UPF1 regulates mRNA stability by sensing poorly translated coding sequences.

Post-transcriptional mRNA regulation shapes gene expression, yet how cis-elements and mRNA translation interface to regulate mRNA stability is poorly understood. We find that the strength of translation initiation, upstream open reading frame (uORF) content, codon optimality, AU-rich elements, microRNA binding sites, and open reading frame (ORF) length function combinatorially to regulate mRNA stability. Machine-learning analysis identifies ORF length as the most important conserved feature regulating mRNA decay. We find that Upf1 binds poorly translated and untranslated ORFs, which are associated with a higher decay rate, including mRNAs with uORFs and those with exposed ORFs after stop codons. Our study emphasizes Upf1's converging role in surveilling mRNAs with exposed ORFs that are poorly translated, such as mRNAs with long ORFs, ORF-like 3' UTRs, and mRNAs containing uORFs. We propose that Upf1 regulation of poorly/untranslated ORFs provides a unifying mechanism of surveillance in regulating mRNA stability and homeostasis in an exon-junction complex (EJC)-independent nonsense-mediated decay (NMD) pathway that we term ORF-mediated decay (OMD).

Chromatin expansion microscopy reveals nanoscale organization of transcription and chromatin.

Nanoscale chromatin organization regulates gene expression. Although chromatin is notably reprogrammed during zygotic genome activation (ZGA), the organization of chromatin regulatory factors during this universal process remains unclear. In this work, we developed chromatin expansion microscopy (ChromExM) to visualize chromatin, transcription, and transcription factors in vivo. ChromExM of embryos during ZGA revealed how the pioneer factor Nanog interacts with nucleosomes and RNA polymerase II (Pol II), providing direct visualization of transcriptional elongation as string-like nanostructures. Blocking elongation led to more Pol II particles clustered around Nanog, with Pol II stalled at promoters and Nanog-bound enhancers. This led to a new model termed "kiss and kick", in which enhancer-promoter contacts are transient and released by transcriptional elongation. Our results demonstrate that ChromExM is broadly applicable to study nanoscale nuclear organization.

The scientific director: A complimentary model for academic leadership.

The current model for academic leadership places unique demands on scientists with highly active research programs. A complimentary model with a dedicated scientific director could remove this strain and allow a greater institutional investment in the community via a partnership. This article explores the rationale and framework of this model.

linc-mipep and linc-wrb encode micropeptides that regulate chromatin accessibility in vertebrate-specific neural cells.

Thousands of long intergenic non-coding RNAs (lincRNAs) are transcribed throughout the vertebrate genome. A subset of lincRNAs enriched in developing brains have recently been found to contain cryptic open-reading frames and are speculated to encode micropeptides. However, systematic identification and functional assessment of these transcripts have been hindered by technical challenges caused by their small size. Here, we show that two putative lincRNAs (linc-mipep, also called lnc-rps25, and linc-wrb) encode micropeptides with homology to the vertebrate-specific chromatin architectural protein, Hmgn1, and demonstrate that they are required for development of vertebrate-specific brain cell types. Specifically, we show that NMDA receptor-mediated pathways are dysregulated in zebrafish lacking these micropeptides and that their loss preferentially alters the gene regulatory networks that establish cerebellar cells and oligodendrocytes - evolutionarily newer cell types that develop postnatally in humans. These findings reveal a key missing link in the evolution of vertebrate brain cell development and illustrate a genetic basis for how some neural cell types are more susceptible to chromatin disruptions, with implications for neurodevelopmental disorders and disease.

Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing.

RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

Stage Specific Transcriptomic Analysis and Database for Zebrafish Oogenesis.

Oogenesis produces functional eggs and is essential for fertility, embryonic development, and reproduction. The zebrafish ovary is an excellent model to study oogenesis in vertebrates, and recent studies have identified multiple regulators in oocyte development through forward genetic screens, as well as reverse genetics by CRISPR mutagenesis. However, many developmental steps in oogenesis, in zebrafish and other species, remain poorly understood, and their underlying mechanisms are unknown. Here, we take a genomic approach to systematically uncover biological activities throughout oogenesis. We performed transcriptomic analysis on five stages of oogenesis, from the onset of oocyte differentiation through Stage III, which precedes oocyte maturation. These transcriptomes revealed thousands of differentially expressed genes across stages of oogenesis. We analyzed trends of gene expression dynamics along oogenesis, as well as their expression in pair-wise comparisons between stages. We determined their functionally enriched terms, identifying uniquely characteristic biological activities in each stage. These data identified two prominent developmental phases in oocyte differentiation and traced the accumulation of maternally deposited embryonic regulator transcripts in the developing oocyte. Our analysis provides the first molecular description for oogenesis in zebrafish, which we deposit online as a resource for the community. Further, the presence of multiple gene paralogs in zebrafish, and the exclusive curation by many bioinformatic tools of the single paralogs present in humans, challenge zebrafish genomic analyses. We offer an approach for converting zebrafish gene name nomenclature to the human nomenclature for supporting genomic analyses generally in zebrafish. Altogether, our work provides a valuable resource as a first step to uncover oogenesis mechanisms and candidate regulators and track accumulating transcripts of maternal regulators of embryonic development.

A group approach to growing as a principal investigator.

Greco et al. describe their experience learning to be more effective and humane PIs. The key to their growth was regular and consistent work with a diverse group of their peers aided by the guidance of an organizational psychologist.

Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes.

The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. Here we study the nuclease activity of SpG and SpRY by targeting 40 sites in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm could help to predict SpG and SpRY targets with high activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.

The landscape of pioneer factor activity reveals the mechanisms of chromatin reprogramming and genome activation.

Upon fertilization, embryos undergo chromatin reprogramming and genome activation; however, the mechanisms that regulate these processes are poorly understood. Here, we generated a triple mutant for Nanog, Pou5f3, and Sox19b (NPS) in zebrafish and found that NPS pioneer chromatin opening at >50% of active enhancers. NPS regulate acetylation across core histones at enhancers and promoters, and their function in gene activation can be bypassed by recruiting histone acetyltransferase to individual genes. NPS pioneer chromatin opening individually, redundantly, or additively depending on sequence context, and we show that high nucleosome occupancy facilitates NPS pioneering activity. Nucleosome position varies based on the input of different transcription factors (TFs), providing a flexible platform to modulate pioneering activity. Altogether, our results illuminate the sequence of events during genome activation and offer a conceptual framework to understand how pioneer factors interpret the genome and integrate different TF inputs across cell types and developmental transitions.

A proteomics approach identifies novel resident zebrafish Balbiani body proteins Cirbpa and Cirbpb.

The Balbiani body (Bb) is the first marker of polarity in vertebrate oocytes. The Bb is a conserved structure found in diverse animals including insects, fish, amphibians, and mammals. During early zebrafish oogenesis, the Bb assembles as a transient aggregate of mRNA, proteins, and membrane-bound organelles at the presumptive vegetal side of the oocyte. As the early oocyte develops, the Bb appears to grow slowly, until at the end of stage I of oogenesis it disassembles and deposits its cargo of localized mRNAs and proteins. In fish and frogs, this cargo includes the germ plasm as well as gene products required to specify dorsal tissues of the future embryo. We demonstrate that the Bb is a stable, solid structure that forms a size exclusion barrier similar to other biological hydrogels. Despite its central role in oocyte polarity, little is known about the mechanism behind the Bb's action. Analysis of the few known protein components of the Bb is insufficient to explain how the Bb assembles, translocates, and disassembles. We isolated Bbs from zebrafish oocytes and performed mass spectrometry to define the Bb proteome. We successfully identified 77 proteins associated with the Bb sample, including known Bb proteins and novel RNA-binding proteins. In particular, we identified Cirbpa and Cirbpb, which have both an RNA-binding domain and a predicted self-aggregation domain. In stage I oocytes, Cirbpa and Cirbpb localize to the Bb rather than the nucleus (as in somatic cells), indicating that they may have a specialized function in the germ line. Both the RNA-binding domain and the self-aggregation domain are sufficient to localize to the Bb, suggesting that Cirbpa and Cirbpb interact with more than just their mRNA targets within the Bb. We propose that Cirbp proteins crosslink mRNA cargo and proteinaceous components of the Bb as it grows. Beyond Cirbpa and Cirbpb, our proteomics dataset presents many candidates for further study, making it a valuable resource for building a comprehensive mechanism for Bb function at a protein level.

Giving translation a hand.

A cell's identity is commonly regarded as its transcriptomic profile. In this issue of Developmental Cell, Fujii et al. (2021) show that a global translation factor subunit acts differentially on transcripts to modulate morphogen signaling levels, revealing a global mechanism of transcript-specific translational control in development.

Satb2 acts as a gatekeeper for major developmental transitions during early vertebrate embryogenesis.

Zygotic genome activation (ZGA) initiates regionalized transcription underlying distinct cellular identities. ZGA is dependent upon dynamic chromatin architecture sculpted by conserved DNA-binding proteins. However, the direct mechanistic link between the onset of ZGA and the tissue-specific transcription remains unclear. Here, we have addressed the involvement of chromatin organizer Satb2 in orchestrating both processes during zebrafish embryogenesis. Integrative analysis of transcriptome, genome-wide occupancy and chromatin accessibility reveals contrasting molecular activities of maternally deposited and zygotically synthesized Satb2. Maternal Satb2 prevents premature transcription of zygotic genes by influencing the interplay between the pluripotency factors. By contrast, zygotic Satb2 activates transcription of the same group of genes during neural crest development and organogenesis. Thus, our comparative analysis of maternal versus zygotic function of Satb2 underscores how these antithetical activities are temporally coordinated and functionally implemented highlighting the evolutionary implications of the biphasic and bimodal regulation of landmark developmental transitions by a single determinant.

Bicc1 and Dicer regulate left-right patterning through post-transcriptional control of the Nodal inhibitor Dand5.

Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells at the left LRO margin. Ciliary activity of the calcium channel Pkd2 is crucial for flow sensing. How this flow signal is further processed and relayed to the laterality-determining Nodal cascade in the left lateral plate mesoderm (LPM) is largely unknown. We previously showed that flow down-regulates mRNA expression of the Nodal inhibitor Dand5 in left sensory cells. De-repression of the co-expressed Nodal, complexed with the TGFß growth factor Gdf3, drives LPM Nodal cascade induction. Here, we show that post-transcriptional repression of dand5 is a central process in symmetry breaking of Xenopus, zebrafish and mouse. The RNA binding protein Bicc1 was identified as a post-transcriptional regulator of dand5 and gdf3 via their 3'-UTRs. Two distinct Bicc1 functions on dand5 mRNA were observed at pre- and post-flow stages, affecting mRNA stability or flow induced translational inhibition, respectively. To repress dand5, Bicc1 co-operates with Dicer1, placing both proteins in the process of flow sensing. Intriguingly, Bicc1 mediated translational repression of a dand5 3'-UTR mRNA reporter was responsive to pkd2, suggesting that a flow induced Pkd2 signal triggers Bicc1 mediated dand5 inhibition during symmetry breakage.

Quantifying the effect of experimental perturbations at single-cell resolution.

Current methods for comparing single-cell RNA sequencing datasets collected in multiple conditions focus on discrete regions of the transcriptional state space, such as clusters of cells. Here we quantify the effects of perturbations at the single-cell level using a continuous measure of the effect of a perturbation across the transcriptomic space. We describe this space as a manifold and develop a relative likelihood estimate of observing each cell in each of the experimental conditions using graph signal processing. This likelihood estimate can be used to identify cell populations specifically affected by a perturbation. We also develop vertex frequency clustering to extract populations of affected cells at the level of granularity that matches the perturbation response. The accuracy of our algorithm at identifying clusters of cells that are enriched or depleted in each condition is, on average, 57% higher than the next-best-performing algorithm tested. Gene signatures derived from these clusters are more accurate than those of six alternative algorithms in ground truth comparisons.

Ythdf m6A Readers Function Redundantly during Zebrafish Development.

During the maternal-to-zygotic transition (MZT), multiple mechanisms precisely control massive decay of maternal mRNAs. N6-methyladenosine (m6A) is known to regulate mRNA decay, yet how this modification promotes maternal transcript degradation remains unclear. Here, we find that m6A promotes maternal mRNA deadenylation. Yet, genetic loss of m6A readers Ythdf2 and Ythdf3 did not impact global maternal mRNA clearance, zygotic genome activation, or the onset of gastrulation, challenging the view that Ythdf2 alone is critical to developmental timing. We reveal that Ythdf proteins function redundantly during zebrafish oogenesis and development, as double Ythdf2 and Ythdf3 deletion prevented female gonad formation and triple Ythdf mutants were lethal. Finally, we show that the microRNA miR-430 functions additively with methylation to promote degradation of common transcript targets. Together these findings reveal that m6A facilitates maternal mRNA deadenylation and that multiple pathways and readers act in concert to mediate these effects of methylation on RNA stability.

A Functional Non-coding RNA Is Produced from xbp-1 mRNA.

The xbp-1 mRNA encodes the XBP-1 transcription factor, a critical part of the unfolded protein response. Here we report that an RNA fragment produced from xbp-1 mRNA cleavage is a biologically active non-coding RNA (ncRNA) essential for axon regeneration in Caenorhabditis elegans. We show that the xbp-1 ncRNA acts independently of the protein-coding function of the xbp-1 transcript as part of a dual output xbp-1 mRNA stress response axis. Structural analysis indicates that the function of the xbp-1 ncRNA depends on a single RNA stem; this stem forms only in the cleaved xbp-1 ncRNA fragment. Disruption of this stem abolishes the non-coding, but not the coding, function of the endogenous xbp-1 transcript. Thus, cleavage of the xbp-1 mRNA bifurcates it into a coding and a non-coding pathway; modulation of the two pathways may allow neurons to fine-tune their response to injury and other stresses.

LabxDB: versatile databases for genomic sequencing and lab management.

SUMMARY: Experimental laboratory management and data-driven science require centralized software for sharing information, such as lab collections or genomic sequencing datasets. Although database servers such as PostgreSQL can store such information with multiple-user access, they lack user-friendly graphical and programmatic interfaces for easy data access and inputting. We developed LabxDB, a versatile open-source solution for organizing and sharing structured data. We provide several out-of-the-box databases for deployment in the cloud including simple mutant or plasmid collections and purchase-tracking databases. We also developed a high-throughput sequencing (HTS) database, LabxDB seq, dedicated to storage of hierarchical sample annotations. Scientists can import their own or publicly available HTS data into LabxDB seq to manage them from production to publication. Using LabxDB's programmatic access (REST API), annotations can be easily integrated into bioinformatics pipelines. LabxDB is modular, offering a flexible framework that scientists can leverage to build new database interfaces adapted to their needs. AVAILABILITY AND IMPLEMENTATION: LabxDB is available at https://gitlab.com/vejnar/labxdb and https://labxdb.vejnar.org for documentation. LabxDB is licensed under the terms of the Mozilla Public License 2.0. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Brd4 and P300 Confer Transcriptional Competency during Zygotic Genome Activation.

The awakening of the genome after fertilization is a cornerstone of animal development. However, the mechanisms that activate the silent genome after fertilization are poorly understood. Here, we show that transcriptional competency is regulated by Brd4- and P300-dependent histone acetylation in zebrafish. Live imaging of transcription revealed that genome activation, beginning at the miR-430 locus, is gradual and stochastic. We show that genome activation does not require slowdown of the cell cycle and is regulated through the translation of maternally inherited mRNAs. Among these, the enhancer regulators P300 and Brd4 can prematurely activate transcription and restore transcriptional competency when maternal mRNA translation is blocked, whereas inhibition of histone acetylation blocks genome activation. We conclude that P300 and Brd4 are sufficient to trigger genome-wide transcriptional competency by regulating histone acetylation on the first zygotic genes in zebrafish. This mechanism is critical for initiating zygotic development and developmental reprogramming.

Genome wide analysis of 3' UTR sequence elements and proteins regulating mRNA stability during maternal-to-zygotic transition in zebrafish.

Posttranscriptional regulation plays a crucial role in shaping gene expression. During the maternal-to-zygotic transition (MZT), thousands of maternal transcripts are regulated. However, how different cis-elements and trans-factors are integrated to determine mRNA stability remains poorly understood. Here, we show that most transcripts are under combinatorial regulation by multiple decay pathways during zebrafish MZT. By using a massively parallel reporter assay, we identified cis-regulatory sequences in the 3' UTR, including U-rich motifs that are associated with increased mRNA stability. In contrast, miR-430 target sequences, UAUUUAUU AU-rich elements (ARE), CCUC, and CUGC elements emerged as destabilizing motifs, with miR-430 and AREs causing mRNA deadenylation upon genome activation. We identified trans-factors by profiling RNA-protein interactions and found that poly(U)-binding proteins are preferentially associated with 3' UTR sequences and stabilizing motifs. We show that this activity is antagonized by C-rich motifs and correlated with protein binding. Finally, we integrated these regulatory motifs into a machine learning model that predicts reporter mRNA stability in vivo.

Genetic compensation triggered by mutant mRNA degradation.

Genetic robustness, or the ability of an organism to maintain fitness in the presence of harmful mutations, can be achieved via protein feedback loops. Previous work has suggested that organisms may also respond to mutations by transcriptional adaptation, a process by which related gene(s) are upregulated independently of protein feedback loops. However, the prevalence of transcriptional adaptation and its underlying molecular mechanisms are unknown. Here, by analysing several models of transcriptional adaptation in zebrafish and mouse, we uncover a requirement for mutant mRNA degradation. Alleles that fail to transcribe the mutated gene do not exhibit transcriptional adaptation, and these alleles give rise to more severe phenotypes than alleles displaying mutant mRNA decay. Transcriptome analysis in alleles displaying mutant mRNA decay reveals the upregulation of a substantial proportion of the genes that exhibit sequence similarity with the mutated gene's mRNA, suggesting a sequence-dependent mechanism. These findings have implications for our understanding of disease-causing mutations, and will help in the design of mutant alleles with minimal transcriptional adaptation-derived compensation.