Antonio Giraldez

• Yale University

The subcellular localization of mRNAs plays a pivotal role in biological processes, including cell migration. For instance, β-actin mRNA and its associated RNA-binding protein (RBP), ZBP1/IGF2BP1, are recruited to focal adhesions (FAs) to support localized β-actin synthesis, crucial for cell migration. However, whether other mRNAs and RBPs also loc...

The cis-regulatory elements encoded in an mRNA determine its stability and translational output. While there has been a considerable effort to understand the factors driving mRNA stability, the regulatory frameworks governing translational control remain more elusive. We have developed a novel massively parallel reporter assay (MPRA) to measure mRN...

A fertilized egg is initially transcriptionally silent and relies on maternally provided factors to initiate development. For embryonic development to proceed, the oocyte-inherited cytoplasm and the nuclear chromatin need to be reprogrammed to create a permissive environment for zygotic genome activation (ZGA). During this maternal-to-zygotic trans...

Maternally-loaded factors in the egg accumulate during oogenesis and are essential for the acquisition of oocyte and egg developmental competence to ensure the production of viable embryos. However, their molecular nature and functional importance remain poorly understood. Here, we present a collection of 9 recessive maternal-effect mutants identif...

The capacity for embryonic cells to differentiate relies on a large-scale reprogramming of the oocyte and sperm nucleus into a transient totipotent state. In zebrafish, this reprogramming step is achieved by the pioneer factors Nanog, Pou5f3, and Sox19b (NPS). Yet, it remains unclear whether cells lacking this reprogramming step are directed toward...

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) le...

Probing endogenous protein localization and function in vivo remains challenging due to laborious gene targeting and monofunctional alleles. Here, we develop a multifunctional, universal, and adaptable toolkit based on genetically encoded affinity reagents (GEARs). GEARs use nanobodies and single chain variable fragments (scFv), which recognize sma...

The cis -regulatory elements encoded in a mRNA determine its stability and translational output. While there has been a considerable effort to understand the factors driving mRNA stability, the regulatory frameworks governing translational control remain elusive. We have developed a novel massively parallel reporter assay (MPRA) to measure mRNA tra...

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...

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 hav...

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...

Thousands of long intergenic non-coding RNAs (lincRNAs) are transcribed throughout the vertebrate genome. A subset of lincRNAs enriched in developing brains has 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...

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. Howe...

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 n...

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...

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.

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 th...

Messenger RNA (mRNA) compartmentalization within the cytosol is well-recognized as a key mechanism of local translation-mediated regulation of protein levels, but whether such localization could be a means of exercising non-coding mRNA function is unknown. Here, we explore non-coding functions for mRNAs associated with focal adhesions (FAs), cellul...

RNA polyadenylation plays a central role in RNA maturation, fate and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translatability and stability of mRNAs. Here we develop Nano3P-seq, a novel method that relies on nanopore sequencing to simultaneously quantify RNA abundance and tail length dynamics at per-r...

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 mesod...

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 PAM, 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. We have characterize...

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 re...

Zygotic genome activation (ZGA) initiates regionalized transcription responsible for the acquisition of distinct cellular identities. ZGA is dependent upon dynamic chromatin architecture sculpted by conserved DNA-binding proteins. However, whether the tissue-specific transcription is mechanistically linked with the onset of ZGA is unknown. Here, we...

Genome-wide chromatin reprogramming is a fundamental requirement for establishing developmental competence in the newly-formed zygote. In zebrafish, Nanog, Pou5f3 and Sox19b play partially redundant roles in zygotic genome activation, however their interplay in establishing chromatin competency, the context in which they do so and their mechanism o...

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-codin...

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...

Genome-wide RNA structure maps have recently become available through the coupling of in vivo chemical probing reagents with next-generation sequencing. Initial analyses relied on the identification of truncated reverse transcription reads to identify the chemically modified nucleotides, but recent studies have shown that mutational signatures can...

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) in Caenorhabditis elegans neurons, providing the first example of ncRNA derived from mRNA cleavage. We show that the xbp-1 ncRN...

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 co...

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 tha...

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. Ho...

[This corrects the article DOI: 10.1038/s41526-017-0033-9.].

RNA folding plays a crucial role in RNA function. However, knowledge of the global structure of the transcriptome is limited to cellular systems at steady state, thus hindering the understanding of RNA structure dynamics during biological transitions and how it influences gene function. Here, we characterized mRNA structure dynamics during zebrafis...

The awakening of the zygote genome, signaling the transition from maternal transcriptional control to zygotic control, is a watershed in embryonic development, but the factors and mechanisms controlling this transition are still poorly understood. By combining CRISPR-Cas9-mediated live imaging of the first transcribed genes (miR-430), chromatin and...

Pre-mRNA splicing is a critical step of gene expression in eukaryotes. Transcriptome-wide splicing patterns are complex and primarily regulated by a diverse set of recognition elements and associated RNA-binding proteins. The retention and splicing (RES) complex is formed by three different proteins (Bud13p, Pml1p and Snu17p) and is involved in spl...

bud13 knock-down show stronger cell death phenotype. (A) Lateral view of WT embryos injected with 0.6mM of morpholino antisense oligonucleotide against bud13 mRNA (MObud13) showing different levels of developmental defects (types I to III). Phenotypes are fully rescue with human hBUD13 mRNA. (scale bar: 0.5mm). WT: represent phenotypically wild typ...

Gene ontology and gene expression analysis of retained introns. (A) DAVID cluster analysis of enriched GO annotations for genes that contain introns with highly increased retention (∆PIR>15) in at least two of the RES mutants. (B) Boxplots showing fold change in expression (FC-expr) for genes containing increased retention (∆PIR>15) compared with t...

RES complex is required during zebrafish development. (A) Scheme illustrating the Cas9-nanos 3′-UTR strategy [20]. The nanos' 3′-UTR concentrates the expression of Cas9 in the germ cells (green circles). (B-C) Bright field microscopy of RES mutant embryos and their corresponding phenotypically wild type sibling (WT), treated with PTU to avoid melan...

CNS molecular markers show mild differences in bud13Δ7/Δ7. In situ hybridization showing expression pattern of shh (notochord and floor plate), krox20 (egr2a; rhombomere 3 and 5) and pax2a (anterior midbrain-hindbrain boundary and hindbrain neurons) in WT (top) and mutant (bottom) embryos. (TIF)

Differentially retained introns detected by RT-PCR. Sequencing read density across the ptch2 (A) and wdr26b (B) loci (upper panels). RNA- seq signal increases strongly (∆PIR>15) in RES mutants only on specific introns (dark blue). RT-PCR assays validating the increased retention (dotted square box) in bud13, rbmx2 and snip1 mutants compared with th...

Transcript expression levels and Gene ontology analysis. (A) Biplot comparing transcript expression levels in RES mutants and their corresponding phenotypically WT siblings. Genes up- or down-regulation were defined as having a fold change in expression of at least 1.5 in all three mutants and at least 2 for two out the three control vs. mutant ind...

Intron enrichment. Stacked bar plot showing enrichment for last introns (A) and introns predicted not to trigger NMD upon inclusion (B) among the RES-dependent (RES-dep) introns. On the contrary, introns that are not affected by RES depletion (Ctr) are depleted for these types of introns. P-values were calculated using Fisher exact test. (C) Stacke...

Extended features of the RES-dependent introns. (A) Boxplots showing the distribution of the median nucleotide (nt) distance from the top 3 predicted branch points (BP) to the 3’ splice site for each intron category. (B) Boxplots of the highest score for the human SF1 position weight matrix (PWM) in the 3’ intronic region (see Methods for details)....

Intron length and GC content genome wide analysis. (A) Boxplots showing the degree of change in intron retention (∆PIR) according to intron length. Number of introns per nucleotide bin: ≤100 = 13,059; 101–300 = 13,332; 301–1,000 = 11,808; 1,001–2500 = 18,164; >2500 = 16,563. (B) Boxplots illustrating the degree of change in intron retention (∆PIR)...

RES-dependent and independent introns in vivo validation. (A) Intron predicted as RES independent were spliced in bud13∆7/∆7 (col1a2 and serpinb1l3) while RES dependent intron wdr26b, but not ptch2, was retained as predicted for the logistic regression model. (B) Scheme of the vector used in the validation assay. PCR product and primers used are de...

Contribution of each feature to reduction of null deviance. (A) Logistic regression models were learned to discriminate between RESdep and Ctr introns with each feature individually and the fraction of the null deviance that was reduced was recorded. Values were averaged over 10,000 repeated holdout experiments. Training data sets consisted of 1,26...

Validation of logistic regression model. (A) ∆PIR values for the top 100 introns based on their likelihood to be bud13-dependent and -independent, as predicted by the regression model. Only introns with no read coverage across the six RNA-seq samples were used for this analysis (108,470 introns). False positive rate (FPR) values are defined for ret...

The CRISPR-Cas9 system biotechnological impact has recently broadened the genome editing toolbox available to different model organisms further with the addition of new efficient CRISPR-based endonucleases. We have recently optimized CRISPR-Cpf1 (renamed Cas12a) system in zebrafish. We showed that i) in the absence of Cpf1 protein, crRNAs are unsta...

Genetic compensation by transcriptional modulation of related gene(s) (also known as transcriptional adaptation) has been reported in numerous systems; however, whether and how such a response can be activated in the absence of protein feedback loops is unknown. Here, we develop and analyze several models of transcriptional adaptation in zebrafish...

Post-transcriptional regulation is crucial to shape gene expression. During the Maternal-to-Zygotic Transition (MZT), thousands of maternal transcripts are regulated upon fertilization and genome activation. Transcript stability can be influenced by cis- elements and trans- factors, but how these inputs are integrated to determine the overall mRNA...

This corrects the article DOI: 10.1038/nature18614.

RNA folding plays a crucial role in RNA function. However, our knowledge of the global structure of the transcriptome is limited to steady-state conditions, hindering our understanding of how RNA structure dynamics influences gene function. Here, we have characterized mRNA structure dynamics during zebrafish development. We observe that on a global...

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 tempera...

As the range and duration of human ventures into space increase, it becomes imperative that we understand the effects of the cosmic environment on astronaut health. Molecular technologies now widely used in research and medicine will need to become available in space to ensure appropriate care of astronauts. The polymerase chain reaction (PCR) is t...

Pre-mRNA splicing is a critical step of gene expression in eukaryotes. Transcriptome-wide splicing patterns are complex and primarily regulated by a diverse set of recognition elements and associated RNA-binding proteins. The retention and splicing (RES) complex is formed by three different proteins (Bud13p, Pml1p and Snu17p) and is involved in spl...

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 temper...

Proper functioning of an organism requires cells and tissues to behave in uniform, well-organized ways. How this optimum of phenotypes is achieved during the development of vertebrates is unclear. Here, we carried out a multi-faceted and single-cell resolution screen of zebrafish embryonic blood vessels upon mutagenesis of single and multi-gene mic...

Gene expression is extensively regulated at the levels of mRNA stability, localization and translation. However, decoding functional RNA-regulatory features remains a limitation to understanding post-transcriptional regulation in vivo. Here, we developed RNA-element selection assay (RESA), a method that selects RNA elements on the basis of their ac...

This protocol describes how to generate and genotype mutants using an optimized CRISPR-Cas9 genome-editing system in zebrafish (CRISPRscan). Because single guide RNAs (sgRNAs) have variable efficiency when targeting specific loci, our protocol starts by explaining how to use the web tool CRISPRscan to design highly efficient sgRNAs. The CRISPRscan...

The CRISPR-Cas9 system uncovered in bacteria has emerged as a powerful genome-editing technology in eukaryotic cells. It consists of two components—a single guide RNA (sgRNA) that directs the Cas9 endonuclease to a complementary DNA target site. Efficient targeting of individual genes requires highly active sgRNAs. Recent efforts have made signific...

Cellular transitions require dramatic changes in gene expression that are supported by regulated mRNA decay and new transcription. The maternal-to-zygotic transition is a conserved developmental progression during which thousands of maternal mRNAs are cleared by post-transcriptional mechanisms. Although some maternal mRNAs are targeted for degradat...

Vascular and haematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs and thus play critical roles in development and disease. At the top of the haemato-vascular specification cascade lies cloche, a gene that when mutated in zebrafish leads to the striking phenotype of loss of mos...

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 analys...

Autism spectrum disorders (ASDs) are a group of devastating neurodevelopmental syndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD risk genes, the mechanisms underlying ASDs remain unknown. Homozygous loss-of-function mutations in Contactin Associated Protein-like 2 (CNTNAP2) are strongly linked to ASDs. Here...

MicroRNAs have emerged as critical regulators of gene expression. Originally shown to regulate developmental timing, microRNAs have since been implicated in a wide range of cellular functions including cell identity, migration and signaling. miRNA-430, the earliest expressed microRNA during zebrafish embryogenesis, is required to undergo morphogene...

Cellular transitions occur at all stages of organismal life from conception to adult regeneration. Changing cellular state involves three main features: activating gene expression necessary to install the new cellular state, modifying the chromatin status to stabilize the new gene expression program, and removing existing gene products to clear out...

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 increa...

Early development depends heavily on accurate con-trol of maternally inherited mRNAs, and yet it remains unknown how maternal microRNAs are regulated during maternal-to-zygotic transition (MZT). We here find that maternal microRNAs are highly adeny-lated at their 3 0 ends in mature oocytes and early em-bryos. Maternal microRNA adenylation is widely...

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 transf...

Background: Whole-exome sequencing has rapidly expanded the list of ASD-associated genes, while co-expression network analysis is beginning to reveal points of spatio-temporal convergence among these genes. Moreover, these new ASD risk genes are elucidating novel biological mechanisms, such as chromatin modification (CHD8) and ion channels (SCN2A),...

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...

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 vertebrat...