STRUCTURE LRP130+RNA · The Structure and RNA Binding Specificity of LRP130, a PPR Motif Protein.
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2005-09-01 → 2008-08-31
- EU contribution
- €80,000
- Participants
- 1
- Scheme
- IRG
Lines connect the coordinator with its partners.
Results in brief
Final Activity Report Summary - STRUCTURE LRP130+RNA (The Structure and RNA Binding Specificity of LRP130, a PPR Motif Protein)
The POZ/BTB domain is an evolutionarily conserved motif found in approximately 40 zinc-finger transcription factors (POZ-ZF factors). Several POZ-ZF factors are implicated in human cancer, and POZ domain interaction interfaces represent an attractive target for therapeutic intervention. Miz-1 (Myc-interacting zinc-finger protein) is a POZ-ZF factor that regulates DNA-damage-induced cell cycle arrest and plays an important role in human cancer by virtue of its interaction with the c-Myc and BCL6 oncogene products. The Miz-1 POZ domain mediates both self-association and the recruitment of non-POZ partners. POZ-ZF factors generally function as homodimers, although higher-order associations and hetero-meric interactions are known to be physiologically important; crucially, the interaction interfaces in such large complexes have not been characterised. We have reported the crystal structure of the Miz-1 POZ domain up to 2.1 A resolution. The tetrameric organisation of Miz-1 POZ reveals two types of interaction interface between subunits; an interface of alpha-helices resembles the dimerisation interface of reported POZ domain structures, whereas a novel beta-sheet interface directs the association of two POZ domain dimers. We show that the beta-sheet interface directs the tetra-merisation of the Miz-1 POZ domain in solution and therefore represents a newly described candidate interface for the higher-order homo- and hetero-oligomerisation of POZ-ZF proteins in vivo. Pumilio controls a number of processes in eukaryotes, including the translational repression of hunchback (hb) mRNA in early Drosophila embryos. The Pumilio Puf domain binds to a pair of 32 nucleotide (nt) Nanos Response Elements (NRE1 and NRE2) within the 3 untranslated region of hb mRNA. Despite the elucidation of structures of human Pumilio Puf domain in complex with hb RNA elements, the nature of hb mRNA recognition remains unclear. In particular, the site that mediates regulation in vivo is significantly larger than the 8-10 nt RNA elements bound to single Puf molecules in crystal structures. Here we present biophysical and biochemical data that partially resolve the paradox. We show that each NRE is composed of two binding sites and that two Puf domains can co-occupy a single NRE. The two Puf domains bind independently/cooperatively to NREs, and there is a higher affinity for the 3' site than for the 5' site. We suggest that the 2 Pumilio:1 NRE complex is the functional regulatory unit in vivo.
Data: CORDIS, © European Union
Project objective
This proposal aims to reveal the structure of the RNA binding domain of the human LRP130 protein, along with the RNA binding specificity for this protein, and the structure of the RNA bound complex.LRP130 is an RNA binding protein that associates with spli ced mRNAs. The protein does not contain any sequence homology with known RNA binding motifs, and therefore is likely to bind RNA through a novel RNA binding fold. LRP130 contains several pentatricopeptide repeat (PPR) motifs; other PPR containing proteins have been shown to bind RNA, and several have been implicated in organellar RNA metabolism. In vivo the majority of the total LRP130 protein is located within mitochondria, where it is directly bound to polyadenylated RNAs. There is also a small fraction w ithin nuclei, bound to mRNAs. In vitro, the RNA binding activity maps to the C-terminal region, and when expressed in E.coli, this protein displays a preferential affinity for polypyrimidines. The RNA binding domain of LRP130 will be expressed in E.coli, p urified by standard chromatographic techniques, and the structure solved by X-ray crystallography. In addition, the RNA binding specificity of this protein will be determined using in vitro evolution (SELEX) experiments. This will provide a high affinity l igand for crystallisation in complex with the protein, as well as suggesting a consensus sequence for RNA binding that can be used to identify sites of interaction in potential target mRNAs. The structure of the protein-RNA complex will then be solved, aga in by crystallography.LRP130 is the first identified mammalian protein directly bound to mitochondrial RNA in vivo. The structure of the RNA binding domain, with and without RNA bound, will be an important step towards understanding the structural basis fo r the functions of PPR motif proteins, and will potentially provide a molecular explanation for the recently described association of mutations in LRP130 with cytochrome c oxidase deficiency in humans.
Original text from CORDIS.
Participants
- UNIVERSITY OF LEEDS · LEEDSCoordinatorUnited Kingdom
Links
Data: CORDIS, © European Union