Regulation of gene expression is fundamental in establishing cellular diversity and a target of natural selection. Untranslated mRNA regions (UTRs) are key mediators of post‐transcriptional ...regulation. Previous studies have predicted thousands of ORFs in 5′ UTRs, the vast majority of which have unknown function. Here, we present a systematic analysis of the translation and function of upstream open reading frames (uORFs) across vertebrates. Using high‐resolution ribosome footprinting, we find that (i) uORFs are prevalent within vertebrate transcriptomes, (ii) the majority show signatures of active translation, and (iii) uORFs act as potent regulators of translation and RNA levels, with a similar magnitude to miRNAs. Reporter experiments reveal clear repression of downstream translation by uORFs/oORFs. uORF number, intercistronic distance, overlap with the CDS, and initiation context most strongly influence translation. Evolution has targeted these features to favor uORFs amenable to regulation over constitutively repressive uORFs/oORFs. Finally, we observe that the regulatory potential of uORFs on individual genes is conserved across species. These results provide insight into the regulatory code within mRNA leader sequences and their capacity to modulate translation across vertebrates.
Synopsis
Upstream open reading frames (uORFs) are found in many genes, but their functional impact is unclear. This study explores their abundant translation, conservation, and potent role in shaping the vertebrate translational landscape.
uORFs are pervasive and actively translated throughout vertebrate transcriptomes.
uORFs and oORFs act as potent regulators of downstream translation and mRNA stability.
Sequence features such as AUG context and number modulate the activity of uORFs in vivo.
uORF regulation is conserved across vertebrate species.
Upstream open reading frames (uORFs) are found in many genes, but their functional impact is unclear. This study explores their abundant translation, conservation, and potent role in shaping the vertebrate translational landscape.
Embryogenesis depends on a highly coordinated cascade of genetically encoded events. In animals, maternal factors contributed by the egg cytoplasm initially control development, whereas the zygotic ...nuclear genome is quiescent. Subsequently, the genome is activated, embryonic gene products are mobilized, and maternal factors are cleared. This transfer of developmental control is called the maternal-to-zygotic transition (MZT). In this review, we discuss recent advances toward understanding the scope, timing, and mechanisms that underlie zygotic genome activation at the MZT in animals. We describe high-throughput techniques to measure the embryonic transcriptome and explore how regulation of the cell cycle, chromatin, and transcription factors together elicits specific patterns of embryonic gene expression. Finally, we illustrate the interplay between zygotic transcription and maternal clearance and show how these two activities combine to reprogram two terminally differentiated gametes into a totipotent embryo.
CRISPR-Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We analyzed the ...molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observed that guanine enrichment and adenine depletion increased sgRNA stability and activity, whereas differential sgRNA loading, nucleosome positioning and Cas9 off-target binding were not major determinants. We also identified sgRNAs truncated by one or two nucleotides and containing 5' mismatches as efficient alternatives to canonical sgRNAs. On the basis of these results, we created a predictive sgRNA-scoring algorithm, CRISPRscan, that effectively captures the sequence features affecting the activity of CRISPR-Cas9 in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos 3' UTR led to the generation of maternal-zygotic mutants, as well as increased viability and decreased somatic mutations. These results identify determinants that influence Cas9 activity and provide a framework for the design of highly efficient sgRNAs for genome targeting in vivo.
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.
MicroRNAs (miRNAs) are typically generated as ∼22-nucleotide double-stranded RNAs via the processing of precursor hairpins by the ribonuclease III enzyme Dicer, after which they are loaded into ...Argonaute (Ago) proteins to form an RNA-induced silencing complex (RISC). However, the biogenesis of miR-451, an erythropoietic miRNA conserved in vertebrates, occurs independently of Dicer and instead requires cleavage of the 3′ arm of the pre-miR-451 precursor hairpin by Ago2. The 3′ end of the Ago2-cleaved pre-miR-451 intermediate is then trimmed to the mature length by an unknown nuclease. Here, using a classical chromatographic approach, we identified poly(A)-specific ribonuclease (PARN) as the enzyme responsible for the 3′–5′ exonucleolytic trimming of Ago2-cleaved pre-miR-451. Surprisingly, our data show that trimming of Ago2-cleaved precursor miRNAs is not essential for target silencing, indicating that RISC is functional with miRNAs longer than the mature length. Our findings define the maturation step in the miRNA biogenesis pathway that depends on Ago2-mediated cleavage.
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•PARN is responsible for trimming of Ago2-cleaved pre-miR-451•Trimming of Ago2-cleaved pre-miRNAs is dispensable for target silencing•RISC can thus be functional with miRNAs that exceed mature length
Most microRNAs are processed by Dicer. However, processing of miR-451, a conserved microRNA linked to erythropoiesis, is Dicer independent and requires Argonaute2 to cleave the precursor microRNA hairpin (pre-miR-451). The cleaved intermediate is then trimmed to the mature length by an unknown nuclease. Here, Giraldez, Tomari, and colleagues identify PARN as the enzyme responsible for 3′–5′ exonucleolytic trimming of pre-miR-451. They also show that, surprisingly, the trimming of Argonaute2-cleaved pre-miRNAs is not essential for target silencing and that RISC can thus function with miRNAs that exceed mature length.
Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. ...Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species.
Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of ...development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide‐encoding genes in vertebrates, providing an entry point to define their function in vivo.
Synopsis
The combination of ORFscore and micPDP enable high confidence prediction of many, small translated ORFs that were functionally not appreciated or previously annotated as lincRNAs.
“ORFScore” explores high‐resolution footprinting with ribosome phasing to identify novel, translated micropeptides in genes previously thought to lack coding potential.
“micPDP” is a new computational pipeline that identifies micropeptides under negative selection across species.
The combination of these techniques enabled high confidence prediction of numerous small translated ORFs suitable for functional characterization.
The combination of ORFscore and micPDP enable high confidence prediction of many, small translated ORFs that were functionally not appreciated or previously annotated as lincRNAs.
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.
After fertilization, maternal factors direct development and trigger zygotic genome activation (ZGA) at the maternal-to-zygotic transition (MZT). In zebrafish, ZGA is required for gastrulation and ...clearance of maternal messenger RNAs, which is in part regulated by the conserved microRNA miR-430. However, the factors that activate the zygotic program in vertebrates are unknown. Here we show that Nanog, Pou5f1 (also called Oct4) and SoxB1 regulate zygotic gene activation in zebrafish. We identified several hundred genes directly activated by maternal factors, constituting the first wave of zygotic transcription. Ribosome profiling revealed that nanog, sox19b and pou5f1 are the most highly translated transcription factors pre-MZT. Combined loss of these factors resulted in developmental arrest before gastrulation and a failure to activate >75% of zygotic genes, including miR-430. Our results demonstrate that maternal Nanog, Pou5f1 and SoxB1 are required to initiate the zygotic developmental program and induce clearance of the maternal program by activating miR-430 expression.
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.
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