Specialised ribosomes in cancer: structural, biochemical and functional characterisation

Summary

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.

Objectives:

1. Determine the contribution of ribosomal alternative subunits RPL22L1 and RPL9 to the oncogenic phenotype
2. Isolate of ribosomes from PTCH1 WT and mutant cells to investigate their subunit composition and identify mRNAs that are preferentially translated by different oncoribosomes.
3. Investigate changes in the macromolecular structure of specialised ribosomes by cryo-EM.

Novelty/Timeliness:

The existence of specialised “oncoribosomes” 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.

Experimental approach:

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.

References

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