Biology subjects

Machne, R.

Publications and source records attributed to Machne, R..

4 recordsLinked to original sources

bioRxiv2024

Alternate RNA decoding results in stable and abundant proteins in mammals

Amino acid substitutions may substantially alter protein stability and function, but the contribution of substitutions arising from alternate translation (deviations from the genetic code) is unknown. To explore it, we analyzed deep proteomic and transcriptomic data from over 1,000 human samples, including 6 cancer types and 26 healthy human tissues. This global analysis identified 60,803 fragmentation spectra corresponding to 8,801 unique substitution sites in proteins derived from 1,782 genes, including 2,000 confidently localized sites. Some substitutions are shared across samples, while others exhibit strong tissue-type and cancer specificity. Surprisingly, products of alternate translation are more abundant than their canonical counterparts for hundreds of proteins, suggesting sense codon recoding. Recoded proteins include transcription factors, proteases, signaling proteins, and proteins associated with neurodegeneration. Mechanisms contributing to substitution abundance include protein stability, codon frequency, codon-anticodon mismatches, and RNA modifications. We characterize how alternatively translated proteoform ratios vary across protein domains, tissue types and cancers. The substitution ratios are positively associated with intrinsically disordered regions and genetic polymorphisms in gnomAD, though the polymorphisms cannot account for the substitutions. The sequence, relative abundance, and the tissue-specificity of alternatively translated proteins are conserved between human and mouse. These results demonstrate the contribution of alternate translation to diversifying mammalian proteomes, and its association with protein stability, tissue-specific proteomes, and diseases.

molecular biology↗

bioRxiv2021

Uncoupling of the Diurnal Growth Program by Artificial Genome Relaxation in Synechocystis sp. PCC 6803

In cyanobacteria DNA supercoiling varies over the diurnal light/dark cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knock-down of gyrase subunits and overexpression of topoisomerase I (TopoI), and characterized the phenotypes. Cell division was blocked, most likely due to inhibition of genomic but not plasmid DNA replication. Cell growth continued to 4-5x of the wildtype cell volume, and metabolic flux was redirected towards glycogen in the TopoI overexpression strain. TopoI induction initially lead to down-regulation of GC-rich and up-regulation of AT-rich genes. The response quickly bifurcated and four diurnal co-expression cohorts (dawn, noon, dusk and night) all responded differently, in part with a circadian ({approx} 24 h) pattern. A GC-rich region - 50 bp of transcription start sites is differentially enriched in these four cohorts. We suggest a model where energy- and gyrase-gated transcription of growth genes at the dark/light transition (dawn) generates DNA supercoiling which then facilitates DNA replication and initiates the diurnal transcriptome program.

microbiology↗

bioRxiv2021

Plasmid supercoiling decreases during the dark phase in cyanobacteria: a clarification of the interpretation of chloroquine-agarose gels.

In cyanobacteria DNA supercoiling varies over the diurnal light/dark cycle and is integrated with the circadian transcription program and (Woelfle et al. [2007], Vijayan et al. [2009], PNAS). Specifically, Woelfle et al. have reported that DNA supercoiling of an endogenous plasmid became progressively higher during prolonged dark phases in Synechococcus elongatus PCC 7942. This is counterintuitive, since higher levels of negative DNA supercoiling are commonly associated with exponential growth and high metabolic flux. Vijayan et al. then have reverted the interpretation of plasmid mobility on agarose gels supplemented with chloroquine diphosphate (CQ), but not further discussed the differences. Here, we set out to clarify this open issue in cyanobacterial DNA supercoiling dynamics. We first re-capitulate Kellers band counting method (1975, PNAS) using CQ instead of ethidium bromide as the intercalating agent. A 500x-1000x higher CQ concentration is required in the DNA relaxation reaction (topoisomerase I) than in the agarose gel buffer to quench all negative supercoiling of pUC19 extracted from Escherichia coli. This is likely due to the dependence of both, the DNA binding affinity of CQ and the induced DNA unwinding angle, on the ionic strength of the buffer. Lower levels of CQ were required to fully relax in vivo pUC19 supercoiling than were used by Woelfle et al. Next, we analyzed the in vivo supercoiling of endogenous plasmids of Synechocystis sp. PCC 6803, at two different CQ concentrations. These experiments indicate that negative supercoiling of plasmids does not increase but decreases in the dark phase, and progressively decreases further in prolonged darkness.

microbiology↗

bioRxiv2021

Spatio-temporal Coordination of Transcription During the Cell Growth Cycle in Budding Yeast.

Metabolic oscillations are characterized by alternating phases of high and low respiratory activity, associated with transcription of genes involved in biosynthetic pathways and growth, and in catabolism and stress response. However, the functional consequences of transcriptome oscillations remain unclear, since most proteins are too stable to be affected by oscillatory transcript abundances. In this work, we investigate a transcriptome time series during an unstable state of the oscillation. Our analyses confirm previous suggestions that the relative times spent in the alternative transcription states are coupled to growth rate. This pulse-width modulation of transcription provides a simple mechanism for the long-standing question of how cells adjust their ribosome content and growth rate to environmental conditions. A mathematical model of this idea reproduces both the almost linear relation of transcript and protein abundances and the non-linear relation of oscillation periods to growth rate.

cell biology↗