TY - JOUR A1 - Saffert, Paul A1 - Adamla, Frauke A1 - Schieweck, Rico A1 - Atkins, John F. A1 - Ignatova, Zoya T1 - An Expanded CAG Repeat in Huntingtin Causes+1 Frameshifting JF - The journal of biological chemistry N2 - Maintenance of triplet decoding is crucial for the expression of functional protein because deviations either into the -1 or +1 reading frames are often non-functional. We report here that expression of huntingtin (Htt) exon 1 with expanded CAG repeats, implicated in Huntington pathology, undergoes a sporadic +1 frameshift to generate from the CAG repeat a trans-frame AGC repeat-encoded product. This +1 recoding is exclusively detected in pathological Htt variants, i.e. those with expanded repeats with more than 35 consecutive CAG codons. An atypical +1 shift site, UUC C at the 5 end of CAG repeats, which has some resemblance to the influenza A virus shift site, triggers the +1 frameshifting and is enhanced by the increased propensity of the expanded CAG repeats to form a stem-loop structure. The +1 trans-frame-encoded product can directly influence the aggregation of the parental Htt exon 1. KW - aggregation KW - Huntington disease KW - translation KW - translation regulation KW - trinucleotide repeat disease KW - frameshifting KW - seeding Y1 - 2016 U6 - https://doi.org/10.1074/jbc.M116.744326 SN - 0021-9258 SN - 1083-351X VL - 291 SP - 18505 EP - 18513 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Girstmair, Hannah A1 - Saffert, Paul A1 - Rode, Sascha A1 - Czech, Andreas A1 - Holland, Gudrun A1 - Bannert, Norbert A1 - Ignatova, Zoya T1 - Depletion of Cognate Charged Transfer RNA Causes Translational Frameshifting within the Expanded CAG Stretch in Huntingtin JF - Cell reports N2 - Huntington disease (HD), a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG-encoded polyglutamine (polyQ) repeat in huntingtin (Htt), displays a highly heterogeneous etiopathology and disease onset. Here, we show that the translation of expanded CAG repeats in mutant Htt exon 1 leads to a depletion of charged glutaminyl-transfer RNA (tRNA) Gln-CUG that pairs exclusively to the CAG codon. This results in translational frameshifting and the generation of various transframe-encoded species that differently modulate the conformational switch to nucleate fibrillization of the parental polyQ protein. Intriguingly, the frameshifting frequency varies strongly among different cell lines and is higher in cells with intrinsically lower concentrations of tRNA Gln-CUG. The concentration of tRNA Gln-CUG also differs among different brain areas in the mouse. We propose that translational frameshifting may act as a significant disease modifier that contributes to the cell-selective neurotoxicity and disease course heterogeneity of HD on both cellular and individual levels. Y1 - 2013 U6 - https://doi.org/10.1016/j.celrep.2012.12.019 SN - 2211-1247 VL - 3 IS - 1 SP - 148 EP - 159 PB - Cell Press CY - Cambridge ER - TY - GEN A1 - Bentele, Kajetan A1 - Saffert, Paul A1 - Rauscher, Robert A1 - Ignatova, Zoya A1 - Bluethgen, Nils T1 - Efficient translation initiation dictates codon usage at gene start T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The genetic code is degenerate; thus, protein evolution does not uniquely determine the coding sequence. One of the puzzles in evolutionary genetics is therefore to uncover evolutionary driving forces that result in specific codon choice. In many bacteria, the first 5-10 codons of protein-coding genes are often codons that are less frequently used in the rest of the genome, an effect that has been argued to arise from selection for slowed early elongation to reduce ribosome traffic jams. However, genome analysis across many species has demonstrated that the region shows reduced mRNA folding consistent with pressure for efficient translation initiation. This raises the possibility that unusual codon usage is a side effect of selection for reduced mRNA structure. Here we discriminate between these two competing hypotheses, and show that in bacteria selection favours codons that reduce mRNA folding around the translation start, regardless of whether these codons are frequent or rare. Experiments confirm that primarily mRNA structure, and not codon usage, at the beginning of genes determines the translation rate. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 912 KW - codon usage KW - mRNA structure KW - translation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-441337 SN - 1866-8372 IS - 912 ER - TY - JOUR A1 - Bentele, Kajetan A1 - Saffert, Paul A1 - Rauscher, Robert A1 - Ignatova, Zoya A1 - Bluethgen, Nils T1 - Efficient translation initiation dictates codon usage at gene start JF - Molecular systems biology N2 - The genetic code is degenerate; thus, protein evolution does not uniquely determine the coding sequence. One of the puzzles in evolutionary genetics is therefore to uncover evolutionary driving forces that result in specific codon choice. In many bacteria, the first 5-10 codons of protein-coding genes are often codons that are less frequently used in the rest of the genome, an effect that has been argued to arise from selection for slowed early elongation to reduce ribosome traffic jams. However, genome analysis across many species has demonstrated that the region shows reduced mRNA folding consistent with pressure for efficient translation initiation. This raises the possibility that unusual codon usage is a side effect of selection for reduced mRNA structure. Here we discriminate between these two competing hypotheses, and show that in bacteria selection favours codons that reduce mRNA folding around the translation start, regardless of whether these codons are frequent or rare. Experiments confirm that primarily mRNA structure, and not codon usage, at the beginning of genes determines the translation rate. KW - codon usage KW - mRNA structure KW - translation Y1 - 2013 U6 - https://doi.org/10.1038/msb.2013.32 SN - 1744-4292 VL - 9 IS - 6 PB - Nature Publ. Group CY - New York ER - TY - JOUR A1 - Hess, Anne-Katrin A1 - Saffert, Paul A1 - Liebeton, Klaus A1 - Ignatova, Zoya T1 - Optimization of Translation Profiles Enhances Protein Expression and Solubility JF - PLoS one N2 - mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein. Y1 - 2015 U6 - https://doi.org/10.1371/journal.pone.0127039 SN - 1932-6203 VL - 10 IS - 5 PB - PLoS CY - San Fransisco ER - TY - GEN A1 - Hess, Anne-Katrin A1 - Saffert, Paul A1 - Liebeton, Klaus A1 - Ignatova, Zoya T1 - Optimization of translation profiles enhances protein expression and solubility T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 518 KW - transfer-RNA genes KW - codon usage KW - Escherichia coli KW - Epoxide hydrolases KW - messenger-RNA KW - sequence KW - elongation KW - Ribosome KW - mechanism KW - Membrane Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-409574 SN - 1866-8372 IS - 518 ER - TY - JOUR A1 - Puri, Pranav A1 - Wetzel, Collin A1 - Saffert, Paul A1 - Gaston, Kirk W. A1 - Russell, Susan P. A1 - Varela, Juan A. Cordero A1 - van der Vlies, Pieter A1 - Zhang, Gong A1 - Limbach, Patrick A. A1 - Ignatova, Zoya A1 - Poolman, Bert T1 - Systematic identification of tRNAome and its dynamics in Lactococcus lactis JF - Molecular microbiology N2 - Transfer RNAs (tRNAs) through their abundance and modification pattern significantly influence protein translation. Here, we present a systematic analysis of the tRNAome of Lactococcus lactis. Using the next-generation sequencing approach, we identified 40 tRNAs which carry 16 different post-transcriptional modifications as revealed by mass spectrometry analysis. While small modifications are located in the tRNA body, hypermodified nucleotides are mainly present in the anticodon loop, which through wobbling expand the decoding potential of the tRNAs. Using tRNA-based microarrays, we also determined the dynamics in tRNA abundance upon changes in the growth rate and heterologous protein overexpression stress. With a fourfold increase in the growth rate, the relative abundance of tRNAs cognate to low abundance codons decrease, while the tRNAs cognate to major codons remain mostly unchanged. Significant changes in the tRNA abundances are observed upon protein overexpression stress, which does not correlate with the codon usage of the overexpressed gene but rather reflects the altered expression of housekeeping genes. Y1 - 2014 U6 - https://doi.org/10.1111/mmi.12710 SN - 0950-382X SN - 1365-2958 VL - 93 IS - 5 SP - 944 EP - 956 PB - Wiley-Blackwell CY - Hoboken ER -