Tma108, a putative M1 aminopeptidase, is a specific nascent chain-associated protein in Saccharomyces cerevisiae

Abstract

The discovery of novel specific ribosome-associated factors challenges the assumption that translation relies on standardized molecular machinery. In this work, we demonstrate that Tma108, an uncharacterized translation machinery-associated factor in yeast, defines a subpopulation of cellular ribosomes specifically involved in the translation of less than 200 mRNAs encoding proteins with ATP or Zinc binding domains. Using ribonucleoparticle dissociation experiments we established that Tma108 directly interacts with the nascent protein chain. Additionally, we have shown that translation of the first 35 amino acids of Asn1, one of the Tma108 targets, is necessary and sufficient to recruit Tma108, suggesting that it is loaded early during translation. Comparative genomic analyses, molecular modeling and directed mutagenesis point to Tma108 as an original M1 metallopeptidase, which uses its putative catalytic peptide-binding pocket to bind the N-terminus of its targets. The involvement of Tma108 in co-translational regulation is attested by a drastic change in the subcellular localization of ATP2 mRNA upon Tma108 inactivation. Tma108 is a unique example of a nascent chain-associated factor with high selectivity and its study illustrates the existence of other specific translation-associated factors besides RNA binding proteins.

Figure 1.

The Translation Machinery-Associated factor Tma108 impacts ATP2 mRNA localization to mitochondria. (A) The impact of different translation machinery-associated factors on ATP2 mRNA localization. Experimental approach (top left). Fluorescent in situ experiments were performed in different mutants using probes to detect ATP2 mRNA and mitochondrial rRNA as a marker for mitochondria position. Typical FISH images (top right) show the impact of TMA108 deletion on ATP2 mRNA localization. In BY4743 strain, the ATP2 mRNA is partially localized to the mitochondria surface. TMA108 deletion led to a drastic increase in ATP2 mRNA localization to the mitochondria: most of the green spots co-localized with the mitochondrial red signal. ATP2 mRNA localization is not perturbed by TMA20, RPL38 or TMA22 deletions. The boxplots (bottom) represent the distributions of the median ATP2 mRNA-mitochondria distances in the analyzed cell populations. Similar FISH analyses were performed on a control mRNA, ATP16, and no significant change in mRNA localization was observed (data not shown). (B) Genome-wide analysis of the impact of Tma108 on the localization of mRNA to mitochondria. Experimental scheme (left) for the analysis of mRNA localization to mitochondria: total mRNA and mRNA associated with the mitochondrial fraction were isolated, reverse transcribed, and labeled for subsequent microarray analysis. Comparative analysis of the mRNA associated with the mitochondrial fraction in BY4743 and TMA108 deleted strain (right). The log2 fold changes in TMA108Δ/Δ relative to BY4743 are plotted: the values for each gene are the means of three biological replicates (see Supplemental Table S5). ATP2 is the single mRNA detected by LIMMA with a significant change in mRNA enrichment factor (P-value = 3.7 × 10⁻³).

Figure 2.

Tma108 interacts with translating ribosomes that are associated with ATP2 mRNA. (A) Experimental approach. Immunoprecipitations were performed on cells grown on rich medium, expressing Tma108-PA and untagged cells (WT) in presence (+) or in absence (−) of 40 mM EDTA. Protein (B, C and E) and RNA (D,F) contents were analyzed from total extract (Input) and immunoprecipitated samples (IP). (B) Input and immunoprecipitated fractions obtained from untagged or Tma108-PA expressing-cells were analyzed by SDS-PAGE followed by silver staining. The position of the Tma108-PA fusion and IgG are indicated. (C) Input and immunoprecipitated fractions obtained from untagged or Tma108-PA expressing-cells were analyzed by SDS-PAGE followed by western-blots using antibodies directed to ribosomal proteins (Rpl1, Rpl3). (D) Following RNA isolation, input and immunoprecipitated fractions obtained from untagged or Tma108-PA expressing-cells were analyzed by agarose gel electrophoresis with ethidium bromide staining (L: 1kb DNA ladder biolabs). The bands corresponding to 28S and 18S rRNA are indicated. (E) Diagram showing the functional distribution of proteins detected by mass spectrometry analysis of Tma108-PA IP in the absence of EDTA. The names of the proteins included in the different sectors are indicated (see Supplementary Table S6). Only proteins reproducibly detected with a MS score superior to the threshold determined with the untagged sample were selected (see Supplementary Figure S1). Most of these proteins are lost when IP are performed in the presence of EDTA (see Figure 5B). (F) Real-time PCR analysis of mRNA immunoprecipitated with Tma108-PA IP. For each analyzed mRNA, the enrichment factor corresponds to the ratio IP/Input normalized using a set of control mRNA (Act1,Trx2,Tpm2,Jen1,Flr1). After normalization, the mean enrichment factor for the control mRNA set is equal to 1. When EDTA is added in the IP, ATP2 mRNA is no longer enriched compared to the other mRNA (see Figure 5B). No specific enrichment of ATP2 mRNA is detected when analyzing IP performed with the untagged strain (data not shown).

Figure 3.

Tma108 targets ribosome translating a subset of the cellular mRNA. (A) Strategy developed to identify mRNA specifically enriched with factors associated with translating ribosomes. Immunoprecipitations were performed from cells expressing proteinA tagged versions of Tma108, Scp160 or Tma46. As a control, the whole set of ribosomes was immunoprecipitated using cells expressing a tagged version of Rpl16a. (B) Hierarchically clustered heat map showed IP/input log2(ratios) for mRNA enriched with at least one of the tested ribosomal partners in two independent experiments (Supplementary Tables S7 and S8). Each line represents an experiment, each column a gene. (C) Analysis of over-enrichment and under-enrichment of GO terms (cellular component) among Tma108 and Scp160 mRNA targets (Supplementary Table S9). The extent of the enrichment for each GO term is expressed as a fold enrichment over genome. Significant over- and under-enrichments are indicated (***P-value < 10⁻³, **P-value < 5 × 10⁻²). (D) Tma108 targets are highly enriched in defined molecular functions. Analysis of enrichment of the GO terms revealed three main categories of over-represented functions: Nucleotide binding (P-value: 2.3 × 10⁻⁸), Zinc ion binding (P-value: 1.5 × 10⁻⁵) and RNA binding (P-value: 9.7 × 10⁻⁵). Over-represented functional categories included in these three GO terms are not represented (Supplementary Table S10). (E) Analysis of Pfam domains over-represented in proteins encoded by Tma108 targets. Eighteen Pfam domains that showed a significant enrichment (P-value < 0.05) in Tma108 dataset were selected (identifiers and P-value in Supplementary Table S11). They were classified according to their belonging to the different GO terms (function) enriched in Tma108 list (e1: Nucleotide binding, e2: RNA binding, e3:Zinc ion binding, e4: Others). The boxplots represent the distribution of mean log2(IP/Input) values obtained in microarray analyses of Tma108 immunoprecipitation for the whole cellular mRNA and for the analyzed GO categories. For each Pfam domain, the values of log₂(IP/Input) measured for each individual gene included in the Pfam dataset are plotted (yellow diamond) overlapped on the boxplot (light gray) of the corresponding GO term (e1–3) or of the total RNA dataset (e4). Pfam domains indicated with green circles correspond to domains with ATPase activity. Dashed lines correspond to the 0.8 threshold that was chosen to determine Tma108 targets.

Figure 4.

Tma108, a new protein of the M1 aminopeptidase family. (A) S. cerevisiae Tma108 (scTma108) was compared to 14 well-characterized M1 aminopeptidases using multiple alignment of primary structures, followed by residue conservation analysis. The alignment shown corresponds to the most conserved region and is restricted to five M1 aminopeptidases. Residues in blue are fully conserved among the 15 M1 aminopeptidases (including Tma108), those in grey are partially conserved (90% of the sequences shown). Residues that are conserved in all the M1 aminopeptidases except in Tma108 are indicated in cyan on Tma108 sequence. Furthermore, Tma108 conservation was analyzed after retrieval of Tma108 orthologs in the diverse sequenced yeast genomes. Eighteen sequences from species (including S. cerevisiae) distributed along the phylogenic tree of the Saccharomycetaceae, were selected to detect residues that are fully conserved. These residues are indicated by color boxes at the top of the alignment (line labeled cons): dark blue boxes indicate residues that are conserved among Tma108 orthologs and similar in the reference aminopeptidases, orange boxes indicate residues that are specific to Tma108 orthologs. Numbers in the boxes indicate residues that were previously shown to be implicated in M1 aminopeptidase activities (based on the crystal structure of aminopeptidase N(42,43)): binding of the first amino acid of the substrate in S1 pocket (1), binding of the second amino acid of the substrate in S'1 pocket (2), binding of the Zn²⁺ cofactor (z), catalysis (c). Frames on the alignment indicate the two sequence signatures of the M1 family, (G/A/H/V)(G/A)MEN and HEXXHX¹⁸E, and the tyrosine involved in the catalytic mechanism. A dashed line indicates the separation between domains 1 and 2 of Tma108 inferred by homology based structure modeling (see below). (B and C) 3D molecular model of Tma108 of S. cerevisiae. Four domains are shown in different colors from light gray to black: NH₂-terminus (1–227), central (228–490), hinge (491–578) and COOH terminal (579–946). Residues underscored by the two conservation analyses (b: conserved residues in all the M1 aminopeptidases, c: conserved residues in all the Tma108 orthologs) described in (a) are colored in the structure (same color code as in a).

Figure 5.

Tma108 directly interacts with proteins encoded by its associated translating complexes. (A) Comparison of the composition of Tma108, Scp160 and Tma46-associated ribosomes with the composition of canonical (Rpl16A-associated) ribosomes. Experimental strategy (top panel). Immunoprecipitations of proteinA-tagged versions of Tma108, Scp160, Tma46 or Rpl16a were followed by mass spectrometry analysis of their protein partners. Venn Diagramm represents the overlap of the four different sets of co-immunoprecipitated proteins: in each sector the number of interactants reproducibly detected in two independent experiments is indicated (Supplementary Table S12). Chart pies show the functional distribution of proteins for shared and Rpl16a specific dataset. The names of the proteins included in the different sectors are indicated. The 3 proteins specifically detected in Tma108 IP are also indicated. (B) Analysis of Tma108 partners following ribosome dissociation with EDTA. Experimental strategy (top panel) used to identify Tma108 interacting partners. Immunoprecipitations (n = 2) were performed on cells expressing Tma108-PA in the presence of 40 mM EDTA (see Figure 2B–D). RNAs (b1) were analyzed by real-time PCR. For each analyzed mRNA, the enrichment factor corresponds to the ratio IP/Input normalized using a set of control mRNA (Act1, Trx2, Jen1, Flr1). Mass spectrometry analysis (b2) of proteins co-immunoprecipitated with Tma108 in the presence of EDTA: only seven proteins were reproducibly detected in that condition. (C) Most of the Tma108-specific proteins partners are translated by Tma108-associated ribosomes. The boxplot represents the distribution of mean log₂(IP/Input) values obtained following microarray analysis of Tma108-associated mRNAs (in the absence of EDTA). Arrows point the values for the mRNAs corresponding to the proteins that were detected as specific Tma108 partners (Figure 5B, panel 2).

Figure 6.

The translation of the N-terminal region of Asn1 is required to recruit Tma108 on the ribosome. Real-time quantitative RT-PCR analyses of the enrichment in Tma108-PA IPs of various ASN1 versions fused to a 13-myc Tag (A, construct 1–3) or a LacZ gene (B, construct 4–5). For construct 5, the experiment was also performed with a mutated version of the Tma108-PA (E296Q) in which the glutamate of the MAMEN motif was replaced by a glutamine. The arrows in bold indicate the positions of the primers used for the Q-PCR. The mean enrichment factors were obtained from two independent immunoprecipitation experiments by calculating the ratio IP/Input normalized using ACT1 and ATP1 as control mRNAs. In each experiment, the enrichment of the endogenous ATP2 mRNA was measured and confirmed that Tma108-PA ribonucleoparticles were efficiently immunoprecipitated (data not shown).

Figure 7.

Model highlighting the co-translational capture of selected nascent chain by Tma108. This study has revealed that Tma108, a putative M1 metallopeptidase, interacts with a subset of ribosomal particles translating mRNA encoding proteins with ATP-binding, RNA-binding and Zinc-binding domains (right). We propose that Tma108 is recruited on selected nascent-chain through interactions involving specific residues located in its catalytic pocket (left). The observation that Tma108 is essential in some cellular contexts (39) strongly suggests that Tma108 could be involved in specific co-translational events.