When rDNA transcription is arrested during mitosis, UBF is still associated with non-condensed rDNA

Abstract

The mechanisms that control inactivation of ribosomal gene (rDNA) transcription during mitosis is still an open question. To investigate this fundamental question, the precise timing of mitotic arrest was established. In PtK1 cells, rDNA transcription was still active in prophase, stopped in prometaphase until early anaphase, and activated in late anaphase. Because rDNA transcription can still occur in prophase and late anaphase chromosomes, the kinetics of rDNA condensation during mitosis was questioned. The conformation of the rDNA was analyzed by electron microscopy from the G2/M transition to late anaphase in the secondary constriction, the chromosome regions where the rDNAs are clustered. Whether at transcribing or non-transcribing stages, non-condensed rDNA was observed in addition to axial condensed rDNA. Thus, the persistence of this non-condensed rDNA during inactive transcription argues in favor of the fact that mitotic inactivation is not the consequence of rDNA condensation. Analysis of the three-dimensional distribution of the rDNA transcription factor, UBF, revealed that it was similar at each stage of mitosis in the secondary constriction. In addition, the colocalization of UBF with non-condensed rDNA was demonstrated. This is the first visual evidence of the association of UBF with non-condensed rDNA. As we previously reported that the rDNA transcription machinery remained assembled during mitosis, the colocalization of rDNA fibers with UBF argues in favor of the association of the transcription machinery with certain rDNA copies even in the absence of transcription. If this hypothesis is correct, it can be assumed that condensation of rDNA as well as dissociation of the transcription machinery from rDNA cannot explain the arrest of rDNA transcription during mitosis. It is proposed that modifications of the transcription machinery occurring in prometaphase could explain the arrest of transcription, while reverse modifications in late anaphase could explain activation.

The cell cycle and cell size influence the rates of global cellular translation and transcription in fission yeast

“…It is also possible that the increase happens at a certain stage of S-phase independent of the amount of DNA since we do not know the extent of S-phase of each cell. We did not observe a reduction in global cellular transcription through mitosis, unlike previous work in mammalian cells (Wansink et al, 1993;G ebrane-Youn es et al, 1997) and budding yeast (Clemente-Blanco et al, 2009). It is possible that undertaking mitosis without breaking down the nuclear envelope (Zheng et al, 2007) prevents the reduction in transcription observed in mammalian cells undertaking open mitosis.…”

Rates of global cellular translation and transcription during cell growth and the cell cycle in fission yeast

“…The dynamic equilibrium model assumes that the increase in the occupancy of RNA polymerases is due to a dynamic equilibrium between free polymerases associating with the DNA and detaching from the DNA. We did not observe a reduction in global cellular transcription through mitosis unlike previous work in mammalian cells [48,49] and budding yeast [50]. It is possible that undertaking mitosis without breaking down the nuclear envelope [51] prevents the reduction in transcription observed in mammalian cells undertaking an open mitosis.…”

Chromatin association and regulation of rDNA transcription by the Ras-family protein RasL11a

“…Only a fraction of the 10 NORs in a diploid cell are active, the others being in a repressed, heterochromatic state (Grummt, 2003; Moss et al , 2007; McStay and Grummt, 2008). On entry into mitosis the nucleolus disassembles and rDNA transcription is silenced, but active NORs retain their open configuration and remain associated with the RPI transcription machinery, including UBF (Roussel et al , 1993, 1996; Jordan et al , 1996; Gebrane‐Younes et al , 1997; Grummt, 2003; Leung et al , 2004; Mais et al , 2005; Moss et al , 2007; McStay and Grummt, 2008). In our immunofluorescence experiments, examination of cells at different stages of mitosis showed that RasL11a and UBF formed prevalent foci on chromosome arms.…”

“…RasL11a co-localizes with UBF not only at nucleoli in interphase, but also on chromosomes in mitotic cells. UBF and the RPI machinery mark a subset of rDNA repeats on chromosome arms (NORs), which correspond to the active repeats (Roussel et al, 1993(Roussel et al, , 1996Jordan et al, 1996;Gebrane-Younes et al, 1997;Leung et al, 2004;Mais et al, 2005). The fact that RasL11a co-localizes on chromosome arms with either UBF (by immunofluorescence) or rDNA (by immuno-FISH) shows that RasL11a also marks active NORs in mitosis.…”