Project description:We measured mRNA levels of two yeast species (S.cerevisiae and S.paradoxus) and their hybrid, at four time-points (0, 20min, 40min, 60min) following transcription arrest using 1,10-Phenantroline (150ug/ml). This data was used to infer mRNA degradation rates of orthologous genes, study the divergence of mRNA degradation rates and the contribution of cis and trans mutations.
Project description:We measured mRNA levels of two yeast species (S.cerevisiae and S.paradoxus) and their hybrid, at four time-points (0, 20min, 40min, 60min) following transcription arrest using 1,10-Phenantroline (150ug/ml). This data was used to infer mRNA degradation rates of orthologous genes, study the divergence of mRNA degradation rates and the contribution of cis and trans mutations. For each of the two biological repeats and each of the four time point, poly(A) mRNAs of the two species was pooled and labeled with cy3 while hybrid poly(A) mRNA was labeled with cy5 and these were hybridized to our custom two-species microarray (Agilent) with four subarrays.
Project description:Bacterial gene expression is thought to involve tightly coupled transcription, translation, and mRNA degradation. However, recent work has indicated that this is not always the case, leaving the generality and regulation of this coordination unclear. Here, we use genetic, kinetic, and spatial analyses in Escherichia coli to show that transcription–translation coupling requires high translational activity and that nearly half of the transcriptome exhibits signatures consistent with partial uncoupling. We find that co-transcriptional mRNA degradation is rare due to membrane localization of RNase E, except for transcripts encoding inner membrane proteins. Our results show that translation efficiency determines the level of premature transcription termination, which in turn shapes mRNA degradation patterns and kinetics. Comparative analyses in Bacillus subtilis and Caulobacter crescentus also reveal gene- and species-specific coordination strategies. This challenges the universality of co-transcriptional coupling and defines how spatial and genetic features coordinate bacterial gene expression.
Project description:During the last decade several examples of coordination between gene transcription and mRNA degradation have been reported. mRNA imprinting by Rpb4 and 7 subunits of RNA polymerase II (RNAPII) and by the Ccr4-Not complex allows controlling its fate during transcription. Transcription regulation by mRNA degradation factors like Xrn1 constitutes a feedback loop that contributes to mRNA homeostasis. Mechanistic details of these phenomena are unclear. Most studies involve measurement of mRNA decay rates, usually by stressing procedures such as transcriptional shut-off or incorporation of modified nucleotides that can lead to biased results. In this work we have used the easily repressible yeast GAL1 gene to perform a genetic analysis of mRNA synthesis and degradation under physiological conditions. We combined this experimental approach with computational multi-agent modelling, testing different possibilities of Xrn1 and Ccr4-Not action in gene transcription. This double strategy brought us to conclude that Xrn1 regulates RNAPII backtracking in a Ccr4-independent manner. We validated this conclusion measuring TFIIS genome-wide recruitment to elongating RNAPII molecules. We found that xrn1∆ and ccr4∆ exhibited very different patterns of TFIIS/RNPAII which confirmed their differential role in controlling transcription elongation.
Project description:Transcription was arrested using 50 ug/ul of rifampin leaving RNA degradation as the sole vector responsible for mRNA abundance. mRNA half-lives were calculated from the decay of signal intensity from T0. Three replicates were sampled before transcription arrest, and after 5 and 10 minutes of mRNA decay for a total of 9 arrays. Five minutes of RNA degradation occurred during the processing of these samples, therefore sample times, indicated in the sample name, represent 0, five, and ten minutes of RNA degradation.
Project description:Synthesis and degradation of cellular constituents must be balanced to maintain cellular homeostasis, especially during adaptation to environmental stress. The role of autophagy in the degradation of proteins and organelles is well-characterized. However, autophagy-mediated RNA degradation in response to stress and the potential preference of specific RNAs to undergo autophagy-mediated degradation have not been examined. In this study, we demonstrate selective mRNA degradation by rapamycin-induced autophagy in yeast. Profiling of mRNAs from the vacuole reveals that subsets of mRNAs, such as those encoding amino acid biosynthesis and ribosomal proteins, are preferentially delivered to the vacuole by autophagy for degradation. We also reveal that autophagy-mediated mRNA degradation is tightly coupled with translation by ribosomes. Genome-wide ribosome profiling suggested a high correspondence between ribosome association and targeting to the vacuole. We propose that autophagy-mediated mRNA degradation is a unique and previously-unappreciated function of autophagy that affords post-transcriptional gene regulation.