Long noncoding RNAs (lncRNAs) comprise a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins. Recent genome-wide studies in humans and the mouse have annotated lncRNAs expressed in cell lines and adult tissues, but a systematic analysis of lncRNAs expressed during vertebrate embryogenesis has been elusive. To identify lncRNAs with potential functions in vertebrate embryogenesis, we performed a time-series of RNA-seq experiments at eight stages during early zebrafish development. We reconstructed 56,535 high-confidence transcripts in 28,912 loci, recovering the vast majority of expressed RefSeq transcripts while identifying thousands of novel isoforms and expressed loci. We defined a stringent set of 1133 noncoding multi-exonic transcripts expressed during embryogenesis. These include long intergenic ncRNAs (lincRNAs), intronic overlapping lncRNAs, exonic antisense overlapping lncRNAs, and precursors for small RNAs (sRNAs). Zebrafish lncRNAs share many of the characteristics of their mammalian counterparts: relatively short length, low exon number, low expression, and conservation levels comparable to that of introns. Subsets of lncRNAs carry chromatin signatures characteristic of genes with developmental functions. The temporal expression profile of lncRNAs revealed two novel properties: lncRNAs are expressed in narrower time windows than are protein-coding genes and are specifically enriched in early-stage embryos. In addition, several lncRNAs show tissue-specific expression and distinct subcellular localization patterns. Integrative computational analyses associated individual lncRNAs with specific pathways and functions, ranging from cell cycle regulation to morphogenesis. Our study provides the first systematic identification of lncRNAs in a vertebrate embryo and forms the foundation for future genetic, genomic, and evolutionary studies.
After fertilization the embryonic genome is inactive until transcription is initiated during the maternal-zygotic transition 1,2,3 . This transition coincides with the formation of pluripotent cells, which in mammals can be used to generate embryonic stem cells. To study the changes in chromatin structure that accompany pluripotency and genome activation, we mapped the genomic locations of histone H3 molecules bearing Lysine trimethylation modifications before and after the maternalzygotic transition in zebrafish. Trimethylation of Lysine 27, which is repressive, and trimethylation of Lysine 4, which is activating, were not detected before the transition. After genome activation, more than 80% of genes were marked by Lysine 4 trimethylation, including many inactive developmental regulatory genes that were also marked by Lysine 27 trimethylation. Sequential chromatin immunoprecipitation demonstrated that the same promoter regions had both trimethylation marks. Such bivalent chromatin domains also exist in embryonic stem cells and are thought to poise genes for activation while keeping them repressed 4,5,6,7,8 . In addition, we found many inactive genes that were uniquely marked by Lysine 4 trimethylation. Despite this activating modification, these monovalent genes were neither expressed nor stably bound by RNA polymerase II. Inspection of published datasets revealed similar monovalent domains in embryonic stem cells. Moreover, Lysine 4 trimethylation marks could form in the absence of both sequence-specific transcriptional activators and stable association of RNA pol II, as indicated by the analysis of an inducible transgene. These results suggest that bivalent and monovalent domains might poise
The development of animal embryos is initially directed by maternal gene products. Then, during the maternal-to-zygotic transition (MZT), developmental control is handed to the zygotic genome. Extensive research in both vertebrate and invertebrate model organisms has revealed that the MZT can be subdivided into two phases, during which very different modes of gene regulation are implemented: initially, regulation is exclusively post-transcriptional and post-translational, following which gradual activation of the zygotic genome leads to predominance of transcriptional regulation. These changes in the gene expression program of embryos are precisely controlled and highly interconnected. Here, we review current understanding of the mechanisms that underlie handover of developmental control during the MZT.
Small RNA molecules participate in a variety of activities in the cell: in a process known as RNA interference (RNAi), double-stranded RNA triggers the degradation of messenger RNA that has a matching sequence; the small RNA intermediates of this process can also modify gene expression in the nucleus. Here we show that a single episode of RNAi in the nematode Caenorhabditis elegans can induce transcriptional silencing effects that are inherited indefinitely in the absence of the original trigger. Our findings may prove useful in the ongoing development of RNAi to treat disease.
Histone modifications influence the interactions of transcriptional regulators with chromatin. Studies in embryos and embryonic stem (ES) cells have uncovered histone modification patterns that are diagnostic for different cell types and developmental stages. For example, bivalent domains consisting of regions of H3 lysine 27 trimethylation (H3K27me3) and H3 lysine 4 trimethylation (H3K4me3) mark lineage control genes in ES cells and zebrafish blastomeres. Such bivalent domains have garnered attention because the H3K27me3 mark might help repress lineage regulatory genes during pluripotency while the H3K4me3 mark could poise genes for activation upon differentiation. Despite the prominence of the bivalent domain concept, studies in other model organisms have questioned its universal nature and the function of bivalent domains has remained unclear. Histone marks are also associated with developmental regulatory genes in sperm. These observations have raised the possibility that specific histone modification patterns might persist from parent to offspring, but it is unclear whether histone marks are inherited or formed de novo. Here, we review the potential roles of H3K4me3 and H3K27me3 marks in embryos and ES cells and discuss how histone marks might be established, maintained and resolved during embryonic development.
Transposon jumps are a major cause of genome instability. In the C. elegans strain Bristol N2, transposons are active in somatic cells, but they are silenced in the germline, presumably to protect the germline from mutations. Interestingly, the transposon-silencing mechanism shares factors with the RNAi machinery. To better understand the mechanism of transposon silencing, we performed a genome-wide RNAi screen for genes that, when silenced, cause transposition of Tc1 in the C. elegans germline. We identified 27 such genes, among which are mut-16, a mutator that was previously found but not identified at the molecular level, ppw-2, a member of the argonaute family, and several factors that indicate a role for chromatin structure in the regulation of transposition. Some of the newly identified genes are also required for cosuppression and therefore represent the shared components of the two pathways. Since most of the newly identified genes have clear homologs in other species, and since transposons are found from protozoa to human, it seems likely that they also protect other genomes against transposon activity in the germline.
In light microscopy, refractive index mismatches between media and sample cause spherical aberrations that often limit penetration depth and resolution. Optical clearing techniques can alleviate these mismatches, but they are so far limited to fixed samples. We present Iodixanol as a non-toxic medium supplement that allows refractive index matching in live specimens and thus substantially improves image quality in live-imaged primary cell cultures, planarians, zebrafish and human cerebral organoids.DOI: http://dx.doi.org/10.7554/eLife.27240.001
Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer is facilitated by terminal repeat (R) elements in the viral genome. This strand-transfer reaction depends on base pairing between the cDNA of the 59R and the 39R. There is accumulating evidence that retroviral R regions contain features other than sequence complementarity that stimulate this critical nucleic acid hybridization step. The R region of the human immunodeficiency virus type 1 (HIV-1) is relatively extended (97 nt) and encodes two wellconserved stem-loop structures, the TAR and poly(A) hairpins. The role of these motifs was studied in an in vitro strand-transfer assay with two separate templates, the 59R donor and the 39R acceptor, and mutants thereof. The results indicate that the upper part of the TAR hairpin structure in the 59R donor is critical for efficient strand transfer. This seems to pose a paradox, as the 59R template is degraded by RNase H before strand transfer occurs. We propose that it is not the RNA hairpin motif in the 59R donor, but rather the antisense motif in the ssDNA copy, which can also fold a hairpin structure, that is critical for strand transfer. Mutation of the loop sequence in the TAR hairpin of the donor RNA, which is copied in the loop of the cDNA hairpin, reduces the transfer efficiency more than fivefold. It is proposed that the natural strand-transfer reaction is enhanced by interaction of the anti-TAR ssDNA hairpin with the TAR hairpin in the 39R acceptor. Base pairing can occur between the complementary loops ("loop-loop kissing"), and strand transfer is completed by the subsequent formation of an extended RNA-cDNA duplex.
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