Structural and functional analysis of Nro1/Ett1: a protein involved in translation termination in S. cerevisiae and in O₂-mediated gene control in S. pombe

Abstract

In Saccharomyces cerevisiae, the putative 2-OG-Fe(II) dioxygenase Tpa1 and its partner Ett1 have been shown to impact mRNA decay and translation. Hence, inactivation of these factors was shown to influence stop codon read-though. In addition, Tpa1 represses, by an unknown mechanism, genes regulated by Hap1, a transcription factor involved in the response to levels of heme and O 2 . The Schizosaccharomyces pombe orthologs of Tpa1 and Ett1, Ofd1, and its partner Nro1, respectively, have been shown to regulate the stability of the Sre1 transcription factor in response to oxygen levels. To gain insight into the function of Nro1/Ett1, we have solved the crystal structure of the S. pombe Nro1 protein deleted of its 54 N-terminal residues. Nro1 unexpectedly adopts a Tetratrico Peptide Repeat (TPR) fold, a motif often responsible for protein or peptide binding. Two ligands, a sulfate ion and an unknown molecule, interact with a cluster of highly conserved amino acids on the protein surface. Mutation of these residues demonstrates that these ligand binding sites are essential for Ett1 function in S. cerevisiae, as investigated by assaying for efficient translation termination.

Structure of the Ribosomal Oxygenase OGFOD1 Provides Insights into the Regio- and Stereoselectivity of Prolyl Hydroxylases

“…Having lost catalytic potential due to redundancy, the CTD may have retained or gained function(s) in substrate and/or binding partner recognition, stabilization, and/or oligomerization. A role for the non-catalytic CTD in the OGFOD1 subfamily in binding to the uS12 substrate, or other binding partners, seems likely; it is reported that the CTDs of both Ofd1 and Tpa1p interact with Nro1 and Ett1, respectively ( Lee et al., 2009; Rispal et al., 2011 ). Non-DSBH domains have been shown to be important in oligomerization and substrate binding selectivity in the case of other 2OG oxygenases, e.g.…”

“…Although the biological roles for uS12 hydroxylation are still emerging, it has been reported that in yeast it can regulate translation in a sequence context dependent manner and that it is involved in stress responses ( Saito et al., 2010; Katz et al., 2014; Loenarz et al., 2014; Singleton et al., 2014 ). Ofd1, a homolog of OGFOD1/Tpa1p from Schizosaccharomyces pombe , binds to the helical repeat protein Nro1 in an O 2 -dependent manner ( Rispal et al., 2011; Yeh et al., 2011 ), thus inhibiting Ofd1 binding to Sre1N, a homolog of the sterol regulatory element binding protein ( Lee et al., 2009 ). In humans, a distinct ROX subfamily, more closely related to the JmjC domain subfamily, comprising MYC-induced nuclear antigen 53 kDa (MINA53) and nucleolar protein 66 kDa (NO66) catalyzes histidyl hydroxylation of human ribosomal proteins uL15 (L27A) and uL2 (L8), respectively.…”

Hypoxia and Gene Expression in Eukaryotic Microbes

“…The role of heme proteins and the potentially important roles of the mitochondria and the respiratory chain remain to be fully explored. Other areas likely to be of future interest include the regulation of translation in hypoxia (109) and the nearly unstudied modulation of the host by fungal pathogens in hypoxic conditions.…”

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

“…89 PTex-enriched proteins contain a WD40 fold; a domain found to directly bind snRNA in Gemin 5 (41), taking part in rRNA biogenesis (Erb1 (42)) and found in RBPs (1). Other enriched domains are: AAA (AT-Pase, see below) fold, tetratrico peptide repeat region (TPR) as found in the yeast Clf1p splicing factor (43), Ski complex (44) and the translation terminator Nro1 (45), and the CH domain which is found among actin-binding proteins (3).…”