Chronic hepatitis B (CHB) is caused by the Hepatitis B virus (HBV) and affects millions of people worldwide. Developing an effective CHB therapy requires using in vivo screening methods, such as mouse models reflecting CHB based on hydrodynamic delivery of plasmid vectors containing a replication-competent HBV genome. However, long-term expression of HBV proteins is accompanied by production of progeny virions, thereby requiring a Biosafety Level (BSL) 3 animal facility. In the present study, we introduced a point mutation in the START codon of the HBV polymerase to develop a mouse model reflecting chronic hepatitis B infection without formation of viral progeny. We induced the mouse model by hydrodynamic injection of adeno-associated virus plasmid vector (pAAV) and minicircle plasmid (pMC) constructs into C57Bl/6 and C3H/HeN mouse strains, monitoring HBV antigens and antibodies in blood by enzyme-linked immunosorbent assay and analyzing liver expression of HBV core antigen by immunohistology. Persisting expression of viral antigens over 140 days (study endpoint) was observed only in the C3H/HeN mouse strain when using pAAV/1.2HBV-A and pMC/1.0HBV-D with pre-C and pre-S recombination sites. In addition, pAAV/1.2HBV-A in C3H/HeN sustained HBV core antigen positivity up to the study endpoint in C3H/HeN mice. Moreover, introducing the point mutation in the START codon of polymerase effectively prevented the formation of viral progeny. Our study establishes an accessible and affordable experimental paradigm for developing a robust mouse model reflecting CHB suitable for preclinical testing of anti-HBV therapeutics in a BSL2 animal facility.

• MeSH
• chronická hepatitida B * genetika   MeSH
• kodon iniciační MeSH
• modely nemocí na zvířatech MeSH
• mutace MeSH
• myši inbrední C3H MeSH
• myši MeSH
• virus hepatitidy B genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Translation reinitiation is a gene-specific translational control mechanism. It is characterized by the ability of short upstream ORFs to prevent full ribosomal recycling and allow the post-termination 40S subunit to resume traversing downstream for the next initiation event. It is well known that variable transcript-specific features of various uORFs and their prospective interactions with initiation factors lend them an unequivocal regulatory potential. Here, we investigated the proposed role of the major initiation scaffold protein eIF4G in reinitiation and its prospective interactions with uORF's cis-acting features in yeast. In analogy to the eIF3 complex, we found that eIF4G and eIF4A but not eIF4E (all constituting the eIF4F complex) are preferentially retained on ribosomes elongating and terminating on reinitiation-permissive uORFs. The loss of the eIF4G contact with eIF4A specifically increased this retention and, as a result, increased the efficiency of reinitiation on downstream initiation codons. Combining the eIF4A-binding mutation with that affecting the integrity of the eIF4G1-RNA2-binding domain eliminated this specificity and produced epistatic interaction with a mutation in one specific cis-acting feature. We conclude that similar to humans, eIF4G is retained on ribosomes elongating uORFs to control reinitiation also in yeast.

• MeSH
• DEAD-box RNA-helikasy genetika   MeSH
• eukaryotický iniciační faktor 3 genetika   MeSH
• eukaryotický iniciační faktor 4E genetika   MeSH
• eukaryotický iniciační faktor 4G genetika   MeSH
• iniciace translace peptidového řetězce genetika   MeSH
• kodon iniciační genetika   MeSH
• lidé MeSH
• otevřené čtecí rámce genetika   MeSH
• proteosyntéza genetika   MeSH
• ribozomy genetika   MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• transkripční faktory bZIP genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.

• MeSH
• 5' nepřekládaná oblast MeSH
• eukaryotický iniciační faktor 2 genetika   metabolismus   MeSH
• eukaryotický iniciační faktor 3 genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• iniciační faktory genetika   metabolismus   MeSH
• kodon iniciační MeSH
• lidé MeSH
• malé podjednotky ribozomu eukaryotické genetika   metabolismus   MeSH
• multiproteinové komplexy genetika   metabolismus   MeSH
• proteosyntéza * MeSH
• ribozomy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• transkripční faktor ATF4 genetika   metabolismus   MeSH
• transkripční faktory bZIP genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Klíčová slova
• irisin,
• MeSH
• bazální metabolismus MeSH
• kodon iniciační metabolismus   MeSH
• lidé MeSH
• membránové proteiny metabolismus   terapeutické užití   MeSH
• modely u zvířat MeSH
• myši MeSH
• obezita * metabolismus   terapie   MeSH
• termogeneze MeSH
• transportní proteiny metabolismus   terapeutické užití   MeSH
• tukové buňky metabolismus   MeSH
• uncoupling protein 1 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH

The complete mitochondrial genome of the ascomycete fungus Phomopsis longicolla was sequenced using Illumina HiSeq platform. It consists of 53,439 bp with high (65.6%) A + T content and includes 14 conserved protein subunits of the mitochondrial oxidative phosphorylation system, two ribosomal RNAs and 25 tRNA genes. To our knowledge, this is the first complete mitochondrial genome of the Diaporthales member.

• MeSH
• Ascomycota genetika   MeSH
• délka genomu MeSH
• genom fungální MeSH
• genom mitochondriální * MeSH
• kodon iniciační MeSH
• mitochondriální geny * MeSH
• molekulární evoluce MeSH
• pořadí genů * MeSH
• sekvenční analýza DNA MeSH
• terminační kodon MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zastoupení bazí MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

V roce 2012 byl v odborné literatuře představen myokin irisin. Lidský irisin svou strukturou zcela odpovídá irisinu myšímu. U myší irisin indukuje formaci hnědých adipocytů ovlivňujících energetický metabolismus zvýšením termogeneze. Irisin se proto dostal do popředí zájmu vědců jako látka, která by zvýšením produkce tepla mohla pomáhat při redukčním režimu obézních jedinců. Článek shrnuje dosavadní poznatky o irisinu a kriticky hodnotí možnost jeho využití pro medicínské účely.

Irisine was introduced in scientific literature in 2012 as an exercise-induced myokine. In humans, its structure is identical to the murine homologue. In mice, irisin induces the formation of brown adipocytes which affect energy metabolism by increasing thermogenesis. That is why irisin became perspective as a substance that, by increasing heat production, could help in obesity treatment. This article summarizes current knowledge about irisin and critically evaluates the possibility of its use in human medicine.

• Klíčová slova
• UCP1,
• MeSH
• cvičení MeSH
• energetický metabolismus MeSH
• fibronektiny * genetika   krev   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• hnědá tuková tkáň * metabolismus   MeSH
• kodon iniciační genetika   MeSH
• lidé MeSH
• obezita * farmakoterapie   MeSH
• termogeneze MeSH
• transportní proteiny mitochondriální membrány fyziologie   MeSH
• tukové buňky metabolismus   MeSH
• uncoupling protein 1 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

• MeSH
• kodon iniciační MeSH
• lidé MeSH
• posttranslační úpravy proteinů fyziologie   MeSH
• RNA biosyntéza   MeSH
• translační biomedicínský výzkum * MeSH
• tRNA-methyltransferasy analýza   MeSH
• Check Tag
• lidé MeSH

We examine the standard genetic code with three stop codons. Assuming that the synchronization period of length 3 in DNA or RNA is violated during the transcription or translation processes, the probability of reading a frameshifted stop codon is higher than if the code would have only one stop codon. Consequently, the synthesis of RNA or proteins will soon terminate. In this way, cells do not produce undesirable proteins and essentially save energy. This hypothesis is tested on the AT-rich Drosophila genome, where the detection of frameshifted stop codons is even higher than the theoretical value. Using the binomial theorem, we establish the probability of reading a frameshifted stop codon within n steps. Since the genetic code is largely redundant, there is still space for some hidden secondary functions of this code. In particular, because stop codons do not contain cytosine, random C → U and C → T mutations in the third position of codons increase the number of hidden frameshifted stops and simultaneously the same amino acids are coded. This evolutionary advantage is demonstrated on the genomes of several simple species, e.g. Escherichia coli.

• MeSH
• DNA genetika   MeSH
• Drosophila genetika   MeSH
• druhová specificita MeSH
• genetický kód genetika   MeSH
• genom hmyzu genetika   MeSH
• hemoglobiny genetika   MeSH
• hmyzí geny genetika   MeSH
• kodon iniciační MeSH
• lidé MeSH
• posunová mutace MeSH
• RNA genetika   MeSH
• terminační kodon MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1(G107R) but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.

• MeSH
• eukaryotický iniciační faktor 1 genetika   MeSH
• eukaryotický iniciační faktor 3 chemie   genetika   metabolismus   MeSH
• fenotyp MeSH
• genová dávka MeSH
• iniciace translace peptidového řetězce MeSH
• kodon iniciační MeSH
• krystalografie rentgenová MeSH
• kvasinky genetika   růst a vývoj   MeSH
• malé podjednotky ribozomu eukaryotické metabolismus   MeSH
• molekulární modely MeSH
• mutace MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In eukaryotes, for a protein to be synthesized, the 40 S subunit has to first scan the 5'-UTR of the mRNA until it has encountered the AUG start codon. Several initiation factors that ensure high fidelity of AUG recognition were identified previously, including eIF1A, eIF1, eIF2, and eIF5. In addition, eIF3 was proposed to coordinate their functions in this process as well as to promote their initial binding to 40 S subunits. Here we subjected several previously identified segments of the N-terminal domain (NTD) of the eIF3c/Nip1 subunit, which mediates eIF3 binding to eIF1 and eIF5, to semirandom mutagenesis to investigate the molecular mechanism of eIF3 involvement in these reactions. Three major classes of mutant substitutions or internal deletions were isolated that affect either the assembly of preinitiation complexes (PICs), scanning for AUG, or both. We show that eIF5 binds to the extreme c/Nip1-NTD (residues 1-45) and that impairing this interaction predominantly affects the PIC formation. eIF1 interacts with the region (60-137) that immediately follows, and altering this contact deregulates AUG recognition. Together, our data indicate that binding of eIF1 to the c/Nip1-NTD is equally important for its initial recruitment to PICs and for its proper functioning in selecting the translational start site.

• MeSH
• eukaryotický iniciační faktor 3 genetika   metabolismus   MeSH
• iniciace translace peptidového řetězce fyziologie   MeSH
• kodon iniciační genetika   metabolismus   MeSH
• malé podjednotky ribozomu eukaryotické genetika   metabolismus   MeSH
• multiproteinové komplexy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH