The gene Tousled of Arabidopsis Thaliana encodes a protein kinase which, when mutated, results in abnormal ower development. From a library of mRNAs that are translationally upregulated by overexpression of the translation initiation factor 4E, we identi®ed a mammalian Tousled Like kinase (TLK1B). The human TLK1B mRNA contains a 5'UTR 1088-nt-long with two upstream AUG codons, and was found to be very inhibitory for translation. The TLK1B protein localizes almost exclusively to the nuclei. TLK1B overexpression in mammalian cells rendered them more resistant to ionizing radiation (IR). Puri®ed TLK1B phosphorylated histone H3 at S 10 with high speci®city both in a mix of core histones and in isolated chromatin, suggesting that histone H3 is a physiological substrate for TLK1B. Moreover, overexpression of TLK1B in transfected cells resulted in a higher degree of H3 phosphorylation. Expression of TLK1B in a yeast strain that harbors a temperature-sensitive mutation of the major H3 kinase, Ipl1, complemented the growth defect; restored normal levels of histone H3 phosphorylation; and increased their resistance to IR. Phosphorylation of H3 has been linked to the activation of the immediate-early genes upon mitogenic stimulation, and to chromatin condensation during mitotic/meiotic events. A possible role for TLK1B in radioprotection is discussed. Oncogene (2001) 20, 726 ± 738.

Ribonucleotide reductase synthesizes dNDPs, a specific and limiting step in DNA synthesis, and can participate in neoplastic transformation when overexpressed. The small subunit (ribonucleotide reductase 2 (RNR2)) was cloned as a major product in a subtraction library from eukaryotic initiation factor 4E (eIF4E)-transformed cells (Chinese hamster ovary-4E (CHO-4E)). CHO-4E cells have 20 -40-fold elevated RNR2 protein, reflecting an increased distribution of RNR2 mRNA to the heavy polysomes. CHO-4E cells display an altered cell cycle with shortened S phase, similar to cells selected for RNR2 overexpression with hydroxyurea. The function of ribonucleotide reductase as a checkpoint component of S progression was studied in yeast in which elevated eIF4E rescued S-arrested rnr2-68 ts cells, by increasing recruitment of its mRNA to polysomes. Crosses between rnr2-68 ts and mutant eIF4E (cdc33-1 ts ) engendered conditional synthetic lethality, with extreme sensitivity to hydroxyurea and the microtubule depolymerizing agent, benomyl. The double mutant (cdc33-1 rnr2-68) also identified a unique terminal phenotype, arrested with small bud and a randomly distributed single nucleus, which is distinct from those of both parental single mutants. This phenotype defines eIF4E and RNR2 as determinants in an important cell cycle checkpoint, in early/mid-S phase. These results also provide a link between protein and DNA synthesis and provide an explanation for cell cycle alterations induced by elevated eIF4E.

The association of G 1 cyclins and Cdc28/cyclindependent protein kinase catalyzes the cell cycle entry (Start) in budding yeast. Activation of Start is presumed to be triggered by a post-transcriptional increase in Cln3 during early G 1 . Cells arrested by mating pheromone show a loss of cyclin-dependent protein kinase activity caused by transcriptional shutoff of cyclins and/or inhibition by Far1. We report that overexpression of eIF4E (Cdc33), a rate-limiting translation initiation factor, causes an increase in CLN3 mRNA translation, which results in increased expression of CLN2 and in slow growth and decreased ␣-factor response. This phenotype was abrogated in a ⌬cln3 or ⌬cln2 background. We isolated the transcription factor MBP1 as a multicopy suppressor of the growth and ␣-factor response defects. Furthermore, elevated MBP1, a transcriptional regulator of cyclins, altered the transcriptional start site in CLN3 mRNA, shifting it 45 nucleotides upstream of the normal. This lengthened 5-untranslated region likely reduces translation efficiency and down-regulates CLN3 protein synthesis, thereby correcting for the excess translation promoted by elevated Cdc33. In addition, the CLN2 mRNA level returned to normal. We propose that regulation of translation initiation by Cdc33 plays a pivotal role in the activation of Start and cell cycle progression in budding yeast.