Tomáš Mašek

Summary: CRISPR-Cas9 has emerged as a powerful tool for targeted genome editing in plants; however, its application in tetraploid potato ( Solanum tuberosum ssp. tuberosum ) remains challenging due to its vegetative propagation and complex highly heterozygous genome. Availability of whole-genome sequence data for the specific genotype is crucial to ensure complete knockout of all alleles of target genes while minimizing off-target mutations. In this study, using the tetraploid potato cultivar Désirée, we report, a complete CRISPR-Cas9-mediated knockout of the BEL5 gene, encoding a transcription factor, known as one of the key regulators driving tuber formation. We employed Agrobacterium -mediated transformation and demonstrated that repeated de novo regeneration could improve editing efficiency by promoting emergence of new mutations. BEL5 knockout plants exhibited a delayed onset of tuberization under inductive short-day conditions in hydroponics; however, their overall tuber yields were comparable to wild type plants. Based on our results, we propose a regulatory role of BEL5 in the timing of tuber onset but, unexpectedly, its dispensability for tuber development in modern cultivated potato. Besides providing functional insight into the BEL5 role in potato, this study includes a methodological approach for efficient CRISPR-Cas9 gene editing in this vegetatively propagated polyploid crop, along with strategies for detecting mutations in genes that lack clear phenotypic manifestation.

The transcription factor StBEL11 is known as a negative regulator of tuberization in the Andean potato (Solanum tuberosum ssp. andigena). This study focuses on its role in the common potato ssp. tuberosum by analysing transgenic lines of cv. Kamýk with down-regulated StBEL11 expression. As expected, these lines showed enhanced tuber induction on nodal segments cultivated in vitro on high-sucrose medium. However, intact plants ex vitro exhibited significantly delayed tuberization and decreased yield, which surprisingly applied especially for lines with the most enhanced in vitro tuberization. Interestingly, reduced leaf StBEL11 expression was accompanied by decreased leaf expression of the positive tuberization regulator StBEL5, but not of SP6A tuberigen. The transgenics showed increased photosynthetic activity, which was not, however, associated with non-structural carbohydrate accumulation. Unexpectedly, StBEL11 down-regulation altered assimilate allocation in favour of roots over shoots and stolons before onset of tuberization and, in lines with the strongest phenotype, also during the early stage of tuberization, which probably explains their reduced tuber yields. The unexpected differences in tuber induction and yield between andigena and tuberosum lines with down-regulated StBEL11 indicate changes in the strength/hierarchy of individual components of the tuberization regulatory network and point to the impossibility of easy knowledge transfer from Andean to cultivated potato.

STUDY QUESTION: Which actively translated maternal transcripts are differentially regulated between clinically relevant in vitro and in vivo maturation (IVM) conditions in mouse oocytes and zygotes?SUMMARY ANSWER: Our findings uncovered significant differences in the global transcriptome as well as alterations in the translation of specific transcripts encoding components of energy production, cell cycle regulation, and protein synthesis in oocytes and RNA metabolism in zygotes.WHAT IS KNOWN ALREADY: Properly regulated translation of stored maternal transcripts is a crucial factor for successful development of oocytes and early embryos, particularly due to the transcriptionally silent phase of meiosis.STUDY DESIGN, SIZE, DURATION: This is a basic science study utilizing an ICR mouse model, best suited for studying in vivo maturation. In the treatment group, fully grown germinal vesicle oocytes from stimulated ovaries were in vitro matured to the metaphase II (MII) stage either as denuded without gonadotropins (IVM DO), or as cumulus–oocyte complexes (IVM COC) in the presence of 0.075 IU/ml recombinant FSH (rFSH) and 0.075 IU/ml recombinant hCG (rhCG). To account for changes in developmental competence, IVM COC from non-stimulated ovaries (IVM COC−) were included. In vivo matured MII oocytes (IVO) from stimulated ovaries were used as a control after ovulation triggering with rhCG. To simulate standard IVM conditions, we supplemented media with amino acids, vitamins, and bovine serum albumin. Accordingly, in vitro pronuclear zygotes (IMZ) were generated by IVF from IVM DO, and were compared to in vivo pronuclear zygotes (IVZ). All experiments were performed in quadruplicates with samples collected for both polyribosome fractionation and total transcriptome analysis. Samples were collected over three consecutive months.PARTICIPANTS/MATERIALS, SETTING, METHODS: All ICR mice were bred under legal permission for animal experimentation (no. MZE-24154/2021-18134) obtained from the Ministry of Agriculture of the Czech Republic. Actively translated (polyribosome occupied) maternal transcripts were detected in in vitro and in vivo matured mouse oocytes and zygotes by density gradient ultracentrifugation, followed by RNA isolation and high-throughput RNA sequencing. Bioinformatic analysis was performed and subsequent data validation was done by western blotting, radioactive isotope, and mitotracker dye labelling.MAIN RESULTS AND THE ROLE OF CHANCE: Gene expression analysis of acquired polysome-derived high-throughput RNA sequencing data revealed significant changes (RPKM ≥ 0.2; P ≤ 0.005) in translation between in vitro and in vivo matured oocytes and respectively produced pronuclear zygotes. Surprisingly, the comparison between IVM DO and IVM COC RNA-seq data of both fractionated and total transcriptome showed very few transcripts with more than a 2-fold difference. Data validation by radioactive isotope labelling revealed a decrease in global translation bof20% in IVM DO and COC samples in comparison to IVO samples. Moreover, IVM conditions compromised oocyte energy metabolism, which was demonstrated by both changes in polysome recruitment of each of 13 mt-protein-coding transcripts as well as by validation using mitotracker red staining.LARGE SCALE DATA: The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE241633 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241633)LIMITATIONS, REASONS FOR CAUTION: It is extremely complicated to achieve in vivo consistency in animal model systems such as porcine or bovine. To achieve a high reproducibility of in vivo stimulations, the ICR mouse model was selected. However, careful interpretation of our findings with regard to assisted reproductive techniques has to be made by taking into consideration intra-species differences between the mouse model and humans. Also, the sole effect of the cumulus cells’ contribution could not be adequately addressed by comparing IVM COC and IVM DO, because the IVM DO were matured without gonadotropin supplementation.WIDER IMPLICATIONS OF THE FINDINGS: Our findings confirmed the inferiority of standard IVM technology compared with the in vivo approach. It also pointed at compromised biological processes employed in the critical translational regulation of in vitro matured MII oocytes and pronuclear zygotes. By highlighting the importance of proper translational regulation during in vitro oocyte maturation, this study should prompt further clinical investigations in the context of translation.STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Czech Grant Agency (22-27301S), Charles University Grant Agency (372621), Ministry of Education, Youth and Sports (EXCELLENCE CZ.02.1.01/0.0/0.0/15_003/0000460 OP RDE), and Institutional Research Concept RVO67985904. No competing interest is declared.

In recent years, numerous evidence has been accumulated about the extent of A-to-I editing in human RNAs and the key role ADAR1 plays in the cellular editing machinery. It has been shown that A-to-I editing occurrence and frequency are tissue-specific and essential for some tissue development, such as the liver. To study the effect of ADAR1 function in hepatocytes, we have created Huh7.5 ADAR1 KO cell lines. Upon IFN treatment, the Huh7.5 ADAR1 KO cells show rapid arrest of growth and translation, from which they do not recover. We analyzed translatome changes by using a method based on sequencing of separate polysome profile RNA fractions. We found significant changes in the transcriptome and translatome of the Huh7.5 ADAR1 KO cells. The most prominent changes include negatively affected transcription by RNA polymerase III and the deregulation of snoRNA and Y RNA levels. Furthermore, we observed that ADAR1 KO polysomes are enriched in mRNAs coding for proteins pivotal in a wide range of biological processes such as RNA localization and RNA processing, whereas the unbound fraction is enriched mainly in mRNAs coding for ribosomal proteins and translational factors. This indicates that ADAR1 plays a more relevant role in small RNA metabolism and ribosome biogenesis.

Translation is critical for development as transcription in the oocyte and early embryo is silenced. To illustrate the translational changes during meiosis and consecutive two mitoses of the oocyte and early embryo, we performed a genome-wide translatome analysis. Acquired data showed significant and uniform activation of key translational initiation and elongation axes specific to M-phases. Although global protein synthesis decreases in M-phases, translation initiation and elongation activity increases in a uniformly fluctuating manner, leading to qualitative changes in translation regulation via the mTOR1/4F/eEF2 axis. Overall, we have uncovered a highly dynamic and oscillatory pattern of translational reprogramming that contributes to the translational regulation of specific mRNAs with different modes of polysomal occupancy/translation that are important for oocyte and embryo developmental competence. Our results provide new insights into the regulation of gene expression during oocyte meiosis as well as the first two embryonic mitoses and show how temporal translation can be optimized. This study is the first step towards a comprehensive analysis of the molecular mechanisms that not only control translation during early development, but also regulate translation-related networks employed in the oocyte-to-embryo transition and embryonic genome activation.

Background: p38-MAPKs are stress-activated kinases necessary for placental development and nutrient and oxygen transfer during murine post-implantation development. In preimplantation development, p38-MAPK activity is required for blastocyst formation. Additionally, we have previously reported its role in regulating specification of inner cell mass (ICM) towards primitive endoderm (PrE), although a comprehensive mechanistic understanding is currently limited. Adopting live embryo imaging, proteomic and transcriptomic approaches, we report experimental data that directly address this deficit. Results: Chemical inhibition of p38-MAPK activity during blastocyst maturation causes impaired blastocyst cavity expansion, most evident between the third and tenth hours post inhibition onset. We identify an overlapping minimal early blastocyst maturation window of p38-MAPKi inhibition (p38-MAPKi) sensitivity, that is sufficient to impair PrE cell fate by the late blastocyst (E4.5) stage. Comparative proteomic analyses reveal substantial downregulation of ribosomal proteins, the mRNA transcripts of which are also significantly upregulated. Ontological analysis of the differentially expressed transcriptome during this developmental period reveals “translation” related gene transcripts as being most significantly, yet transiently, affected by p38-MAPKi. Moreover, combined assays consistently report concomitant reductions in de novo translation that are associated with accumulation of unprocessed rRNA precursors. Using a phosphoproteomic approach, ± p38-MAPKi, we identified Mybpp1a, an rRNA transcription and processing regulator gene, as a potential p38-MAPK effector. We report that siRNA mediated clonal knockdown of Mybpp1a is associated with significantly diminished PrE contribution. Lastly, we show that defective PrE specification caused by p38-MAPKi (but not MEK/ERK signalling inhibition) can be partially rescued by activating the archetypal mTOR mediated translation regulatory pathway. Conclusions: Activated p38-MAPK controls blastocyst maturation in an early and distinctly transient developmental window by regulating gene functionalities related to translation, that creates a permissive environment for appropriate specification of ICM cell fate.

Vaccinia virus (VACV) is a prototypical poxvirus originally used for eradication of smallpox. Investigation into VACV mRNAs carried out almost half a century ago substantially contributed to the fundamental discovery of the 5’ mRNA cap, a hallmark of all eukaryotic and many viral mRNAs. VACV research also facilitated the identification and understanding of the general mechanism of 5’ mRNA cap synthesis. We characterized the VACV transcripts at the individual mRNA molecule level and found that vaccinia postreplicative mRNAs, containing nontemplated 5’ poly(A) leaders, surprisingly lack the 5’ cap structure in vivo . We also show that the lengths of the nontemplated leaders and the presence or absence of cap structures at the 5’ mRNA ends are controlled by the initiator sequence of the VACV postreplicative promoters. One Sentence Summary: The promoter sequence determines the synthesis of the 5’ cap and poly(A) leaders in vaccinia virus postreplicative mRNAs.

Stress granules (SGs), hallmarks of the cellular adaptation to stress, promote survival, conserve cellular energy, and are fully dissolved upon the cessation of stress treatment. Different stresses can initiate the assembly of SGs, but arsenite and heat are the best studied of these stresses. The composition of SGs and posttranslational modifications of SG proteins differ depending on the type and severity of the stress insult, methodology used, cell line, and presence of overexpressed and tagged proteins. A group of 18 proteins showing differential localization to SGs in heat‐ and arsenite‐stressed mammalian cell lines is described. Upon severe and prolonged stress, physiological SGs transform into more solid protein aggregates that are no longer reversible and do not contain mRNA. Similar pathological inclusions are hallmarks of neurodegenerative diseases. SGs induced by heat stress are less dynamic than SGs induced by arsenite and contain a set of unique proteins and linkage‐specific polyubiquitinated proteins. The same types of ubiquitin linkages have been found to contribute to the development of neurodegenerative disorders such as Parkinson disease, Alzheimer disease, and amyotrophic lateral sclerosis (ALS). We propose heat stress‐induced SGs as a possible model of an intermediate stage along the transition from dynamic, fully reversible arsenite stress‐induced SGs toward aberrant SGs, the hallmark of neurodegenerative diseases. Stress‐ and methodology‐specific differences in the compositions of SGs and the transition of SGs to aberrant protein aggregates are discussed.This article is categorized under:RNA in Disease and Development > RNA in DiseaseRNA Interactions with Proteins and Other Molecules > RNA–Protein ComplexesRNA Export and Localization > RNA Localization

Meiotic maturation of oocyte relies on pre-synthesised maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol that demonstrates a combination of the sucrose gradient ultracentrifugation in small SW55Ti tubes with the qRT-PCR-based quantification of 18S and 28S rRNAs in fractionated polysome profile. This newly optimised method, named Scarce Sample Polysome Profiling (SSP-profiling), is suitable for both scarce and conventional sample sizes and is compatible with downstream RNA-seq to identify polysome associated transcripts. Utilising SSP-profiling we have assayed the translatome of mouse oocytes at the onset of nuclear envelope breakdown (NEBD)—a developmental point, the study of which is important for furthering our understanding of the molecular mechanisms leading to oocyte aneuploidy. Our analyses identified 1847 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteasomal components, glycolytic and amino acids synthetic enzymes, proteins involved in cytoskeleton organization plus RNA-binding and translation initiation factors. In addition to transcripts encoding known players of meiotic progression, we also identified several mRNAs encoding proteins of unknown function. Polysome profiles generated using SSP-profiling were more than comparable to those developed using existing conventional approaches, being demonstrably superior in their resolution, reproducibility, versatility, speed of derivation and downstream protocol applicability.

The rate of chromosome segregation errors that emerge during meiosis I in the mammalian female germ line are known to increase with maternal age; however, little is known about the underlying molecular mechanism. The objective of this study was to analyze meiotic progression of mouse oocytes in relation to maternal age. Using the mouse as a model system, we analyzed the timing of nuclear envelope breakdown and the morphology of the nuclear lamina of oocytes obtained from young (2 months old) and aged females (12 months old). Oocytes obtained from older females display a significantly faster progression through meiosis I compared to the ones obtained from younger females. Furthermore, in oocytes from aged females, lamin A/C structures exhibit rapid phosphorylation and dissociation. Additionally, we also found an increased abundance of MPF components and increased translation of factors controlling translational activity in the oocytes of aged females. In conclusion, the elevated MPF activity observed in aged female oocytes affects precocious meiotic processes that can multifactorially contribute to chromosomal errors in meiosis I.