The control of mRNA fate during cellular stress
Zusammenfassung der Projektergebnisse
During various forms of cellular stress, bulk protein synthesis is severely reduced or even abolished due to the impairment of the initiation step of mRNA translation. This is mainly facilitated by an upregulation of eIF2α phosphorylation or the cleavage of eIF4G, the deregulation of two key translation initiation factors. Notably, in various if not all eukaryotic cells, translationally stalled transcripts are not degraded but recruited to transiently forming cytoplasmic stress granules (SGs). In previous studies we showed that IGF2BP1, also termed ZBP1, is recruited to stress granules where it prevents the turnover of its target mRNAs. Accordingly, we proposed that the selective stabilization of mRNAs by RNA-binding proteins (RBPs) is facilitated via stress granules and provides a functional screening criterion for the identification of physiologically relevant RNA-substrates of SG-recruited RBPs. Thus, the major aim of the proposed studies was to evaluate how mRNA fate is controlled during cellular stress. Specifically we aimed at analyzing: 1) if the formation of stress granules is essential for bulk mRNA stabilization during cellular stress; 2) the role of the IGF2 mRNA binding protein family (IGF2BP) during cellular stress; 3) the protein composition and dynamics of SGs in further detail. (1) Studies addressing the role of SGs in preventing mRNA degradation were substantially slowed down due to the fact that various reported strategies for preventing or impairing SG-formation were barely or not reproducible. However, we finally managed to establish two strategies which allow preventing stress granule formation without affecting stress signaling. These enabled us to validate that SG-formation is dispensable for the stabilization of bulk mRNA during cellular stress. Along these studies, we also observed that the putative prion-like proteins like TIA1 or G3BP1, which were proposed to serve as scaffolds facilitating SG-assembly, are rapidly exchanged between cytoplasm and SGs. On the contrary, RBPs like IGF2BPs or YBX1 are barely if at all turned over in SGs (Bley et al., in preparation). These findings contradict the current view of a prion-based assembly of SGs. In view of the proposed involvement of SGs or SG-like aggregates in human diseases, in particular neurodegenerative diseases, we consider this finding important and will address protein dynamics in stressed cells in further detail. (2) Studies on the role of IGF2BPs during the cellular stress response revealed that all three members of the protein family are recruited to SGs in a RNA-binding dependent manner. Moreover, we successfully used the observed IGF2BP-dependent stabilization of target mRNAs during cellular stress to identify novel target transcripts of IGF2BP1, including MAPK4, PTEN and LEF1. This did set the stage for three recently published studies in which we identified and characterized IGF2BP1 to act as a key promigratory post-transcriptional regulator of actin dynamics and pro-mesenchymal gene expression in tumorderived cells. By controlling the expression of MAPK4 and PTEN, IGF2BP1 enhances directed cell migration and pro-migratory cell-matrix adhesion. By promoting the expression of LEF1, IGF2BP1 enhances promesenchymal gene expression including the upregulation of FN1 and SLUG synthesis. (3) Finally, we aimed at characterizing the protein composition of SGs by establishing purification procedures of transiently forming stress granules. In cells stably expressing GFP-IGF2BP1, we attempted to isolate stress granules by combining density centrifugation and immunopurification. At first, we evaluated protocols without cross-linking prior to cell lysis. This allowed purifying mRNP-like complexes which however were much smaller than the expected SGs suggesting that these disassembled during or post lysis. Next, we attempted to stabilize SGs before cell lysis by formaldehyde or chemical cross-linking. This, however, prevented the successful extraction of SGs, even when combining cross-linking with perturbing cytoskeletal integrity. These findings together with FRAP (Fluorescence Recovery After Photobleaching) studies of key SG-localized proteins provided further evidence that SGs are transient assemblies of rather stable mRNPs without any significant intrinsic prion-like aggregation of RBPs. In summary our studies allowed us to: 1) validate that stress granules are not important for the stabilization of bulk mRNA during cellular stress; 2) establish a functional screening protocol, termed SIRL (Stress-based Isolation of RNA Ligands), allowing the identification of target transcripts of RNA-binding proteins; 3) characterize the role IGF2BP1 in tumor cell migration and adhesion based on SIRL-identified target mRNAs.
Projektbezogene Publikationen (Auswahl)
- (2006) ZBP1 regulates mRNA stability during cellular stress. J Cell Biol, 175, 527-534
Stohr, N., Lederer, M., Reinke, C., Meyer, S., Hatzfeld, M., Singer, R.H. and Huttelmaier, S.
- (2012) IGF2BP1 promotes cell migration by regulating MK5 and PTEN signaling. Genes Dev, 26, 176-189
Stohr, N., Kohn, M., Lederer, M., Glass, M., Reinke, C., Singer, R.H. and Huttelmaier, S.
(Siehe online unter https://doi.org/10.1101/gad.177642.111) - (2012) IGF2BP1: a post-transcriptional "driver" of tumor cell migration. Cell Adh Migr, 6, 312-318
Stohr, N. and Huttelmaier, S.
- (2013) IGF2BP1 promotes mesenchymal cell properties and migration of tumor-derived cells by enhancing the expression of LEF1 and SNAI2 (SLUG). Nucleic Acids Res, 41, 6618-6636
Zirkel, A., Lederer, M., Stohr, N., Pazaitis, N. and Huttelmaier, S.
(Siehe online unter https://doi.org/10.1093/nar/gkt410) - (2013) Subcellular localization and RNP formation of IGF2BPs (IGF2 mRNA-binding proteins) is modulated by distinct RNA-binding domains. Biol Chem, 394, 1077-1090
Wachter, K., Kohn, M., Stohr, N. and Huttelmaier, S.
(Siehe online unter https://doi.org/10.1515/hsz-2013-0111) - Stress granules are dispensable for mRNA stabilization during cellular stress. Nucleic Acids Research, Volume 43, Issue 4, 27 February 2015, Pages e26
Nadine Bley, Marcell Lederer, Birgit Pfalz, Claudia Reinke, Tommy Fuchs, Markus Glaß, Birgit Möller, Stefan Hüttelmaier
(Siehe online unter https://doi.org/10.1093/nar/gku1275)
