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. 2020 Jan 3;367(6473):100-104.
doi: 10.1126/science.aaz4352. Epub 2019 Nov 14.

TTC5 mediates autoregulation of tubulin via mRNA degradation

Zhewang Lin  1 , Ivana Gasic  2 , Viswanathan Chandrasekaran  1 , Niklas Peters  1 , Sichen Shao  3 , Timothy J Mitchison  2 , Ramanujan S Hegde  4
Affiliations

TTC5 mediates autoregulation of tubulin via mRNA degradation

Zhewang Lin et al. Science. .

Abstract

Tubulins play crucial roles in cell division, intracellular traffic, and cell shape. Tubulin concentration is autoregulated by feedback control of messenger RNA (mRNA) degradation via an unknown mechanism. We identified tetratricopeptide protein 5 (TTC5) as a tubulin-specific ribosome-associating factor that triggers cotranslational degradation of tubulin mRNAs in response to excess soluble tubulin. Structural analysis revealed that TTC5 binds near the ribosome exit tunnel and engages the amino terminus of nascent tubulins. TTC5 mutants incapable of ribosome or nascent tubulin interaction abolished tubulin autoregulation and showed chromosome segregation defects during mitosis. Our findings show how a subset of mRNAs can be targeted for coordinated degradation by a specificity factor that recognizes the nascent polypeptides they encode.

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. TTC5 interacts with the N-termini of nascent tubulins.
(A) Experimental strategy to detect interaction partners close to the N-terminus of nascent tubulin. The UV-activated crosslinking amino acid p-benzoyl-l-phenylalanine (Bpa) is introduced site-specifically at position 7 using amber suppression (see fig. S1). (B) Photo-crosslinking analysis of 35S-labelled 94-amino acid long ribosome-nascent chain complexes (RNCs) of human β-tubulin and mutants (indicated in red) in the N-terminal MREI motif. The positions of non-crosslinked tRNA-associated nascent chain (NC-tRNA) and a crosslinking partner specific to wild type tubulin (red asterisk) are indicated. Other nascent chain crosslinks agnostic to the MREI motif are indicated by black asterisks. (C) Quantitative mass spectrometry of proteins co-purified with wild type (WT) versus MHQV mutant (mut) β-tubulin RNCs plotted by molecular weight. (D) Photo-crosslinking and immunopreciptation (IP) analysis of 35S-labelled 94-amino acid long RNCs of α- or β-tubulin compared to the indicated mutants.
Fig. 2
Fig. 2. Mechanism of ribosome-nascent chain engagement by TTC5.
(A) Overview of the cryo-EM derived structure of the complex between TTC5 and a ribosome containing the first 64 amino acids of β-tubulin. (B) Close-up view of the TTC5 interaction with 28S rRNA. Three conserved lysine residues in TTC5 within electrostatic interaction distance of the rRNA backbone are indicated. (C) The surface of TTC5 that interacts with ribosomal protein uL24 is indicated in orange. The 28S-interacting residues from panel B are shown in yellow. The N-terminal 8 amino acids of the β-tubulin nascent chain is shown in red within its binding groove of TTC5. (D) Close-up view of the N-terminal 8 amino acids of nascent β-tubulin (MREIVHIQ) within TTC5. Yellow spheres denote C-β atoms for the indicated side chains (not modelled). R147 is within salt-bridge distance of E3, and D225 and E259 are within salt-bridge distance of R2. F194 on the ‘floor’ of the binding groove, shown in Fig. 3A to crosslink with nascent β-tubulin, is indicated.
Fig. 3
Fig. 3. Avidity-based RNC binding imparts specificity to TTC5.
(A) Photo-crosslinking analysis of 35S-labelled 94-amino acid long ribosome-nascent chain complexes (RNCs) of human β-tubulin and N-terminal mutants (indicated in red) with recombinant StrepII-tagged TTC5 containing the photo-crosslinking residue Bpa at position F194. The nascent chain crosslink to TTC5 is indicated (TTC5-XL) and verified by pulldown via the StrepII tag (bottom panel). (B) Photo-crosslinking analysis using 35S-labelled 64-amino acid long ribosome-nascent chain complexes (RNCs) of human β-tubulin or the N-terminal MRQI mutant. Wild type or mutant recombinant StrepII-tagged TTC5 was included in the assay as indicated. The photo-crosslinking residue Bpa is at position 7 of the β-tubulin nascent chain. An aliquot of the total translation reaction was analyzed to verify equal levels of nascent chain (NC) synthesis by autoradiography and equal levels of recombinant TTC5 (SII-TTC5) by immunoblotting for the StrepII tag. The remainder was UV irradiated and TTC5 crosslinks were recovered via the StrepII tag and visualized by autoradiography (bottom panel). (C) Summary of interaction analysis between the indicated recombinant TTC5 proteins and the indicated synthetic peptides in a thermal shift denaturation assay (see fig. S7). (D) Wild type or mutant StrepII-tagged TTC5 was included during in vitro translation of wild type or mutant 64-mer β-tubulin RNCs as indicated. Equal translation of 35S-labelled nascent chain synthesis was verified (NC total). The remainder of each translation was affinity purified via the StrepII tag and analyzed by staining of total proteins (bottom panel).
Fig. 4
Fig. 4. TTC5 is required for tubulin autoregulation and accurate mitosis.
(A) The indicated HEK293 cell lines were either left untreated or treated for 3 h with colchicine. The relative amounts of the indicated mRNAs or pre-mRNAs were quantified by RT-qPCR and normalized to a control ribosomal RNA. Plotted is the mean ± SD from three replicates. Similar results are seen in HeLa cells and with different microtubule destabilizing agents (fig. S8). (B) Pre-formed RNC-TTC5 complexes (see fig. S10) were mixed with buffer or cytosol from TTC5 knockout cells that had been pre-treated (+col) or not (-col) with colchicine for 3 hours. All samples were subjected to UV crosslinking to monitor the nascent chain interactions. The positions of the non-crosslinked tRNA linked nascent chain (NC-tRNA) and TTC5 crosslink (TTC5-XL) are indicated. (C) TTC5-knockout HEK293 cells were pretreated for the indicated times with colchicine and used to prepare lysates. One of the control samples included colchicine added after cell lysis (indicated as 0*). The products recovered by binding to recombinant TTC5 were analyzed for β-tubulin mRNA by quantitative RT-PCR. The relative recoveries are plotted (mean ± SD from three replicates). Similar results were seen for α-tubulin and when nocodazole was used instead of colchicine to trigger autoregulation (fig. S11). (D) Diagram (left) and examples (right) of accurate (top) and erroneous (bottom) chromosome alignment and segregation visualized with SirDNA dye during mitosis in HeLa cell lines (see fig. S13). Scale bar = 5 µm. (E) Quantification of errors in chromosome alignment and segregation in the indicated HeLa cell lines. Plotted is mean ± SEM from 4-6 independent biological replicates (dots) with 200-400 analyzed cells per replicate. Not significant (n.s.) and p<0.001 (**) in paired Student’s t-tests are indicated.
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