Biology subjects

Masek, T.

Publications and source records attributed to Masek, T..

4 recordsLinked to original sources

bioRxiv2026

CRISPR-Cas9 Induced Knockout of BEL5 in Tetraploid Potato: Optimized Methodology via Repeated de novo Regeneration and Impact on Tuberization

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 Desiree, 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.

plant biology↗

bioRxiv2020

p38-MAPK mediated rRNA processing and translation regulation enables PrE differentiation during mouse blastocyst maturation.

Backgroundp38-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. ResultsChemical 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. ConclusionsActivated 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.

developmental biology↗

bioRxiv2020

Transcripts of vaccinia virus postreplicative genes do not contain a 5' methylguanosine cap

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 SummaryThe promoter sequence determines the synthesis of the 5 cap and poly(A) leaders in vaccinia virus postreplicative mRNAs.

molecular biology↗

bioRxiv2018

Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Linear plasmids with almost identical compact genetic organization have been found in the cytoplasm of yeast species from nine genera. We employed pGKL1,2 plasmids from Kluyveromyces lactis as a model to investigate the previously unstudied transcriptome of yeast cytoplasmic linear plasmids. We performed 5 and 3 RACE analysis of all the pGKL1,2 mRNAs and found them not 3 polyadenylated and containing mostly uncapped 5 poly(A) leaders that are not complementary to the plasmid DNA. The degree of 5 capping and/or 5 polyadenylation is specific to each gene and is controlled by the corresponding promoter regions. We refined the description of the pGKL1,2 promoters and found new alternative promoters of several genes. We also provide evidence that K2ORF3 encodes an mRNA cap guanine-N7-methyltransferase and that 5 capped pGKL1,2 transcripts contain N7-methylated caps. Translation of pGKL1,2 transcripts is enhanced in Ism1{Delta} and pab1{Delta} strains and is independent of eIF4E and Pab1 translation factors. We suggested a model of a primitive regulation of pGKL1,2 plasmids gene expression where degree of 5 mRNA capping, degree of 5 non-template polyadenylation and presence of negative regulators as PAB1 and Lsm1 play an important role. Our data also suggest a close relationship between linear plasmids and poxviruses.

molecular biology↗