Mitochondrial biogenesis and neural differentiation of human iPSC is modulated by idebenone in a developmental stage-dependent manner.

Idebenone, the synthetic analog of coenzyme Q10 can improve electron transport in mitochondria. Therefore, it is used in the treatment of Alzheimer's disease and other cognitive impairments. However, the mechanism of its action on neurodevelopment is still to be elucidated. Here we demonstrate that the cellular response of human induced pluripotent stem cells (hiPSC) to idebenone depends on the stage of neural differentiation. When: neural stem cells (NSC), early neural progenitors (eNP) and advanced neural progenitors (NP) have been studied a significant stimulation of mitochondrial biogenesis was observed only at the eNP stage of development. This coexists with the enhancement of cell viability and increase in total cell number. In addition, we report novel idebenone properties in a possible regulation of neural stem cells fate decision: only eNP stage responded with up-regulation of both neuronal (MAP2), astrocytic (GFAP) markers, while at NSC and NP stages significant down-regulation of MAP2 expression was observed, promoting astrocyte differentiation. Thus, idebenone targets specific stages of hiPSC differentiation and may influence the neural stem cell fate decision.

Sensitivity of hiPSC-derived neural stem cells (NSC) to Pyrroloquinoline quinone depends on their developmental stage.

Pyrroloquinoline quinone (PQQ) is a factor influencing on the mitochondrial biogenesis. In this study the PQQ effect on viability, total cell number, antioxidant capacity, mitochondrial biogenesis and differentiation potential was investigated in human induced Pluripotent Stem Cells (iPSC) - derived: neural stem cells (NSC), early neural progenitors (eNP) and neural progenitors (NP). Here we demonstrated that sensitivity to PQQ is dependent upon its dose and neural stage of development. Induction of the mitochondrial biogenesis by PQQ at three stages of neural differentiation was evaluated at mtDNA, mRNA and protein level. Changes in NRF1, TFAM and PPARGC1A gene expression were observed at all developmental stages, but only at eNP were correlated with the statistically significant increase in the mtDNA copy numbers and enhancement of SDHA, COX-1 protein level. Thus, the "developmental window" of eNP for PQQ-evoked mitochondrial biogenesis is proposed. This effect was independent of high antioxidant capacity of PQQ, which was confirmed in all tested cell populations, regardless of the stage of hiPSC neural differentiation. Furthermore, a strong induction of GFAP, with down regulation of MAP2 gene expression upon PQQ treatment was observed. This indicates a possibility of shifting the balance of cell differentiation in the favor of astroglia, but more research is needed at this point.

Developmental stage dependent neural stem cells sensitivity to methylmercury chloride on different biofunctional surfaces.

Sensitivity of neural stem cells viability, proliferation and differentiation upon exposure to methylmercury chloride (MeHgCl) was investigated on different types of biofunctional surfaces. Patterns of biodomains created by microprinting/microspotting of poly-l-lysine or extracellular matrix proteins (fibronectin and vitronectin) allowed for non-specific electrostatic or specific, receptor mediated interactions, respectively, between stem cells and the surface. The neural stem cell line HUCB-NSC has been previously shown to be susceptible to MeHgCl in developmentally dependent manner. Here we demonstrated that developmental sensitivity of HUCB-NSC to MeHgCl depends upon the type of adhesive biomolecules and the geometry of biodomains. Proliferation of HUCB-NSC was diminished in time and MeHgCl concentration dependent manner. In addition, the response to MeHgCl was found to be cell-type dependent. Undifferentiated cells were the most sensitive independently of the type of bioactive domain. Significant decrease of GFAP+ cells was detected among cells growing on poly-l-lysine, while on fibronectin and vitronectin, this effect was observed only in the highest (1µM) concentration of MeHgCl. ß-Tubulin III expressing cells were most sensitive on fibronectin domains. In addition, limited bioactive domains to µm in size, as compared to non-patterned larger area of the same adhesive substrate, exerted protective role. Thus, the surface area and type of cell/biofunctional surface interaction exerted significant influence on developmental stage and cell-type specific response of HUCB-NSC to MeHgCl.

A human putative Suv3-like RNA helicase is conserved between Rhodobacter and all eukaryotes.

We have cloned and sequenced a cDNA of the human homologue of the Saccharomyces cerevisiae Suv3 putative RNA helicase which is indispensable for mitochondrial function in yeast. The human Suv-3-like protein has a typical mitochondrial leader sequence. Northern blot data and analysis of ESTs in the data banks indicate that this human gene (SUPV3L1) is expressed in practically all tissues, though at different levels. Sequence homology analysis has shown a strong conservation of the protein in a number of eukaryotic organisms -- plants, mammals and fungi, but no close homologues exist in bacteria with the exception of the purple bacterium Rhodobacter sphaeroides. This gene is thus ubiquitously present in all eukaryotic organisms.

The yeast nuclear gene DSS1, which codes for a putative RNase II, is necessary for the function of the mitochondrial degradosome in processing and turnover of RNA.

The yeast nuclear gene DSS1 codes for a mitochondrial protein containing regions of homology to bacterial RNase II and can act as a multicopy suppressor of a deletion of the SUV3 gene, which encodes an RNA helicase. In order to establish the function of the DSS1 gene in mitochondrial biogenesis we studied RNA metabolism in yeast strains disrupted for SUV3 or DSS1. The results indicate that in the absence of DSS1 the in vitro activity of 3'-5' exoribonuclease is abolished and mitochondrial translation is blocked. In disruption strains harboring intronless mitochondrial genomes steady-state levels of COB mRNA and 16S rRNA were very low, while in the presence of a mitochondrial genome containing the omega intron in the 21S rRNA gene the excised intron accumulates. Moreover we observed an accumulation of precursors of 21S rRNA and the VAR1 mRNA. All these phenotypes are virtually identical to those of strains in which SUV3 is disrupted. We suggest that the DSS1 gene product, like the SUV3 gene product, is a subunit of the yeast mitochondrial degradosome (mtEXO), and that this protein complex participates in intron-independent turnover and processing of mitochondrial transcripts. In addition our studies exclude any role for the NUC1 nuclease in these phenomena.

Yeast nuclear PET127 gene can suppress deletions of the SUV3 or DSS1 genes: an indication of a functional interaction between 3' and 5' ends of mitochondrial mRNAs.

Saccharomyces cerevisiae nuclear genes SUV3 and DSS1 encode putative RNA helicase and RNase II, respectively, which are subunits of the mitochondrial degradosome (mtEXO): a three-protein complex which has a 3' to 5' exoribonuclease activity and plays a major role in regulating stability of mitochondrial RNA. Lack of either of the two gene products results in a respiratory negative phenotype, while on the molecular level it causes a total block of mitochondrial translation, loss of the in vitro exoribonuclease activity and changes in stability and processing of many mtRNAs. We have found that the yeast nuclear gene PET127 present on a low or high copy number vector can effectively suppress the effects of the SUV3 or DSS1 gene disruptions. Since the product of the PET127 gene is involved in processing of the 5' ends of mitochondrial mRNAs, we suggest that there is a functional coupling between the 5' and 3' ends of mitochondrial mRNAs.

A novel restriction endonuclease UnbI, a neoschizomer of Sau96I from an unidentified psychrofilic bacterium from Antarctica is inhibited by phosphate ions.

A novel type II restriction endonuclease UnbI was isolated from an unidentified psychrofilic bacterial strain from Antarctica. UnbI recognizes and cleaves the sequence 5'-GGNCC-3', producing 5 nucleotide long sticky ends. In this respect it differs from its neoschizomer Sau96I and all other restriction enzymes recognizing this sequence. UnbI has a relatively low temperature optimum of 15 degrees C to 20 degrees C and its activity is completely inhibited by inorganic phosphate.

The S. cerevisiae nuclear gene SUV3 encoding a putative RNA helicase is necessary for the stability of mitochondrial transcripts containing multiple introns.

The product of the nuclear gene SUV3 is implicated in a variety of post-transcriptional processes in yeast mitochondria. We have analysed the effect of SUV3 gene-disruption on the expression of intron-containing alleles of the mitochondrial cytb and cox1 genes. We have constructed several strains with mitochondrial genomes containing different combinations of cytb and cox1 introns, and associated these genomes with the disruption of SUV3. The resulting strains were tested for their respiratory competence and spectral cytochrome content. All the strains containing only two or three introns showed normal expression of cytb and cox1, whereas the strains containing more introns were unable to express the appropriate gene. The analysis of mitochondrial RNAs by Northern hybridisation showed that the loss of respiratory competence in the strains containing more introns is due to the decrease of mRNA level with no over-accumulation of high-molecular-weight precursors. However, the transcription of the genes was not affected. These results led us to the notion that SUV3 is required for the stability of intron-containing cytb and cox1 transcripts in a cumulative way, not dependent on any particular intron.

The novel nuclear gene DSS-1 of Saccharomyces cerevisiae is necessary for mitochondrial biogenesis.

A previously unknown nuclear gene DSS-1 from Saccharomyces cerevisiae was cloned and sequenced. The gene was isolated as a multicopy suppressor of a disruption of the SUV-3 gene coding for a DEAD/H box protein involved in processing and turnover of mitochondrial transcripts. The DSS-1 gene codes for a 970 amino-acid protein of molecular weight 111 kDa and is necessary for mitochondrial biogenesis. Amino-acid sequence analysis indicates the presence of motifs characteristic for Escherichia coli RNase II, the dis3 protein from Schizosaccharomyces pombe, the cyt4 protein participating in RNA processing and turnover in Neurospora crassa mitochondria, and the vacB protein from Shigella flexneri. We suggest that the DSS-1 protein may be a component of the mitochondrial 3'-5' exoribonuclease complex.

The suv3 nuclear gene product is required for the in vivo processing of the yeast mitochondrial 21s rRNA transcripts containing the r1 intron.

We have constructed a yeast mitochondrial genome containing only one group-I intron, r1, from the 21s rRNA gene and introduced this genome into a strain bearing a disruption of the suv3 gene. The presence of the r1 intron alone causes a block in respiration, while the isogenic strain containing the intronless genome is respiratory competent. Northern analysis indicates that the functional suv3 protein is necessary for the yeast cell in order to process the r1-containing transcripts: in the absence of the suv3 protein the hybridization pattern of the excised r1 intron is altered and the amount of mature 21s rRNA is 50-fold lower. We suggest that the multifunctional suv3 protein, which displays motifs of ATP-dependent RNA helicases, is necessary for the in vivo pathway leading to formation of mature 21s rRNA from the transcripts containing the r1 intron in mitochondria of Saccharomyces cerevisiae.

Evolutionary conservation of the transcribed spacer sequences of the rDNA repeat unit in three species of the genus Aspergillus.

We have cloned and sequenced the two intervening transcribed spacers in the rDNA repeat unit of three Aspergillus species--A. nidulans, A. awamori and A. wentii. The A. wentii and A. awamori spacers are almost identical and share a high degree of homology with the A. nidulans spacers. All spacers have a high G-C content (66%-76%) and the potential of forming complex secondary structures, which may indicate that they play a role in the maturation of pre-rRNA molecules.

The yeast nuclear gene suv3 affecting mitochondrial post-transcriptional processes encodes a putative ATP-dependent RNA helicase.

Mitochondrial gene expression is controlled largely through the action of products of the nuclear genome. The yeast nuclear gene suv3 has been implicated in a variety of mitochondrial posttranscriptional processes and in translation and, thus, represents a key control element in nuclear-mitochondrial interactions. We have exploited a property of a mutant allele of suv3, SUV3-1, that causes, among other effects, a massive increase in the abundance of excised group I introns to clone the wild-type gene by a strategy of colony Northern hybridization. We have determined that the 84-kDa deduced protein product of the suv3 gene, which maps to chromosome XVI, has a typical mitochondrial targeting presequence and additional sequence motifs that suggest that it belongs to a family of ATP-dependent RNA helicases, enzymes whose importance in post-transcriptional and translational events has recently become apparent. We have identified the SUV3-1 mutation as a G----T transversion that creates a Val----Leu substitution in a 10-amino acid block that is highly conserved among ATP-dependent RNA helicases. We discuss some implications of this mutation on the effects of the SUV3-1 allele on mitochondrial RNA metabolism.

Generation of genetic recombinants in Trichosporon cutaneum by spontaneous segregation of protoplast fusants.

Auxotrophic mutants were isolated in two strains of Trichosporon cutaneum. Complementing auxotrophs were hybridized by protoplast fusion. Some of the fusants were apparently transient diploids and segregated to give recombinant marker combinations.

Structure and evolution of 5S rRNA genes and pseudogenes in the genus Aspergillus.

We have cloned and determined the nucleotide sequence of 18 DNA fragments hybridizing to 5S rRNA from two Aspergillus species--A. wentii and A. awamori. Four of the analyzed sequences were pseudogenes. The gene sequences of these two species were very similar and differed from Aspergillus nidulans at both constant and microheterogeneous sites.

Unusual evolutionary conservation of 5S rRNA pseudogenes in Aspergillus nidulans: similarity of the DNA sequence associated with the pseudogenes with the mouse immunoglobulin switch region.

All Aspergillus nidulans 5S rRNA pseudogenes known so far are the result of integration of an approx. 0.2-kbp-long DNA sequence into the 5S rRNA genes. This sequence, called block C, is present in at least five copies in the A. nidulans genome and seems to be associated either with 5S rRNA genes or pseudogenes. In contrast to the 78% sequence conservation of the C-block in pseudogenes, the truncated 5' halves of the pseudogenes are very highly conserved (96.9-100%). We postulate that the 5S rRNA pseudogenes are still a subject of concerted evolution. The C-block sequence shows similarity to the switch region of the mouse heavy chain immunoglobulin gene. A characteristic motif GGGTGAG is repeated several times in both sequences; the sequence conservation is 63%.

Synthesis of human insulin gene. VIII. Construction of expression vectors for fused proinsulin production in Escherichia coli.

We have constructed two families of plasmids suitable for the cloning of genes and for directing the synthesis of large amounts of fused proteins in Escherichia coli. The plasmids include the E. coli lac promoter and a portion of the coding sequence for beta-galactosidase, which can code for approx. 590 or 450 amino acids. The truncated beta-galactosidase gene ends with a poly-linker region at the 3' end, which can be cleaved by any one of the eight common restriction enzymes and joined to the gene coding for any desired protein. Each family includes three plasmids that enable fusion to be made in all three of the translational reading frames. We have cloned a synthetic human proinsulin gene into these plasmids, and 30% of the total E. coli protein was represented by the 590 amino acid-long truncated beta-galactosidase fused to proinsulin. The yield of proinsulin in this system is more than twice the amount produced by using a 1007 amino acid-long beta-galactosidase gene for fusion.