Biology subjects

Vejnar, C. E.

Publications and source records attributed to Vejnar, C. E..

8 recordsLinked to original sources

bioRxiv2026

Beyond codon optimality: codon pairs regulate mRNA stability dependent on translation

The coding sequence of an mRNA directs its own decay, yet how codons, codon context, and amino acids collectively regulate mRNA stability remains poorly understood. Here we use a massively parallel reporter assay to decode this regulatory layer in zebrafish embryos. We find that codon pairs and amino acid pairs regulate mRNA stability beyond the level of individual codons. This regulation depends on the identity and order of neighboring codons and their encoded amino acids in a translation-dependent manner. The relative contributions of codon and amino acid combinations to mRNA stability can be quantified using machine learning. We further found that endogenous mRNAs are regulated by codon pairs, thereby governing developmental gene regulation and biological function. This codon context-dependent decay requires deadenylation and decapping by Cnot7 and Dcp2, with Upf1 acting on long non-optimal ORFs. Together, our results redefine codon optimality as a context-dependent, pair-level code with implications for RNA biology and therapeutic mRNA design.

molecular biology↗

bioRxiv2022

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 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 been hindered by technical challenges caused by their small size. Here we show that two putative lincRNAs (linc-mipep 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 highlight the power of screening for unexplored micropeptide functions by revealing a key missing link in the evolution of vertebrate brain cell development and illustrating a genetic basis for how some neural cell types are more susceptible to chromatin disruptions, with implications for neurodevelopmental disorders and disease.

developmental biology↗

bioRxiv2021

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 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 characterized and optimized the activity of SpG and SpRY 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 efficient mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm can predict SpG and SpRY 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.

genetics↗

bioRxiv2020

Synergistic activity of Nanog, Pou5f3, and Sox19b establishes chromatin accessibility and developmental competence in a context-dependent manner

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 of action remain poorly defined. Here, we generated a triple maternal-zygotic nanog-/-;pou5f3-/-;sox19b-/- mutant and assessed the causal relationship between transcription factor (TF) occupancy, chromatin accessibility and genome activation. Analyses of this triple mutant and combinatorial rescues revealed highly synergistic and context-dependent activity of Nanog, Pou5f3, and Sox19b (NPS) in establishing chromatin competency at >50% of active enhancers. Motif analysis revealed a network of TFs that depend on NPS for establishing chromatin accessibility, including the endodermal determinant Eomesa, whose binding we show is regulated by NPS pioneer-like activity. Finally, we demonstrated that NPS play an essential role in establishing H3K27ac and H3K18ac at enhancers and promoters, and that their function in transcriptional activation can be bypassed by targeted recruitment of histone acetyltransferases to individual genes. Altogether, our findings reveal a large network of TFs that function to establish developmental competency, many of which depend on the synergistic and highly context-dependent role of NPS in establishing chromatin accessibility and regulating histone acetylation in order to activate the genome.

developmental biology↗

bioRxiv2018

Brd4 and P300 regulate zygotic genome activation through histone acetylation

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 transcription analysis during zebrafish embryogenesis, we observed that genome activation is gradual and stochastic, and the active state is inherited in daughter cells. We discovered that genome activation is regulated through both translation of maternal mRNAs and the effects of these factors on the chromatin. We show that chemical inhibition of H3K27Ac writer (P300) and reader (Brd4) block genome activation, while induction of a histone acetylation prematurely activates transcription, and restore genome activation in embryos where translation of maternal mRNAs is impaired, demonstrating that they are limiting factors for the activation of the genome. In contrast to current models, we do not observe triggering of genome activation by a reduction of the nuclear-cytoplasmic (N/C) ratio or slower cell division. We conclude that genome activation is controlled by a time-dependent mechanism involving the translation of maternal mRNAs and the regulation of histone acetylation through P300 and Brd4. This mechanism is critical to initiating zygotic development and developmental reprogramming.

developmental biology↗

bioRxiv2018

A post-transcriptional regulatory code for mRNA stability during the zebrafish maternal-to-zygotic transition

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 stability is unclear. Here, we show that most transcripts are under combinatorial regulation by multiple decay pathways during zebrafish MZT. To identify cis-regulatory elements, we performed a massively parallel reporter assay for stability-influencing sequences, which revealed that 3-UTR poly-U motifs are associated with mRNA stability. In contrast, miR-430 target sequences, UAUUUAUU AU-rich elements (ARE), CCUC and CUGC elements emerged as the main destabilizing motifs in the embryo, with miR-430 and AREs causing mRNA deadenylation in a genome activation-dependent manner. To identify the trans-factors interacting with these cis-elements, we comprehensively profiled RNA-protein interactions and their associated regulatory activities across the transcriptome during the MZT. We find that poly-U binding proteins are preferentially associated with 3-UTR sequences and stabilizing motifs, and that antagonistic sequence contexts for poly-C and poly-U binding proteins shape the binding landscape and magnitude of regulation across the transcriptome. Finally, we integrate these regulatory motifs into a machine learning model that accurately predicts the stability of mRNA reporters in vivo. Our findings reveal how mechanisms of post-transcriptional regulation are coordinated to direct changes in mRNA stability within the early zebrafish embryo.

genomics↗

bioRxiv2018

mRNA structure dynamics identifies the embryonic RNA regulome

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 level, translation guides structure rather than structure guiding translation. We detect a decrease in structure in translated regions, and we identify the ribosome as a major remodeler of RNA structure in vivo. In contrast, we find that 3-UTRs form highly folded structures in vivo, which can affect gene expression by modulating miRNA activity. Furthermore, we find that dynamic 3-UTR structures encode RNA decay elements, including regulatory elements in nanog and cyclin A1, key maternal factors orchestrating the maternal-to-zygotic transition. These results reveal a central role of RNA structure dynamics in gene regulatory programs.

molecular biology↗

bioRxiv2017

CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing

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.

genetics↗