Specialised ribosomes in cancer: structural, biochemical and functional characterisation

The concept of specialised ribosomes with distinct protein and RNA composition and functional diversity has been recently proposed. Rapid proliferating cancer cells have an increased demand for protein synthesis, which can be accomplished by changes in ribosome subunit composition and selective translation of transcripts. It has been proposed that these “oncoribosomes” are key players in cancer. The primary supervisor recently found profound changes in expression of 2 large ribosomal subunits in cells with mutations in the tumour suppressor PTCH1. Cancer cells that carry those alterations have a more aggressive oncogenic behaviour. This led us to hypothesise that ribosome subunit composition changes in response to loss of a tumour suppressor function could contribute to changes in translation that promote an oncogenic phenotype.

<h5>Objectives:&nbsp;</h5> <ol> <li>Determine the contribution of ribosomal alternative subunits RPL22L1 and RPL9 to the oncogenic phenotype&nbsp;</li> <li>Isolate of ribosomes from PTCH1 WT and mutant cells to investigate their subunit composition and identify mRNAs that are preferentially translated by different oncoribosomes.</li> <li>Investigate changes in the macromolecular structure of specialised ribosomes by cryo-EM.</li> </ol> <h5>Novelty/Timeliness:</h5> <p>The existence of specialised &ldquo;oncoribosomes&rdquo; that confer enhanced cell fitness has been recently suggested. Our study will demonstrate how changes in ribosomal composition, structure and function after inactivation of a tumour suppressor contribute to the acquisition of a malignant phenotype.</p> <h5>Experimental approach:</h5> <p>This study is highly cross-disciplinary and will provide the PGR with a unique and enabling skill set. The student will apply CRISPR/Cas9 technology to inactivate selected ribosomal isoforms that are upregulated in our cancer cell model and use transient transfection to characterise oncogenic properties in cell-based assays. This will confer a strong expertise in in vitro models of cancer cell biology provided by the primary supervisor. The student will also perform purification of ribosomes expressing alternative tagged-RPL22 and RPL9 isoforms by affinity purification, separation of polysomes bound to mRNA by preparative gradient centrifugation and analysis of mRNA transcripts specifically enriched (actively translated) by poly-Ribo-Seq. This is extremely relevant to cancer research because most studies perform RNA-seq of single or bulk tumour cells but there are many reports of discordance between mRNA and protein expression. The student will compare the transcriptome with the translatome (RNA-seq vs poly-Ribo-seq) through combination of state-of-the-art biochemical techniques and computational biology pipelines with the secondary supervisor. Finally, the student will develop cryo-EM structural biology skills by characterising structural changes in ribosomes expressing alternative isoforms with the third supervisor.</p> <h5>References</h5> <ol> <li>Riobo NA, Lu K, Ai X, Haines GM, Emerson CP Jr. (2006) Phosphoinositide 3-kinase and Akt are essential for Sonic Hedgehog signaling. Proc. Natl. Acad. Sci. USA 103(12):4505-10</li> <li>Riobo NA, Saucy B, Dilizio C, Manning DR (2006) Activation of heterotrimeric G proteins by Smoothened. Proc. Natl. Acad. Sci. USA 103(33):12607-12</li> <li>Riobo NA, Haines GM, Emerson CP Jr. Protein kinase C-delta and mitogen-activated protein/extracellular signal-regulated kinase-1 control Gli activation in Hedgehog signaling. Cancer Res. 66(2): 839-845 (2006).</li> <li>Robbins DJ, Fei DL, Riobo NA (2012) The Hedgehog signal transduction network Science Signal.4(200):pt7</li> <li>AH Polizio, P Chinchilla, X Chen, DR Manning and NA Riobo. Sonic Hedgehog activates the GTPases Rac1 and RhoA in a Gli-independent manner via coupling of Smoothened to Gi proteins. Sci. Signal. 7(pt4) (2011)</li> <li>XL Chen, P Chinchilla, J Fombonne, L Ho, C Guix, J Keen, P Mehlen and NA Riobo. Patched-1 pro-apoptotic activity is downregulated by modification of K1413 by the E3 ubiquitin-protein ligase Itchy homolog. Mol. Cell. Biol. &nbsp;34(20): 3855-66 (2014)</li> <li>Chen X, Morales-Alcala CC, Riobo-Del Galdo NA* (2018) Autophagic flux is regulated by interaction between the C-terminal domain of PATCHED1 and ATG101. Mol. Cancer Res. 16(5):909-19&nbsp;</li> <li>Brennan-Crispi DM, Overmiller AM, Tamayo-Orrego L, Marous MR, Sahu J, McGuinn KP, Cooper F, Georgiou I, Frankfurter M, Salas-Alanis JC, Charron F, Millar SE, Mahoney MG, Riobo-Del Galdo NA* (2019) Overexpression of Desmoglein2 in a mouse model of Gorlin syndrome enhances spontaneous basal cell carcinoma formation through STAT3-mediated Gli1 expression. J. Invest. Dermatol. 139(2): 300-7</li> <li>Mellis D, Staines KA, Peluso S, Georgiou ICh, Dora N, Kubiak M, Grillo M, Farquharson C, Kinsella E, Thronburn A, Ralston SH, Salter DM, Riobo-Del Galdo NA*, Hill RE*, Ditzel M*. (2021) Ubiquitin-protein ligase Ubr5 cooperates with Hedgehog signalling to promote skeletal tissue homeostasis. PLoS Genetics 17(4):e1009275</li> <li>Hopes T, Norris K, Agapiou M, McCarthy CGP, Lewis PA, O&rsquo;Connell MJ, Fontana J, Aspden JL. (2022) Ribosome heterogeneity in Drosophila melanogaster gonads through paralog-switching. Nucleic Acids Research 50:2240-57.</li> <li>Norris K, Hopes T, Aspden JL (2021) Ribosome heterogeneity and specialization in development. Wiley Interdiscip rev RNA 12:e1644.</li> <li>Hopkins FR, &Aacute;lvarez-Rodr&iacute;guez B, Heath GR, Panayi K, Hover S, Edwards TA, Barr JN, Fontana J. (2022) The native orthobunyavirus ribonucleoprotein possesses a helical architecture. MBio 13:e0140522</li> </ol>

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