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Gene-specific transcript buffering revealed by perturbation of coactivator complexes

ABSTRACT: Transcript buffering entails reciprocal modulation of mRNA synthesis and degradation to maintain stable RNA levels under varying cellular conditions. Current models depict a global connection between mRNA synthesis and degradation, but underlying mechanisms remain unclear. Here we show that changes in RNA metabolism following depletion of TIP60/KAT5, the acetyltransferase subunit of the NuA4 transcriptional coactivator complex, reveal that transcript buffering occurs at a gene-specific level. By combining RNA sequencing of nuclear, cytoplasmic, and newly synthesised transcript fractions with biophysical modelling in mouse embryonic stem cells, we demonstrate that transcriptional changes caused by TIP60 depletion are offset by corresponding changes in RNA nuclear export and cytoplasmic stability, indicating gene-specific buffering. Disruption of the unrelated ATAC coactivator complex also causes gene-specific transcript buffering. We propose that cells dynamically adjust RNA splicing, export, and degradation in response to individual RNA synthesis alterations, thereby sustaining cellular homeostasis.

ORGANISM(S): Mus musculus

PROVIDER: GSE291187 | GEO | 2025/03/10

REPOSITORIES: GEO

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Project description:The rates of mRNA synthesis and degradation determine cellular mRNA levels and can be monitored by comparative Dynamic Transcriptome Analysis (cDTA) that uses non-perturbing metabolic RNA labeling. Here we present cDTA data for 46 yeast strains lacking genes involved in mRNA degradation and metabolism. In these strains, changes in mRNA degradation rates are generally compensated by changes in mRNA synthesis rates, resulting in a buffering of mRNA levels. We show that buffering of mRNA levels requires the RNA exonuclease Xrn1. The buffering is rapidly established when mRNA synthesis is impaired, but is delayed when mRNA degradation is impaired, apparently due to Xrn1-dependent transcription repressor induction. Cluster analysis of the data defines the general mRNA degradation machinery, reveals different substrate preferences for the two mRNA deadenylase complexes Ccr4-Not and Pan2-Pan3, and unveils an interwoven cellular mRNA surveillance network. For this purpose, cDTA was carried out as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111). S. cerevisiae cultures were grown at 30¡ãC in 50 ml aliquots of YPD medium. S. pombe cultures were grown at 32¡ãC in YES medium. For inhibitor perturbation, BY4741 cells (OD600=0.1) were inoculated from a saturated overnight culture in two 100 ml aliquots of YPD liquid medium, incubated at 30¡ãC and grown to early-log phase (OD600=0.8). Cells were labeled for 6 minutes (sample 0 min). Then cycloheximide was added and cells were labeled for 10 minutes and 60 minutes. Cells were harvested and treated as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111). 1,10-Phenanthroline was added to the culture to a final concentration of 25 _g/ml as described (Dori-Bachash et al., 2012), and labeled for 6 minutes after 18 minutes treatment. Cells were treated as above. For the anchor-away analysis, S. cerevisiae cells (OD600=0.1) were cultured in two 200 ml aliquots of YPD containing 1_g/ml rapamycin and incubated at 30¡ãC untill OD600 reached 0.8. Due to the instability of rapamycin, we added 1_g/ml rapamycin every two hours. Then a 20 ml sample was taken and labeled with 4sU. The cells were then treated as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111).

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Gene-specific transcript buffering revealed by perturbation of coactivator complexes

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