Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediating PHAS precursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

Reproductive phasiRNAs are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely (i) not triggered by microRNAs, (ii) not loaded by AGO18 proteins, and (iii) not capable of mediating cis-cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

Lost in space: what single-cell RNA sequencing cannot tell you.

Plant scientists are rapidly integrating single-cell RNA sequencing (scRNA-seq) into their workflows. Maximizing the potential of scRNA-seq requires a proper understanding of the spatiotemporal context of cells. However, positional information is inherently lost during scRNA-seq, limiting its potential to characterize complex biological systems. In this review we highlight how current single-cell analysis pipelines cannot completely recover spatial information, which confounds biological interpretation. Various strategies exist to identify the location of RNA, from classical RNA in situ hybridization to spatial transcriptomics. Herein we discuss the possibility of utilizing this spatial information to supervise single-cell analyses. An integrative approach will maximize the potential of each technology, and lead to insights which go beyond the capability of each individual technology.

Bookend: precise transcript reconstruction with end-guided assembly.

We developed Bookend, a package for transcript assembly that incorporates data from different RNA-seq techniques, with a focus on identifying and utilizing RNA 5' and 3' ends. We demonstrate that correct identification of transcript start and end sites is essential for precise full-length transcript assembly. Utilization of end-labeled reads present in full-length single-cell RNA-seq datasets dramatically improves the precision of transcript assembly in single cells. Finally, we show that hybrid assembly across short-read, long-read, and end-capture RNA-seq datasets from Arabidopsis thaliana, as well as meta-assembly of RNA-seq from single mouse embryonic stem cells, can produce reference-quality end-to-end transcript annotations.

Innate, translation-dependent silencing of an invasive transposon in Arabidopsis.

Co-evolution between hosts' and parasites' genomes shapes diverse pathways of acquired immunity based on silencing small (s)RNAs. In plants, sRNAs cause heterochromatinization, sequence degeneration, and, ultimately, loss of autonomy of most transposable elements (TEs). Recognition of newly invasive plant TEs, by contrast, involves an innate antiviral-like silencing response. To investigate this response's activation, we studied the single-copy element EVADÉ (EVD), one of few representatives of the large Ty1/Copia family able to proliferate in Arabidopsis when epigenetically reactivated. In Ty1/Copia elements, a short subgenomic mRNA (shGAG) provides the necessary excess of structural GAG protein over the catalytic components encoded by the full-length genomic flGAG-POL. We show here that the predominant cytosolic distribution of shGAG strongly favors its translation over mostly nuclear flGAG-POL. During this process, an unusually intense ribosomal stalling event coincides with mRNA breakage yielding unconventional 5'OH RNA fragments that evade RNA quality control. The starting point of sRNA production by RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6), exclusively on shGAG, occurs precisely at this breakage point. This hitherto-unrecognized "translation-dependent silencing" (TdS) is independent of codon usage or GC content and is not observed on TE remnants populating the Arabidopsis genome, consistent with their poor association, if any, with polysomes. We propose that TdS forms a primal defense against EVD de novo invasions that underlies its associated sRNA pattern.

Gene expression variation in Arabidopsis embryos at single-nucleus resolution.

Soon after fertilization of egg and sperm, plant genomes become transcriptionally activated and drive a series of coordinated cell divisions to form the basic body plan during embryogenesis. Early embryonic cells rapidly diversify from each other, and investigation of the corresponding gene expression dynamics can help elucidate underlying cellular differentiation programs. However, current plant embryonic transcriptome datasets either lack cell-specific information or have RNA contamination from surrounding non-embryonic tissues. We have coupled fluorescence-activated nuclei sorting together with single-nucleus mRNA-sequencing to construct a gene expression atlas of Arabidopsis thaliana early embryos at single-cell resolution. In addition to characterizing cell-specific transcriptomes, we found evidence that distinct epigenetic and transcriptional regulatory mechanisms operate across emerging embryonic cell types. These datasets and analyses, as well as the approach we devised, are expected to facilitate the discovery of molecular mechanisms underlying pattern formation in plant embryos. This article has an associated 'The people behind the papers' interview.

MicroRNA Dynamics and Functions During Arabidopsis Embryogenesis.

MicroRNAs (miRNAs) are short noncoding RNAs that mediate the repression of target transcripts in plants and animals. Although miRNAs are required throughout plant development, relatively little is known regarding their embryonic functions. To systematically characterize embryonic miRNAs in Arabidopsis (Arabidopsis thaliana), we developed or applied high-throughput sequencing-based methods to profile hundreds of miRNAs and associated targets throughout embryogenesis. We discovered dozens of miRNAs that dynamically cleave and repress target transcripts, including 30 that encode transcription factors. Transcriptome analyses indicated that these miRNA:target interactions have profound effects on embryonic gene expression programs. Moreover, we demonstrated that the miRNA-mediated repression of six transcription factors are individually required for proper division patterns of various embryonic cell lineages. These data indicate that the miRNA-directed repression of multiple transcription factors is critically important for the establishment of the plant body plan, and they provide a foundation to further investigate how miRNAs contribute to these initial cellular differentiation events.

The embryonic transcriptome of Arabidopsis thaliana.

KEY MESSAGE: Arabidopsis embryos possess unique transcriptomes relative to other plant tissues including somatic embryos, and can be partitioned into four transcriptional phases with characteristic biological processes. Cellular differentiation is associated with changes in transcript populations. Accurate quantification of transcriptomes during development can thus provide global insights into differentiation processes including the fundamental specification and differentiation events operating during plant embryogenesis. However, multiple technical challenges have limited the ability to obtain high-quality early embryonic transcriptomes, namely the low amount of RNA obtainable and contamination from surrounding endosperm and seed-coat tissues. We compared the performance of three low-input mRNA sequencing (mRNA-seq) library preparation kits on 0.1 to 5 nanograms (ng) of total RNA isolated from Arabidopsis thaliana (Arabidopsis) embryos and identified a low-cost method with superior performance. This mRNA-seq method was then used to profile the transcriptomes of Arabidopsis embryos across eight developmental stages. By comprehensively comparing embryonic and post-embryonic transcriptomes, we found that embryonic transcriptomes do not resemble any other plant tissue we analyzed. Moreover, transcriptome clustering analyses revealed the presence of four distinct phases of embryogenesis which are enriched in specific biological processes. We also compared zygotic embryo transcriptomes with publicly available somatic embryo transcriptomes. Strikingly, we found little resemblance between zygotic embryos and somatic embryos derived from late-staged zygotic embryos suggesting that somatic and zygotic embryo transcriptomes are distinct from each other. In addition to the biological insights gained from our systematic characterization of the Arabidopsis embryonic transcriptome, we provide a data-rich resource for the community to explore.

NanoPARE: parallel analysis of RNA 5' ends from low-input RNA.

Diverse RNA 5' ends are generated through both transcriptional and post-transcriptional processes. These important modes of gene regulation often vary across cell types and can contribute to the diversification of transcriptomes and thus cellular differentiation. Therefore, the identification of primary and processed 5' ends of RNAs is important for their functional characterization. Methods have been developed to profile either RNA 5' ends from primary transcripts or the products of RNA degradation genome-wide. However, these approaches either require high amounts of starting RNA or are performed in the absence of paired gene-body mRNA-seq data. This limits current efforts in RNA 5' end annotation to whole tissues and can prevent accurate RNA 5' end classification due to biases in the data sets. To enable the accurate identification and precise classification of RNA 5' ends from standard and low-input RNA, we developed a next-generation sequencing-based method called nanoPARE and associated software. By integrating RNA 5' end information from nanoPARE with gene-body mRNA-seq data from the same RNA sample, our method enables the identification of transcription start sites at single-nucleotide resolution from single-cell levels of total RNA, as well as small RNA-mediated cleavage events from at least 10,000-fold less total RNA compared to conventional approaches. NanoPARE can therefore be used to accurately profile transcription start sites, noncapped RNA 5' ends, and small RNA targeting events from individual tissue types. As a proof-of-principle, we utilized nanoPARE to improve Arabidopsis thaliana RNA 5' end annotations and quantify microRNA-mediated cleavage events across five different flower tissues.

Widespread Contamination of Arabidopsis Embryo and Endosperm Transcriptome Data Sets.

A major goal of global gene expression profiling in plant seeds has been to investigate the parental contributions to the transcriptomes of early embryos and endosperm. However, consistency between independent studies has been poor, leading to considerable debate. We have developed a statistical tool that reveals the presence of substantial RNA contamination from maternal tissues in nearly all published Arabidopsis thaliana endosperm and early embryo transcriptomes generated in these studies. We demonstrate that maternal RNA contamination explains the poor reproducibility of these transcriptomic data sets. Furthermore, we found that RNA contamination from maternal tissues has been repeatedly misinterpreted as epigenetic phenomena, which has resulted in inaccurate conclusions regarding the parental contributions to both the endosperm and early embryo transcriptomes. After accounting for maternal RNA contamination, no published genome-wide data set supports the concept of delayed paternal genome activation in plant embryos. Moreover, our analysis suggests that maternal and paternal genomic imprinting are equally rare events in Arabidopsis endosperm. Our publicly available software (https://github.com/Gregor-Mendel-Institute/tissue-enrichment-test) can help the community assess the level of contamination in transcriptome data sets generated from both seed and non-seed tissues.

Diverse Roles of Axonemal Dyneins in Drosophila Auditory Neuron Function and Mechanical Amplification in Hearing.

Much like vertebrate hair cells, the chordotonal sensory neurons that mediate hearing in Drosophila are motile and amplify the mechanical input of the ear. Because the neurons bear mechanosensory primary cilia whose microtubule axonemes display dynein arms, we hypothesized that their motility is powered by dyneins. Here, we describe two axonemal dynein proteins that are required for Drosophila auditory neuron function, localize to their primary cilia, and differently contribute to mechanical amplification in hearing. Promoter fusions revealed that the two axonemal dynein genes Dmdnah3 (=CG17150) and Dmdnai2 (=CG6053) are expressed in chordotonal neurons, including the auditory ones in the fly's ear. Null alleles of both dyneins equally abolished electrical auditory neuron responses, yet whereas mutations in Dmdnah3 facilitated mechanical amplification, amplification was abolished by mutations in Dmdnai2. Epistasis analysis revealed that Dmdnah3 acts downstream of Nan-Iav channels in controlling the amplificatory gain. Dmdnai2, in addition to being required for amplification, was essential for outer dynein arms in auditory neuron cilia. This establishes diverse roles of axonemal dyneins in Drosophila auditory neuron function and links auditory neuron motility to primary cilia and axonemal dyneins. Mutant defects in sperm competition suggest that both dyneins also function in sperm motility.

Physiological, anatomical, and behavioral changes after acoustic trauma in Drosophila melanogaster.

Noise-induced hearing loss (NIHL) is a growing health issue, with costly treatment and lost quality of life. Here we establish Drosophila melanogaster as an inexpensive, flexible, and powerful genetic model system for NIHL. We exposed flies to acoustic trauma and quantified physiological and anatomical effects. Trauma significantly reduced sound-evoked potential (SEP) amplitudes and increased SEP latencies in control genotypes. SEP amplitude but not latency effects recovered after 7 d. Although trauma produced no gross morphological changes in the auditory organ (Johnston's organ), mitochondrial cross-sectional area was reduced 7 d after exposure. In nervana 3 heterozygous flies, which slightly compromise ion homeostasis, trauma had exaggerated effects on SEP amplitude and mitochondrial morphology, suggesting a key role for ion homeostasis in resistance to acoustic trauma. Thus, Drosophila exhibit acoustic trauma effects resembling those found in vertebrates, including inducing metabolic stress in sensory cells. This report of noise trauma in Drosophila is a foundation for studying molecular and genetic sequelae of NIHL.