(a) Scientific basis
Mutations in ribosomal protein (RP) genes, coding for components of ribosomes (the protein production factories in cells), account for ~75% of Diamond-Blackfan Anaemia (DBA), a rare inherited syndrome characterised by severe anaemia due to a failure in the bone marrow to produce red blood cells. Two important questions arising are: (i) why do mutations in the ubiquitous RP genes specifically affect red blood cells, and (ii) what are the genetic mutations in the ~25% of undiagnosed cases?
We previously showed that GATA1, a key protein controlling all genes in red cells, contacts RP genes in red cells suggesting direct control by GATA1, which may help explain the anaemia caused by RP gene mutations. Moreover, possible mutations in GATA1 contact sites in RP genes, may account for (some of) the ~25% of undiagnosed cases. We thus abolished GATA1 contacts in two RP genes in red cells resulting in their downregulation. We also teamed up with the diagnostic lab at King’s College Hospital and the UK and French DBA Registries, to design and test a new diagnostic panel that includes all GATA1 contact sites in RP genes. In this PhD, the student will (i) characterise fully the cells with abolished GATA1 contacts in the two RP genes and (ii) analyse undiagnosed DBA samples using the new diagnostic panel.
(b) Techniques and skills:
– Cell culture, flow cytometry, molecular and cellular methods
– Polysome profiling by ultra-high performance liquid chromatography (uHPLC)
– Analysis of polysome-associated mRNAs (translatome) and RNA expression profiling by next generation sequencing (NGS)
– Proteomics by mass spectrometry
– Analysis of NGS and mass spectrometry data (bioinformatics)
– Electrophoretic mobility shift assays (EMSAs), CUT&RUN
– Gene editing by CRISPR/Cas9
(c) Overarching aims:
(i) detailed cellular, molecular and proteomic characterisation of cells with abolished GATA1 contacts in two RP genes (namely, RPS19 and RPL11);
(ii) screening of undiagnosed DBA samples for mutations in GATA1 contacts with RP genes, using a novel diagnostic panel;
(iii) Validation of prioritised mutations from (iii) using gene editing in an erythroid cell line.
(d) Objectives per year:
Year 1: Familiarisation with methods required for aim (i). Polysome profiling.
Year 2: Translatome, RNA expression profiling, proteomics; data analysis. Analysis by EMSAs/CUT&RUN of potential mutations from the diagnostic assay.
Year 3: Data analysis completion; first manuscript drafting. Gene editing of prioritised mutations from the diagnostic panel.
Year 4: Analysis of gene-edited cells; thesis writing; second manuscript drafting.
(e) Summary of a potential 3-month rotation project:
The student will culture RPS19 and RPL11 gene-edited cells and will check expression of the two genes at the RNA and protein levels. Will also prepare samples for polysome profiling.