COPY NUMBER, EPIGENETIC STATE AND EXPRESSION OF THE RIBOSOMAL-RNA GENES IN YOUNG AND SENESCENT RAT EMBRYO FIBROBLASTS

The recent cloning of the gene that causes the premature aging in Wern er syndrome patients has evoked speculations that deficits in expressi on of the ribosomal RNA genes could be related to cellular aging in ge neral. Here we compare the state of the rRNA genes and the rRNA metabo lism in young and senescent (aged) rat embryo fibroblasts (REF), South ern blot analysis revealed that the copy number and the methylation st ate of the genes did not change significantly with increasing cumulati ve population doublings (CPD) of the culture. Hence, young (low number s of CPD) acid senescent REF (high numbers of CPD), respectively, have the same repertoire of rDNA units that can be transcribed, The rRNA s ynthesis in these cells was analyzed by incorporation of labeled uridi ne at conditions allowing the measurement of absolute rather than rela tive rRNA synthesis rates, We revealed that the cell density dependenc e of the rRNA synthesis diminishes in senescent cells. Exponentially g rowing young REF exhibited an rRNA synthesis of 16 amol uridine incorp oration per minute acid cell, The rRNA synthesis decreased 10-fold in quiescent cells at saturating cell densities, Exponentially growing RE F near the end of their replicative lifespan exhibited a 2-fold lower rRNA synthesis rate compared to young cells, However, in senescent REP the rRNA synthesis rate decreased only 2-fold with increasing cell de nsities resulting in a 3-fold higher rRNA synthesis rate compared to y oung cells at saturating cell densities. These data could be confirmed by calculating the rRNA synthesis rates from the rRNA content, the rR NA half-life, and the proliferation rate of the cells, Hence, senescen t REF exhibited a higher rRNA synthesis rate when compared to young ce lls at similar growth rates resulting in the generally observed higher rRNA content (and cell size) of senescent cells, We conclude that cel lular senescence of REF is not accompanied by rRNA expression deficien cies.