Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus

Abstract

Nucleoli gained interest for their role as cellular stress sensors and as potential targets for cancer treatment. The effect of DNA double-strand breaks (DSBs) in nucleoli on rRNA transcription or nucleolar organisation appears to depend on the agent used to introduce DSBs, DSB frequency and presence (or not) of DSBs outside the nucleoli. We target nucleoli with carbon ions at the ion microbeam SNAKE to address the controversy. Localized ion irradiation with 1 to 100 carbon ions per point (about 0.3-30 Gy per nucleus) did not lead to overall reduced ribonucleotide incorporation in the targeted nucleolus or other nucleoli of the same cell. However, both 5EU incorporation and Parp1 protein level are locally decreased at the damaged nucleolar chromatin regions marked by γH2AX, suggesting a localized inhibition of rRNA transcription. This locally restricted transcriptional inhibition was not accompanied by nucleolar segregation, a structural reorganisation observed after inhibition of rRNA transcription by treatment with actinomycin D or UV irradiation. The present data indicates that even multiple complex DSBs do not lead to pan-nucleolar response if affecting only a subnucleolar region.