Project Summary
Contact
List of current group members
Collaborating team publications
Factsheet [PDF]
Link to Children's Hospital, University of Heidelberg

Project Summary

Overview
Our team focusses on basic and translational aspects of mRNA metabolism in hereditary diseases. One specific aspect of this general theme is the mechanism of nonsense mediated decay [NMD], one of the cell's key mRNA quality control pathways. The other specific aspect is the mechanism and the regulation of mRNA 3' end processing. The entry points into these two major projects of the group have been the detailed clinical analysis of two common hematological disorders, ß-thalassemia and thrombophilia, respectively. Interestingly, it is now becoming apparent that the principles that have been uncovered by the analysis of these two model conditions are applicable more generally. Furthermore, the results of these projects have illustrated that the detailed analysis of unexplained medical phenomena can result in the identification and characterisation of previously unrecognized basic biological mechanisms with broad implications. With a continued focus on the "disease mechanisms aspect" of our work, future work will build on the maturity that this work has reached towards more translational aspects.

Nonsense mediated decay [NMD]
NMD targets mRNAs with premature translation stop codons for rapid degradation. The phenomenon of NMD was first described in human cells and in yeast almost simultaneously in 1979, which already suggested broad phylogenetic conservation and an important biological role of this intriguing mechanism. The key mechanistic question of NMD is how an abnormal mRNA is recognized, which differs from the normal mRNA only in the length of its open reading frame but not in its overall structure. Significantly, this recognition of translational sense occurs before the mRNA is entering its rounds of translation. The basic signals that are required for NMD to work are [1] the splicing dependent deposition of a multi-protein complex, referred to as exon junction complex [EJC], close to the exon-exon boundaries of the mRNA and [2] the interaction of the EJC with components of the terminating ribosome. The translation apparatus can thus distinguish a proper translation termination event in the 3' terminal exon, which is not followed by a downstream EJC from an improper termination event, which is followed by a downstream EJC.
For our analysis of the NMD mechanism, we used mutation analyses of EJC components and RNAi-mediated depletion of NMD- and EJC-proteins in the context of a functional tethering assay and showed that different EJC components specify distinct routes of NMD with differential cofactor requirements [Gehring et al. 2005]. This work challenged the then predominant paradigm and proposed a concept that has since gained much additional support through work by us and others. Subsequent work functionally analyzed the isoforms of the key NMD factor UPF3 [UPF3a and UPF3b] and found that these isoforms serve clearly separable functions in NMD and in translation, although they are found in EJCs with an otherwise similar composition [Kunz et al. 2006]. In addressing the important question whether different modes of translation may serve distinguishable functions in NMD, we found that NMD can be activated by both, cap-dependent and IRES-mediated translation [Holbrook et al. 2006]. Furthermore, we provided direct evidence that normal endogenous cellular mRNAs can be regulated by alternative NMD pathways [Gehring et al. 2005]. The existence of such alternative NMD pathways conceptually suggested NMD to represent a variable process affecting RNA stability and potentially regulating different classes of mRNAs in different tissues or at different times of development. This new concept significantly widened the previously assumed function of NMD as a constitutive and mechanistically linear "vacuum cleaner" for misprocessed and mutated mRNAs [Neu-Yilik et al. 2004 & 2008].
As a logical next step in further characterising the mechanism of NMD pathways, we studied the interaction of components of the EJC and the translation termination complex. It had previously been shown by others that the post-termination complex consists of at least 4 specific proteins, the release factors eRF1 and eRF3, the helicase UPF1 and the UPF1-specific kinase SMG1. The phosphorylation of UPF1 by SMG1 is an essential step of triggering mRNA degradation and SMG1 has been shown to be activated by an interaction with the EJC in general and its component UPF2 in particular. We have recently found evidence that UPF1 delays termination thus enabling the post-termination complex to either interact with the poly[A] binding protein [PABPC1], likely inducing cycling of the mRNA into the next round of translation, or to interact with the EJC to trigger UPF1 phosphorylation and mRNA decay. We also demonstrated that UPF1 phosphorylation can either be supported by UPF2 or by UPF3b as a biochemical correlate for the previously identified alternative NMD pathways. On the basis of these observations, we propose an integrated model of NMD, where UPF1 delays translation termination and is phosphorylated by SMG1, if the termination-promoting interaction of PABPC1 with eRF3 cannot readily occur. The EJC, with UPF2 or UPF3b as a cofactor, interferes with proper termination and leads to the phosphorylation of UPF1 and subsequent decay. This model integrates previously competing models of NMD and suggests a mechanistic basis for alternative NMD pathways [Ivanov et al. 2008].
The medical perspective of NMD as a key post-transcriptional mechanism limiting abnormal and regulating normal gene expression is a particular focus of our team [Holbrook et al. 2004]. Recently, other groups have shown that NMD can vary between cell types and between individuals. Furthermore, the expression of disease-related genes that carry identical nonsense mutations has been reported to differ and to modulate disease severity. These observations led us to the hypothesis that variations of NMD efficiency may contribute to the phenotypic variability of hereditary and possibly of acquired genetic disorders. It has so far been impossible to test this hypothesis because of the lack of an assay system to reliably quantify NMD efficiency in readily accessible clinical material. We have now established an assay system for the quantification of NMD efficiency, which is based on carefully validated cellular NMD target transcripts. We demonstrated in a HeLa cell model system that NMD efficiency can be remarkably variable and represents a stable characteristic of different subclones of HeLa cells. In one of these lines, low NMD efficiency was shown to be functionally related to the reduced abundance of the exon junction component RNPS1. Cellular concentrations of RNPS1 can thus modify NMD efficiency and cell type specific co-factor availability emerged as a novel principle that controls NMD [Viegas et al. 2007]. In a complementary approach, we designed a cell-based chemiluminescence reporter system that enables us to robustly measure the effect of modifications of the NMD pathway with a high degree of sensitivity and in a format that can be used for high throughput small molecule and/or RNAi screening [Boelz et al., 2007].
The medical dimension of the variability of NMD has been directly addressed in cystic fibrosis [CF] by a collaborative project with the Kerem group at Hebrew University in Jerusalem. Previous work of the Kerem group had shown that nonsense suppression in CF-patients with nonsense mutations can reconstitute CFTR function. However, the readthrough efficiency and the subsequent correction of chloride transport in response to the aminoglycoside gentamicin has been highly variable among a group of CF-patients carrying the same nonsense mutation. We have now demonstrated in nasal epithelial cell lines of such patients that the abundance of nonsense mutated CFTR transcripts differs. A functional role of variable NMD efficiency was implicated by the finding that the differences in CFTR transcript levels correlated with differences in the abundance of normal cellular NMD targets and by experimentally depleting and repleting important NMD factors. Interestingly, chloride transport could be elevated more effectively by nonsense suppression in patients with less efficient NMD than in those with highly efficient NMD [Linde et al, 2007a & 2007b]. More generally, this study further supported the concept that variable NMD efficiency may play a role as a genetic modifier of different cellular functions and may govern the response to treatments that aim to promote readthrough of nonsense mutated transcripts.

3'end processing of prothrombin mRNA and predisposition to thrombosis [thrombophilia].
The entry point of this project was a clinical study that aimed to identify the genetic factors predisposing children to develop thrombosis. At that time, a common mutation of the coagulation factor II gene [also referred to as prothrombin] was found, which affects about 2% of the North-West European population. This mutation increases prothrombin plasma levels approximately 1.5-fold and the risk of thrombosis approximately 5-fold. Interestingly, this mutation affects the 3' terminal nucleotide of the mRNA exactly at the site where the pre-mRNA is cleaved and polyadenylated. This location suggested to us that altered RNA processing might be involved in causing the observed increase of prothrombin gene expression by an unidentified molecular mechanism. In our subsequent functional analysis of this mutation we discovered an increase of 3'end processing efficiency as a novel principle for the pathogenesis of a hereditary disorder [Gehring et al. 2001].
Further studies identified that inefficient 3' end prothrombin mRNA processing is physiological and has been conserved in evolution. Moreover, we discovered that the prothrombin 3' end processing signal is characterized by a delicate balance of positive and negative signals, which can be disturbed by a number of clinically relevant gain-of-function mutations in this area [Danckwardt et al. 2004, 2006a & 2006b]. One of these mutations represents the first example of a pathologically relevant mutation affecting the CstF binding site in the 3' flanking sequence of a human gene [Danckwardt et al., 2004].
One important component of the unusual architecture of the prothrombin 3' end processing site is an upstream sequence element [USE] that is characterized by overlapping conserved 3' UTR sequence motifs and which is located at a critical distance to the 3' end processing signal within the 3' UTR. We have subsequently discovered prothrombin to represent the prototype of a novel class of genes, which is characterised by low efficiency 3' end processing signals that are balanced by an activating USE. We therefore addressed the mechanism of USE function and identified proteins that specifically interact with the USE to promote 3' end formation. Interestingly, these factors included splicing proteins [U2AF35 and U2AF65, hnRNPI] that also promote 3' end formation via USEs contained in a variety of other RNAs, indicating a broader functional role of this type of 3' end processing in many other mammalian genes [Danckwardt et al 2007 & 2008].

Contact

Matthias W. Hentze, MD
Associate Director
European Molecular Biology Laboratory
Meyerhofstraße 1
69117 Heidelberg
Tel: +49 6221 387-8501
Fax: +49 6221 387-8518
E-mail: matthias.hentze@embl.de
Group home page

Andreas E. Kulozik, MD, PhD
Head of the Department
Pediatric Oncology, Haematology, Immunology and Pulmonology
Children's Hospital, University of Heidelberg
Im Neuenheimer Feld 153
69120 Heidelberg
Tel: +49 6221 56 4555
Fax: +49 6221 56 4559
E-mail: andreas.kulozik@med.uni-heidelberg.de

List of current group members

Niels Gehring, PhD [senior post-doc]
Sven Danckwardt, MD [senior physician scientist]
Gabriele Neu-Yilik, PhD [senior post-doc]
Joachim Kunz, MD [physician scientist]
Melissa Schlitter, MD [physician scientist, medical faculty post-doc training program]
Stella Lamprinaki [EMBL International PhD Programme]
Anne Gantzert [medical faculty PhD student]

Collaborating team publications

1. Thermann, R., G. Neu-Yilik, A. Deters, U. Frede, K. Wehr, C. Hagemeier, M.W. Hentze, A.E. Kulozik. Binary specification of nonsense codons by splicing and cytoplasmic translation. EMBO Journal, 17, 3484-3494 [1998]
2. Hentze, M.W., A.E. Kulozik. A perfect message: RNA surveillance and Nonsense Mediated Decay. Cell 96: 307-310 [1999]
3. Neu-Yilik, G., N.H. Gehring, R. Thermann, U. Frede, M.W. Hentze, A.E. Kulozik. Splicing and 3' end formation in the definition of NMD competent human b-globin mRNPs. EMBO Journal 20: 532-540 [2001].
4. Gehring, N.H., U. Frede, G. Neu-Yilik, P. Hundsdoerfer, M.W. Hentze, A. E. Kulozik. Increased efficiency of mRNA 3' end formation: a novel genetic mechanism contributing to hereditary thrombophilia. Nature Genetics 28: 389-392 [2001].
5. Brocke, K., G. Neu-Yilik, N.H. Gehring, M.W. Hentze, A. E. Kulozik. The human intronless melanocortin 4-receptor gene is NMD-insensitive. Human Molecular Genetics 11: 331-335 [2002].
6. Danckwardt, S., G. Neu-Yilik, R. Thermann, U. Frede, M.W. Hentze, A. E. Kulozik. Abnormally spliced b-globin mRNAs: A single point mutation generates NMD-sensitive and NMD-insensitive transcripts Blood 99: 1811-1816 [2002].
7. Schell, T., A.E. Kulozik, M.W. Hentze. Integration of splicing, transport and translocation to achieve mRNA quality control by the nonsense-mediated decay pathway. Genome Biology 3: REVIEWS 1006 [2002].
8. Gehring, N.H., G. Neu-Yilik, T. Schell, M.W. Hentze, A.E. Kulozik. Y14 and hUpf3b form an NMD-activating complex. Molecular Cell 11:939-949 [2003].
9. Schell, T., T. Köcher, M. Wilm, B. Seraphin, A.E. Kulozik and M.W. Hentze. Complexes between the nonsense-mediated decay [NMD] factor Hupf1 and essential NMD factors in HeLa cells. Biochemical Journal 373: 775-83 [2003].
10. Baron-Benhamou J, Gehring NH, Kulozik AE, Hentze MW. Using the lambdaN Peptide to Tether Proteins to RNAs. Methods in Molecular Biology 257:135-154 [2004].
11. Neu-Yilik, G., N.H. Gehring, M.W. Hentze, A.E. Kulozik. Nonsense-mediated mRNA decay: from vacuum cleaner to Swiss army knife. Genome Biology 5:218 [2004].
12. Danckwardt, S., N.H. Gehring, G. Neu-Yilik, P. Hundsdoerfer, M. Pförsich, M.W. Hentze, A.E. Kulozik. The prothrombin 3'end formation signal reveals a unique architecture that is sensitive to thrombophilic gain-of-function mutations. Blood 104:428-35. [2004].
13. Holbrook, J., G. Neu-Yilik, M.W. Hentze, A.E. Kulozik. Nonsense mediated decay approaches the clinic. Nature Genetics 36: 801-809 [2004].
14. Gehring, N, J.B. Kunz, G. Neu-Yilik, S. Breit, M. H. Viegas, M. W. Hentze, A.E. Kulozik. Exon-junction components specify distinct routes of nonsense-mediated mRNA decay with differential co-factor requirements. Molecular Cell 20: 65-75 [2005].
15. Danckwardt S., K. Hartmann, N. Gehring, M.W. Hentze, A.E. Kulozik. 3'end processing of the prothrombin mRNA in thrombophilia. Acta Hematologica. 115, 192 - 197 [2006a].
16. Danckwardt S., K. Hartmann, M.W. Hentze, Y. Levy, R. Eichele, V. Deutsch, A.E. Kulozik, O. Ben-Tal. The prothrombin 20209 C®T mutation in Jewish-Maroccan Caucasians: Molecular analysis of gain-of-function of 3’ end processing. Journal of Thrombosis and Haemostasis 4: 1078-1085 [2006b].
17. Stockklausner, C., S. Breit, G. Neu-Yilik, N. Echner, M.W. Hentze, A.E: Kulozik, N. Gehring. The uORF-containing thrombopoietin mRNA escapes Nonsense-Mediated Decay [NMD]. Nucleic Acids Research 34: 2355-2363 [2006].
18. Kunz, J., G. Neu-Yilik, M.W. Hentze, A.E: Kulozik, N. Gehring. Separable functions of hUpf3a and hUpf3b in nonsense-mediated mRNA decay and translation RNA 12:1015-1022 [2006].
19. Holbrook, J., G. Neu-Yilik, N. Gehring, A. E. Kulozik, M. Hentze. Internal Ribosome Entry Sequence [IRES]-mediated translation initiation triggers efficient nonsense mediated decay EMBO Reports 7:722-726 [2006].
20. Boelz; S., G. Neu-Yilik, N.H Gehring, M.W Hentze, A. E. Kulozik. A chemiluminescence-based reporter system to monitor nonsense-mediated mRNA decay. Biochemical and Biophysical Research Communications 349:186-191 [2006].
21. Linde, L., S. Boelz, M. Nissim-Rafima, Y. Oren, M. Wilschanski, Y. Yakoov, D. Virgilis, G. Neu-Yilik, A.E. Kulozik, E. Kerem, B. Kerem. Nonsense-mediated mRNA decay affects the level of CFTR nonsense transcripts and governs the response to aminoglycosides. Journal of Clinical Investigation 117:683-692 [2007a].
22. Linde, L, S. Boelz, G., A.E. Kulozik, B. Kerem. The efficiency of nonsense-mediated mRNA decay is an inherent character and varies among different cells. European Journal of Human Genetics 15: 1156-1162 [2007b].
23. Danckwardt, S., I. Kaufmann, M. Gentzel, K. Förstner, A.S. Gantzert, N.H. Gehring, G. Neu-Yilik, P. Bork, W. Keller, M. Wilm, M.W. Hentze, A.E. Kulozik. Splicing factors stimulate polyadenylation via USEs at non-canonical 3’ end formation signals. EMBO Journal 26:2658-69 [2007]
24. Viegas, M. H., N. H. Gehring, S. Breit, M. W. Hentze, A. E. Kulozik. The abundance of RNPS1, a protein component of the exon junction complex, can determine the 5 variability in efficiency of the Nonsense Mediated Decay Pathway. Nucleic Acids Research 35:4542-4551 [2007].
25. Danckwardt, S, M.W. Hentze, A.E. Kulozik. 3' end mRNA processing: Molecular mechanisms and implications for health and disease. EMBO Journal 27, 482–498 [2008].
26. Ivanov P., N. Gehring, J. Kunz, M.W. Hentze, A.E. Kulozik. Interactions between UPF1, eRFs, PABP and the exon junction complex suggest an integrated model for mammalian NMD pathways. EMBO Journal e-pub ahead of print [2008].

Factsheet [PDF]

Link to Children's Hospital, University of Heidelberg