Function of a/eIF2 in post-transcriptional regulation

The fundamental process of protein synthesis remains poorly understood in the archaeal domain of life. We have begun to analyze the mechanism(s) and function(s) of translation initiation factors (tif) in the model crenarchaeon Sulfolobus solfataricus with particular emphasis on the trimeric eukaryotic ortholog a/eIF2. Besides establishing its requirement for binding of Met-tRNA Met (tRNAi) to the ribosome, these studies revealed that the function of the a/eIF2 subunits deviates from their eukaryotic counterparts. Most strikingly, we observed that a/eIF2, in particular the a/eIF2 -subunit exhibits an additional function with resemblance to the eukaryotic cap-complex. It binds to the 5-triphosphate end of mRNA and protects its 5-part from degradation. In this project we aim at elucidating this unprecedented function of a tif with regard to (i) the extreme lifestyle of S. solfataricus, (ii) possible interaction partners of a/eIF2, (iii) the S. solfataricus mRNA metabolism, (iv) the conservation in different Archaea , and (v) the interaction between a/eIF2 and tRNAi and mRNA, respectively, at atomic resolution. (i) To extend our recent findings we will first use genetic and immunological means to test whether there is -under different growth and stress conditions- a correlation between the level of a/eIF2 or a/eIF2 and the stability of mRNAs. Moreover, using DNA microarrays, we will assess whether particular transcripts, e.g. leaderless mRNAs, are preferentially stabilized by a/eIF2 or a/eIF2. (ii) Since we have shown that a/eIF2 / a/eIF2 bound to the 5-end of a leaderless mRNA can inhibit translation, the question arises how the respective mRNA(s) can be "recycled". We will use immunological, biochemical and biophysical means to search for possible protein interaction partners of a/eIF2 / a/eIF2, and to test for a possible modification of a/eIF2(). (iii) With the exception of 3 5 exosomal decay, mRNA turnover has not been studied in detail in Archaea. As we have shown that a/eIF2 counteracts 5 3 decay, we will attempt to identify the RNase activity involved. S. solfataricus protein fractions obtained by chromatography will be tested in in-gel assays and the candidate proteins will be identified by mass spectrometry. (iv) Based on the known genome sequences of close(r) relatives of S. solfataricus and Euryarchaeota, we will purify a number of a/eIF2 homologues and test their "activity" in terms of binding to the 5`-triphosphate end of mRNAs. These studies are anticipated to shed light on the evolutionary conservation of the RNA-protective function of a/eIF2 within the domain of Archaea. (v) Our recent biochemical studies showed that binding of a/eIF2() to tRNAi and to the 5-end of mRNA is mutually exclusive. However, these studies did not permit a distinction as to whether the binding sites on a/eIF2 for both ligands overlap, or whether binding to the mRNA interferes with structural changes required for tRNAi binding. Together with our long term collaborator Dr. M. Garber, Institute of Protein Research, Pushchino, Russia, we will address this question by attempting to crystallize and study the 3D structures of a/eIF2 complexes with tRNAi and with the 5-triphosphate end of an RNA, respectively.

Post-transcriptional control mechanisms of gene regulation are poorly understood in the archaeal domain of life. In a preceding project we started to analyze the function(s) of translation initiation factors in the model crenarchaeon Sulfolobus solfataricus (Sso) with particular emphasis on the trimeric eukaryotic ortholog aIF2. Besides establishing its requirement for binding of Met-tRNAMet (tRNAi) to the ribosome, we observed that aIF2, in particular the aIF2 ?-subunit exhibits an additional function with resemblance to the eukaryotic cap-complex. It binds to the 5-triphosphate end of mRNA and protects from 5 to 3 directional decay.During the course of this project we made efforts to identify and to characterize the Sso RNase activity involved in 5 to 3 directional decay. By means of zymogram assays and bioinformatics, we have identified a 5? to 3? exoribonuclease, which is affected by the phosphorylation state of the 5?-end of the mRNA. The protein comprises typical signature motifs of the ?-CASP family of metallo-?-lactamases. The enzyme was first termed Sso-RNAse J, and has been reclassified as aCPSF2. Binding of aIF2-? to the 5?-end of mRNA counteracted the 5?-to-3? exoribonucleolytic activity of aCSPF2 in vitro. This study provided the first experimental evidence for a 5? to 3? directional mRNA decay pathway in the crenarchaeal clade of Archaea. The biological significance of aCSPF2 was addressed in S. acidocaldarius (Saci) as this organism is more amenable to genetic manipulation. The Saci-aCPSF2 was functionally characterized. Like Sso-aCPSF2, Saci-aCPSF2 degrades RNA with 5 to 3 directionality in vitro. A deletion mutant was constructed, and the influence of Saci-aCPSF2 on the transcriptome was assessed employing RNAseq. This analysis revealed 560 genes with differential transcript abundance, suggesting a considerable role of this enzyme in RNA metabolism. In addition, bioinformatic analyses revealed several transcripts that are preferentially degraded at the 5end. This was exemplarily verified for two transcripts by Northern-blot analyses, showing for the first time that aCPSF2 proteins play indeed a role in 5' to 3' directional mRNA decay in the crenarchaeal clade of Archaea.We hypothesized that binding of aIF2 to the 5end of mRNAs occurs during unfavorable growth conditions, and that aIF2 is released upon relief of nutrient stress, enabling resumption of translation of 5 end protected mRNAs. Elevated levels of aIF2-? resulted in growth retardation of Sso, and binding of aIF2-? to the 5 end of a leaderless RNA inhibited ribosome binding in vitro. As leaderless mRNAs are prevalent in Sso, we studied the mechanism underlying aIF2 recycling from mRNAs. We identified a protein termed Trf (translation recovery factor) that co-purified with aIF2 during outgrowth of cells from prolonged stationary phase. Subsequent in vitro studies revealed that Trf triggers the release of aIF2 from RNA and that Trf directly interacts with the aIF2-? subunit. The importance of aIF2 release by Trf is further underscored by an impaired protein synthesis during outgrowth from stationary phase in a Sso trf deletion mutant. The unique RNA protection / recycling mechanism resembles mRNA storage in Eukaryotes.