Research

Structural studies of translational control mechanisms

We study mammalian translation control mechanisms that rescue ribosomes that have stalled due to a variety of reasons including, amino acid starvation, codon sub-optimality and mRNA defects. Ribosome stalling triggers degradation of the nascent polypeptide and mRNA chains, and rescue of the ribosome. Our studies have provided important mechanistic insights into how such stalled ribosomes are detected and processed.

Ribosome-associated quality control (RQC)

RQC is a critical cellular response whose mis-regulation is associated with several human diseases including ribosomopathies, neurodegenerative disorders and autism (1, 2). Stalling at aberrant poly(A) sequences can occur in up to 5% of mature mRNAs, which are inappropriately polyadenylated within coding regions. Ribosomes that translate such poly(A) sequences stall and are rescued by RQC. To determine the molecular basis for ribosome stalling, we determined the 2.8 Å cryo-EM structure of rabbit ribosomes stalled on poly(A) sequences (3). When ribosomes stall, trailing ribosomes then catch up and collide with them, and cellular mechanisms have evolved to detect and rescue such collisions. To understand how these collisions can be detected, we determined the cryo-EM structure of the ‘collided di-ribosome’, and showed that this species is competent for signalling ribosome stalling and rescue (4). This discovery has shed light on the mechanisms of translational control and nucleated followup studies in diverse topics including disome profiling (5–7), integrated stress response (ISR) (8–10), mRNA decay (11, 12) and protein folding (13).

Co-translational mRNA decay

TTC5 mediates autoregulation of tubulin via mRNA degradation

Co-translational mRNA decay has recently emerged as a novel mechanism for regulating protein expression, for example in the autoregulatory feedback control of cellular tubulin subunit concentration and in targeted mRNA degradation (14, 15). To understand this process, we identified tetratricopeptide protein 5 (TTC5) as a tubulin-specific ribosome-associating factor that triggers co-translational degradation of tubulin mRNAs in response to excess soluble tubulin (16). My structural analyses revealed how a subset of mRNAs (in this case, tubulins) can be targeted for coordinated degradation by a specificity factor that recognizes the nascent polypeptides they encode

Cryo-EM structure of mammalian ribosomes in complex with the mRNA decay factor CCR4-NOT

We have studied a second co-translational mRNA decay system that involves the CCR4-NOT complex (17, 18). It has recently been reported that this activity also occurs on stalled (or ‘slow-moving’) ribosomes (11, 19). We recently reconstituted and determined the structure of CCR4-NOT in complex with ribosomes stalled on non-optimal codon stretches (20). The structure reveals that the CNOT3 subunit of CCR4-NOT binds to the empty E site of stalled ribosomes, which are then marked by monoubiquitylation of the ribosomal protein eS7 by the associated RING E3 ubiquitin ligase CNOT4.

Mitoribosome-associated Quality Control (mtRQC)

We discovered that mammalian mitochondria evolved a separate RQC system to rescue stalled mitochondrial ribosomes, and dubbed this new pathway ‘mitoribosome-associated quality control’ (mtRQC) (21). To understand how mitoribosomes function, we determined structures of elongating human mitoribosomes bound to tRNAs, nascent polypeptides, the guanosine triphosphatase elongation factors mtEF-Tu and mtEF-G1, and the Oxa1L translocase.

Mitochondrial Ribosome Biogenesis

We identified major steps during the formation of the highly conserved peptidyl transferase centre (PTC) in the large subunit of the human mitoribosome. We discovered that the mitochondrial proteins NSUN4•MTERF4•GTPBP7 act in concert as ribosome biogenesis factors, and demonstrated the importance of their roles in ribosome assembly for mitochondrial and organismal health and fitness using in vivo mutagenesis and RNAseq in the nematode C. elegans (22).

References

1. M. C. Rodrigo-Brenni, R. S. Hegde, Design Principles of Protein Biosynthesis-Coupled Quality Control. Dev. Cell 23, 896–907 (2012).

2. O. Brandman, R. S. Hegde, Ribosome-associated protein quality control. Nat. Struct. Mol. Biol. 23, 7–15 (2016).

3. V. Chandrasekaran, S. Juszkiewicz, J. Choi, J. D. Puglisi, A. Brown, S. Shao, V. Ramakrishnan, R. S. Hegde, Mechanism of ribosome stalling during translation of a poly(A) tail. Nat. Struct. Mol. Biol. 26, 1132–1140 (2019).

4. S. Juszkiewicz, V. Chandrasekaran, Z. Lin, S. Kraatz, V. Ramakrishnan, R. S. Hegde, ZNF598 Is a Quality Control Sensor of Collided Ribosomes. Mol. Cell 72, 469-481.e7 (2018).

5. S. Meydan, N. R. Guydosh, Disome and Trisome Profiling Reveal Genome-wide Targets of Ribosome Quality Control. Mol. Cell 79, 588-602.e6 (2020).

6. P. Han, Y. Shichino, T. Schneider-Poetsch, M. Mito, S. Hashimoto, T. Udagawa, K. Kohno, M. Yoshida, Y. Mishima, T. Inada, S. Iwasaki, Genome-wide Survey of Ribosome Collision. Cell Rep. 31, 107610 (2020).

7. J. Szavits-Nossan, L. Ciandrini, Inferring efficiency of translation initiation and elongation from ribosome profiling. Nucleic Acids Res. 48, 9478–9490 (2020).

8. A. A. Pochopien, B. Beckert, S. Kasvandik, O. Berninghausen, R. Beckmann, T. Tenson, D. N. Wilson, Structure of Gcn1 bound to stalled and colliding 80S ribosomes. bioRxiv, 2020.10.31.363135 (2020).

9. C. C.-C. Wu, A. Peterson, B. Zinshteyn, S. Regot, R. Green, Ribosome Collisions Trigger General Stress Responses to Regulate Cell Fate. Cell 182, 404-416.e14 (2020).

10. L. L. Yan, H. S. Zaher, Ribosome quality control antagonizes the activation of the integrated stress response on colliding ribosomes. Mol. Cell, S1097276520308339 (2020).

11. R. Buschauer, Y. Matsuo, T. Sugiyama, Y.-H. Chen, N. Alhusaini, T. Sweet, K. Ikeuchi, J. Cheng, Y. Matsuki, R. Nobuta, A. Gilmozzi, O. Berninghausen, P. Tesina, T. Becker, J. Coller, T. Inada, R. Beckmann, The Ccr4-Not complex monitors the translating ribosome for codon optimality. Science 368 (2020).

12. C. L. Simms, L. L. Yan, H. S. Zaher, Ribosome Collision Is Critical for Quality Control during No-Go Decay. Mol. Cell 68, 361-373.e5 (2017).

13. M. Bertolini, K. Fenzl, I. Kats, F. Wruck, F. Tippmann, J. Schmitt, J. J. Auburger, S. Tans, B. Bukau, G. Kramer, Interactions between nascent proteins translated by adjacent ribosomes drive homomer assembly. Science 371, 57–64 (2021).

14. D. W. Cleveland, M. A. Lopata, P. Sherline, M. W. Kirschner, Unpolymerized tubulin modulates the level of tubulin mRNAs. Cell 25, 537–546 (1981).

15. I. Gasic, S. A. Boswell, T. J. Mitchison, Tubulin mRNA stability is sensitive to change in microtubule dynamics caused by multiple physiological and toxic cues. PLOS Biol. 17, e3000225 (2019).

16. Z. Lin, I. Gasic, V. Chandrasekaran, N. Peters, S. Shao, T. J. Mitchison, R. S. Hegde, TTC5 mediates autoregulation of tubulin via mRNA degradation. Science 367, 100–104 (2020).

17. J. Chen, Y.-C. Chiang, C. L. Denis, CCR4, a 3′–5′ poly(A) RNA and ssDNA exonuclease, is the catalytic component of the cytoplasmic deadenylase. EMBO J. 21, 1414–1426 (2002).

18. M. A. Collart, The Ccr4-Not complex is a key regulator of eukaryotic gene expression. WIREs RNA 7, 438–454 (2016).

19. A. Radhakrishnan, Y.-H. Chen, S. Martin, N. Alhusaini, R. Green, J. Coller, The DEAD-Box Protein Dhh1p Couples mRNA Decay and Translation by Monitoring Codon Optimality. Cell 167, 122-132.e9 (2016).

20. E. Absmeier, V. Chandrasekaran, F. J. O’Reilly, J. A. W. Stowell, J. Rappsilber, L. A. Passmore, Specific recognition and ubiquitination of translating ribosomes by mammalian CCR4-NOT. Nat. Struct. Mol. Biol. 30, 1314–1322 (2023).

21. N. Desai, H. Yang, V. Chandrasekaran, R. Kazi, M. Minczuk, V. Ramakrishnan, Elongational stalling activates mitoribosome-associated quality control. Science 370, 1105–1110 (2020).

22. V. Chandrasekaran, N. Desai, N. O. Burton, H. Yang, J. Price, E. A. Miska, V. Ramakrishnan, Visualizing formation of the active site in the mitochondrial ribosome. eLife 10, e68806 (2021).

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