Academic journal article Genetics

Contribution of Trf4/5 and the Nuclear Exosome to Genome Stability through Regulation of Histone mRNA Levels in Saccharomyces Cerevisiae

Academic journal article Genetics

Contribution of Trf4/5 and the Nuclear Exosome to Genome Stability through Regulation of Histone mRNA Levels in Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

Balanced levels of histones are crucial for chromosome stability, and one major component of this control regulates histone mRNA amounts. The Saccharomyces cerevisiae poly(A) polymerases Trf4 and Trf5 are involved in a quality control mechanism that mediates polyadenylation and consequent degradation of various RNA species by the nuclear exosome. None of the known RNA targets, however, explains the fact that trf mutants have specific cell cycle defects consistent with a role in maintaining genome stability. Here, we investigate the role of Trf4/5 in regulation of histone mRNA levels. We show that loss of Trf4 and Trf5, or of Rrp6, a component of the nuclear exosome, results in elevated levels of transcripts encoding DNA replication-dependent histones. Suggesting that increased histone levels account for the phenotypes of trf mutants, we find that TRF4 shows synthetic genetic interactions with genes that negatively regulate histone levels, including RAD53. Moreover, synthetic lethality of trf4Δ rad53Δ is rescued by reducing histone levels whereas overproduction of histones is deleterious to trf's and rrp6Δ mutants. These results identify TRF4, TRF5, and RRP6 as new players in the regulation of histone mRNA levels in yeast. To our knowledge, the histone transcripts are the first mRNAs that are upregulated in Trf mutants.

IN eukaryotic cells, the DNA is packaged into nucleosomes, where each nucleosome consists of an octamer composed of two histone H2A-H2B heterodimers and a histone H3-H4 tetramer wrapped with ~146 bp of DNA. The four histones are present in the nucleosome, and therefore within chromosomes, in equimolar stoichiometry with respect to each other and to the DNA. Cells have evolved multiple mechanisms that maintain histone abundance at very precise levels, and any disruption of these mechanisms that leads to a prolonged imbalance in the ratio of the histones to each other or to the amount of DNA leads to chromosome instability. A key feature of the control is that expression of the histone genes is tightly coupled to rates of DNA synthesis in Saccharomyces cerevisiae (HEREFORD et al. 1981, 1982; OSLEY and HEREFORD 1982). Histones are transcribed from four sets of gene pairs (HTA1-HTB1 and HTA2-HTB2 for H2A and H2B, and HHT1-HHF1 and HHT2-HHF2 for H3 and H4), which are divergently transcribed from the respective promoters. Transcription is activated at the G1/S transition and repressed in G1, G2, and M phases of the cell cycle (OSLEY and HEREFORD 1982; OSLEY and LYCAN 1987; CROSS and SMITH 1988; XU et al. 1992; SUTTON et al. 2001). In addition, histone mRNAs are also modulated post-transcriptionally through 3' elements of the genes (LYCAN et al. 1987; XU et al. 1990; CAMPBELL et al. 2002). This complexity has thwarted efforts to fully understand the mechanisms underlying histone mRNA homeostasis. In this work, we describe a previously unanticipated pathway that contributes to modulation of histone mRNA levels.

Despite being initially identified biochemically as DNA polymerases (WANG et al. 2000), it is widely accepted today that TRF4 and TRF5 encode for nuclear poly(A) polymerases in budding yeast, as predicted from their primary sequence (ARAVIND and KOONIN 1999; SAITOH et al. 2002; HARACSKA et al. 2005). In fact, TRF4 has also been designated PAP2 [poly(A) polymerase 2]. The structure and biochemical functions of Trf4, and of Trf5, which is 58% identical to Trf4, are conserved throughout evolution, as orthologs in Schizosaccharomyces pombe (READ et al. 2002; SAITOH et al. 2002; WIN et al. 2006a,b), Caenorhabditis elegans (WANG et al. 2002a), Xenopus (BARNARD et al. 2004), human, and mouse (KWAK et al. 2004) have been associated with poly(A) polymerase activity. There is also functional conservation at the physiological level, since S. pombe cid14 can complement the lethality of S. cerevisiae trf4-ts top17Δ at the restrictive temperature (WIN et al. 2006a).

Although polyadenylation had generally been thought to increase the stability of eukaryotic mRNAs, current studies challenge this view. …

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