Crystal structure of Tpa1 from Saccharomyces cerevisiae, a component of the messenger ribonucleoprotein complex

Abstract

Tpa1 (for termination and polyadenylation) from Saccharomyces cerevisiae is a component of a messenger ribonucleoprotein (mRNP) complex at the 3′ untranslated region of mRNAs. It comprises an N-terminal Fe(II)- and 2-oxoglutarate (2OG) dependent dioxygenase domain and a C-terminal domain. The N-terminal dioxygenase domain of a homologous Ofd1 protein from Schizosaccharomyces pombe was proposed to serve as an oxygen sensor that regulates the activity of the C-terminal degradation domain. Members of the Tpa1 family are also present in higher eukaryotes including humans. Here we report the crystal structure of S. cerevisiae Tpa1 as a representative member of the Tpa1 family. Structures have been determined as a binary complex with Fe(III) and as a ternary complex with Fe(III) and 2OG. The structures reveal that both domains of Tpa1 have the double-stranded β-helix fold and are similar to prolyl 4-hydroxylases. However, the binding of Fe(III) and 2OG is observed in the N-terminal domain only. We also show that Tpa1 binds to poly(rA), suggesting its direct interaction with mRNA in the mRNP complex. The structural and functional data reported in this study support a role of the Tpa1 family as a hydroxylase in the mRNP complex and as an oxygen sensor.

OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation

“…Structures of Tpa1p reveal the 2OG oxygenase characteristic doublestranded β-helix (DSBH) fold domain with typical Fe(II) and 2OG binding residues, but also indicate, as predicted for OGFOD1, the presence of a second DSBH fold domain in the C terminus, likely without a direct role in oxygenase catalysis (7,8). Analysis of the active site of Tpa1p suggests it is a protein hydroxylase with distant similarity to the mammalian HIF prolyl hydroxylase domain (PHD)-containing family of enzymes (7,8). Substitution of predicted active site residues in Ofd1 or Tpa1p abrogates the relevant function (4, 7), implying function is dependent on oxidation of an unassigned substrate.…”

Sudestada1, a Drosophila ribosomal prolyl-hydroxylase required for mRNA translation, cell homeostasis, and organ growth

“…S1 B and C). Given that S. cerevisiae Tpa1p and S. pombe Ofd1 have putative active sites in the N-terminal of their two DSBH domains that possess similarity to PHD2 (15,23), we tested whether Sud1 plays a role in the HIF-dependent transcriptional response to hypoxia by observing the effects of Sud1 knockdown on HIF/Sima-dependent transcription in the embryonic tracheal system. Embryos expressing sud1 RNAi failed to modulate a HIF-dependent transcriptional reporter, whereas, as expected, embryos that express an RNAi targeting the prolyl-4-hydroxylase gene fatiga displayed strong upregulation of the same reporter under mild hypoxic conditions, providing a positive control for the assay (Fig.…”

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

“…In addition, this protein was shown to interact with Pab1, eRF1, and eRF3 and hence proposed to be part of an mRNP complex associated at the mRNA 39 end (Keeling et al 2006). Structure and sequence analysis strongly support that Tpa1 belongs to the 2-oxoglutarate-Fe(II) dioxygenase family and most probably acts as a prolyl hydroxylase (PHD), an enzyme catalyzing the O 2 -dependant hydroxylation of the proline side-chain Kim et al 2010). However, this catalytic activity remains experimentally elusive, most likely because a biologically relevant substrate has not yet been identified.…”