1 PhD project offered in the IPP winter call 2020/2021
To protect cells from dysfunctional and toxic, disease-causing proteins formed by aberrant translation, eukaryotic co-translational quality control pathways detect translation defects and remove aberrant protein products, together with associated faulty mRNA blueprints and sometimes faulty ribosomal components. In the model eukaryote, budding yeast, the important quality control trigger factor Hel2 acts together with the ribosome-associated quality control trigger (RQT) complex to sense ribosome collisions and mark collided ribosomes for downstream processing. We have characterized interactions of Hel2 with RNA using the UV crosslinking and analysis of cDNA (CRAC) technique (Winz et al., 2019). In mammals, the ASC-1 complex has recently been described as an equivalent of the yeast RQT complex, but is also involved in DNA damage repair, where it recruits the demethylase ALKBH3 to sites of DNA damage.
In the first part of this project, using CRAC and selective ribosome profiling, we will test how different members of the RQT complex interact with RNA and ribosomes, and how they cooperate with each other and with Hel2 in triggering quality control in yeast.
In the second part of this project, we will explore whether the ASC-1 complex, together with the mammalian ortholog of Hel2 (ZNF598) can also recruit ALKBH3 to modified or damaged mRNA upon ribosome stalling. This could represent a repair strategy, as an alternative to the degradation usually triggered by ZNF598. In vitro and in vivo we will test the effect of mRNA modifications and damage on translation and on recruitment of the human ASC-1 complex and ALKBH3. Employing sequencing-based analyses of RNA modifications, we will test if ALKBH3 actively removes RNA modification at such sites.
Publications relevant to the project
Cahová*, Winz*, Höfer*, Nübel, Jäschke (2015) NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs. Nature, 519:374- 377. (*: equal contributions).
Hauenschild R, Tserovski L, Schmid K, Thüring K, Winz ML, Sharma S, Entian KD, Wacheul L, Lafontaine DL, Anderson J, Alfonzo J, Hildebrandt A, Jäschke A, Motorin Y, Helm M (2015) The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent. Nucleic Acids Research, 43(20):9950-9964.
Winz, Cahová, Nübel, Frindert, Höfer, Jäschke (2017) Capture and sequencing of NAD-capped RNA sequences with NAD captureSeq. Nature Protocols, 12 (1), 122-149.
Winz, Peil, Turowski, Rappsilber, Tollervey (2019) Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control. Nature Communications, 10(1), 563.