Cellular metabolism of misfolded proteins

The workgroup has previously generated mutants in the Death Inducer Obliterator (DIDO) gene, and has shown a function for this gene in RNA transcription and splicing. DIDO gene mutants show global alterations in RNA splicing patterns, which we attribute to the absence of one of three isoforms. Recent data have shown that the levels of the largest isoform control the production of differentially spliced RNA products (Mora Gallardo et al., 2019), and that its deletion leads to an inflammatory response (Gutierrez et al., 2022). 

We are now studying the downstream effects of the splicing defects on a cellular level. One part of the group is investigating the formation of RNA molecules that directly stimulate cellular immunity, much like viral RNA. A new project will focus on the the translation of misspliced RNA into proteins. Normally, a small proportion of proteins adopts an aberrant conformation and is rapidly eliminated by the cell. In our mutants, however, the natural degradation of "bad" proteins may not be sufficient, leading to the accumulation of large aggregates.

The TFM proposal comprises the analysis of the cellular pathways involved in the metabolism of aberrant proteins, which result form translation of misspliced messenger RNA. The focus will be on the equilibrium between natural degradation and the accumulation in localized (concentrated) aggregates. We plan to use a combination of techniques, including  cell culture, immunofluorescence with specific antibodies, protein identification and western blotting, and (if required) CRISPR suppression of target genes such as interferon-stimulated genes (ISGs). Drug treatments will be used to interfere with natural protein turnover pathways such as ubiquitination, proteasome and trasnport along the cytoskeleton. Final objetive is evaluate and quantify these deleterious effects of protein misfolding on the cell. The relevance of the observations made during the practical work will be studied on the hand of public databases. Finally, the student is expected to finish the project with a written report.