initiate contact with the endometrium to generate a successful pregnancy, will without question have far-reaching influence for many scientific and medical disciplines. Here, I attempt to unravel some of these concepts, combining molecular and developmental biology with genome-wide analysis only recently made possible through advances in techniques with low input. These approaches, in combination, allow us to ask questions about early developmental systems that would not have been possible only years prior.In the oocyte, global transcriptional silencing is a highly conserved mechanism that is essential for the transition from the differentiated oocyte to the totipotent zygote. Here, I report that global transcriptional silencing in the mouse oocyte depends on an mRNA decay activator. By downregulating master regulators of transcription during oocyte growth, particularly a group of mRNAs encoding demethylases for H3K4 and H3K9, ZFP36L2 enables increased histone methylation that is associated with transcriptional silencing. These results uncover a mRNA decay mechanism that acts a developmental switch during growth of the mammalian oocyte, resulting in wide-spread shifts in chromatin modification, and mediating silencing of transcription in the oocyte.The pluripotent population of cells in the blastocyst, the inner cell mass, is established in the mouse embryo approximately three days after fertilization. These cells will undergo gastrulation to form the entire organism and can be maintained in culture as embryonic stem cells. I report here that UPF2, a mRNA decay activator, is needed specifically within the pluripotent inner cell mass of the mouse embryo for maintenance of pluripotency in the embryo in vivo, as well as for embryonic stem cells in vitro. That mRNA decay may underly the establishment or maintenance of this intriguing and complex population of cells, is an exciting possibility.Reproductive success depends on embryo implantation in the uterus, and in fertile and infertile couples alike, failure of the embryo to implant in the wall of the uterus accounts for up to 75% of all lost pregnancies. Here, I provide a qualitative assessment of gene expression and cellular communication networks within the major compartments of the human blastocyst that are most closely associated with successful implantation and ongoing pregnancy. Most strikingly, establishment and/or maintenance of the extraembryonic primitive endoderm lineage—following the second major embryonic differentiation event—most strongly differentiates embryos of high and low implantation potential. Unbiased machine learning identified genes within each embryo compartment most closely associated with implantation. Taken together, this data supports a model in which successful implantation and ongoing pregnancy predominantly depends upon the inner cell mass and highlights a potentially novel role for the extraembryonic primitive endoderm in early pregnancy success

Miniaturization Technologies for Efficient Single-Cell Library Preparation for Next-Generation Sequencing.

As the cost of next-generation sequencing has decreased, library preparation costs have become a more significant proportion of the total cost, especially for high-throughput applications such as single-cell RNA profiling. Here, we have applied novel technologies to scale down reaction volumes for library preparation. Our system consisted of in vitro differentiated human embryonic stem cells representing two stages of pancreatic differentiation, for which we prepared multiple biological and technical replicates. We used the Fluidigm (San Francisco, CA) C1 single-cell Autoprep System for single-cell complementary DNA (cDNA) generation and an enzyme-based tagmentation system (Nextera XT; Illumina, San Diego, CA) with a nanoliter liquid handler (mosquito HTS; TTP Labtech, Royston, UK) for library preparation, reducing the reaction volume down to 2 µL and using as little as 20 pg of input cDNA. The resulting sequencing data were bioinformatically analyzed and correlated among the different library reaction volumes. Our results showed that decreasing the reaction volume did not interfere with the quality or the reproducibility of the sequencing data, and the transcriptional data from the scaled-down libraries allowed us to distinguish between single cells. Thus, we have developed a process to enable efficient and cost-effective high-throughput single-cell transcriptome sequencing

Chromatin Modification and Global Transcriptional Silencing in the Oocyte Mediated by the mRNA Decay Activator ZFP36L2.

Global transcriptional silencing is a highly conserved mechanism central to the oocyte-to-embryo transition. We report the unexpected discovery that global transcriptional silencing in oocytes depends on an mRNA decay activator. Oocyte-specific loss of ZFP36L2 an RNA-binding protein that promotes AU-rich element-dependent mRNA decay prevents global transcriptional silencing and causes oocyte maturation and fertilization defects, as well as complete female infertility in the mouse. Single-cell RNA sequencing revealed that ZFP36L2 downregulates mRNAs encoding transcription and chromatin modification regulators, including a large group of mRNAs for histone demethylases targeting H3K4 and H3K9, which we show are bound and degraded by ZFP36L2. Oocytes lacking Zfp36l2 fail to accumulate histone methylation at H3K4 and H3K9, marks associated with the transcriptionally silent, developmentally competent oocyte state. Our results uncover a ZFP36L2-dependent mRNA decay mechanism that acts as a developmental switch during oocyte growth, triggering wide-spread shifts in chromatin modification and global transcription

The role of the NMD factor UPF3B in olfactory sensory neurons.

The UPF3B-dependent branch of the nonsense-mediated RNA decay (NMD) pathway is critical for human cognition. Here, we examined the role of UPF3B in the olfactory system. Single-cell RNA-sequencing (scRNA-seq) analysis demonstrated considerable heterogeneity of olfactory sensory neuron (OSN) cell populations in wild-type (WT) mice, and revealed that UPF3B loss influences specific subsets of these cell populations. UPF3B also regulates the expression of a large cadre of antimicrobial genes in OSNs, and promotes the selection of specific olfactory receptor (Olfr) genes for expression in mature OSNs (mOSNs). RNA-seq and Ribotag analyses identified classes of mRNAs expressed and translated at different levels in WT and Upf3b-null mOSNs. Integrating multiple computational approaches, UPF3B-dependent NMD target transcripts that are candidates to mediate the functions of NMD in mOSNs were identified in vivo. Together, our data provides a valuable resource for the olfactory field and insights into the roles of NMD in vivo

The Antagonistic Gene Paralogs Upf3a and Upf3b Govern Nonsense-Mediated RNA Decay

Gene duplication is a major evolutionary force driving adaptation and speciation, as it allows for the acquisition of new functions and can augment or diversify existing functions. Here, we report a gene duplication event that yielded another outcome--the generation of antagonistic functions. One product of this duplication event--UPF3B--is critical for the nonsense-mediated RNA decay (NMD) pathway, while its autosomal counterpart--UPF3A--encodes an enigmatic protein previously shown to have trace NMD activity. Using loss-of-function approaches in vitro and in vivo, we discovered that UPF3A acts primarily as a potent NMD inhibitor that stabilizes hundreds of transcripts. Evidence suggests that UPF3A acquired repressor activity through simple impairment of a critical domain, a rapid mechanism that may have been widely used in evolution. Mice conditionally lacking UPF3A exhibit "hyper" NMD and display defects in embryogenesis and gametogenesis. Our results support a model in which UPF3A serves as a molecular rheostat that directs developmental events

The Antagonistic Gene Paralogs Upf3a and Upf3b Govern Nonsense-Mediated RNA Decay

Gene duplication is a major evolutionary force driving adaptation and speciation, as it allows for the acquisition of new functions and can augment or diversify existing functions. Here, we report a gene duplication event that yielded another outcome – the generation of antagonistic functions. One product of this duplication event – UPF3B – is critical for the nonsense-mediated RNA decay (NMD) pathway, while its autosomal counterpart – UPF3A – encodes an enigmatic protein previously shown to have trace NMD activity. Using loss-of-function approaches in vitro and in vivo, we discovered that UPF3A acts primarily as a potent NMD inhibitor that stabilizes hundreds of transcripts. Evidence suggests that UPF3A acquired repressor activity through simple impairment of a critical domain, a rapid mechanism that may have been widely used in evolution. Mice conditionally lacking UPF3A exhibit “hyper” NMD and display defects in embryogenesis and gametogenesis, consistent with UPF3A serving as a molecular rheostat that directs developmental events