During the past year, we have been focusing to develop new methodologies that are needed for studying the structural biology of the 3'UTR RNA of the human VEGF mRNA. VEGF is a single most important protein that is critical to angiogenesis. These methods include GASR (J. Am. Chem. Soc., 2009, 131 (30), pp 10507-10515) and G2G (J. Mol. Biol., 2009, Aug. 8). GASR stands for """"""""derive global architecture from SAXS and RDC"""""""" data, and G2G stands for """"""""derive global structure of large RNAs in solution from global restraints"""""""". In addition, we have applied the G2G method to determine the three dimensional structure of the 3'UTR RNA fragment (102 nt) of the turnip crinkle virus (TCV). This 102-nt RNA plays an important role in enhancing translation initiation and is the prototype of 3'UTR RNAs that involved in regulation of gene expression. Expression of many proteins important to cancer biology, such as VEGF, is also controlled by 3'UTR RNAs. 1. Developing GASR method. Determining global structures of multi-subunit biomacromolecules in solution is a challenging problem. We report here a methodology that determines the global architecture of hetero- or homodimeric systems in solution using the residual dipolar couplings (RDCs) as the global orientation and the small-angle X-ray scattering (SAXS) as the global shape restraints;this methodology is implemented in an efficient program, known as global architecture derived from SAXS and RDC (GASR). We first applied the method to derive the global architecture of HIV protease, a globular homodimeric protein, to test the robustness of the method using simulated data with added noise. We then applied the method to determine the interfaces in the integrin-linked kinase (ILK)-PINCH complex using the experimental data. Without the benefit of SAXS data, the global architecture (and thus the interfaces of this complex) was underdetermined because of a lack of a sufficient number of detectable experimental distance restraints between the subunits of the complex. This method provides a new general approach for determining global structures of macromolecular complexes in solution. We have published the first manuscript on the method for an RNA:RNA complex (JACS, 2008 Mar 19;130(11):3292-3). An article that present a full theoretical and experimental description of the method has been published (J. Am. Chem. Soc., 2009, 131 (30), pp 10507-10515). 2. Developing G2G method. We have developed a novel top-down methodology that uses duplexes global orientations and overall molecular dimension restraints, which were extracted from experimental NMR and small angle X-ray scattering (SAXS) data respectively, to determine global architectures of large RNA molecules. The methodology is implemented in an efficient computational program package, G2G. We demonstrate the efficiency of the method by rapidly determining the global structure of a 71-nucleotide RNA using experimental data. The backbone RMSD of the ensemble of the calculated global structures relative to the X-ray crystal structure is 3.0 0.4 , and this RMSD is only 2.4 0.16 for the three duplexes that were orientation- and translation-restrained during the calculation. The global structure dramatically simplifies interpretation of multi-dimensional nuclear Overhauser spectra for a rapid high resolution structure determination. We anticipate that this top-down method will become the standard routine for determining structures of large RNAs in solution. An article that presents a full account of theory and experimental aspects of the method has been published in JMB (doi:10.1016/j.jmb.2009.08.001 ). 3. Structure determination of the TCV RNA using NMR and small angle X-ray scattering (SAXS). The 3′ UTRs of many plant viral mRNAs have been demonstrated to have tRNA-like activities and have been proposed to fold into a tRNA-like structure. Among these plant viral RNAs, the 3′UTR of the turnip yellow mosaic virus (TYMV) was best characterized biochemically, and a tRNA-like computational model based on tRNA has been proposed. However, a three-dimensional structure has not been determined for any of these UTRs. The experimentally determined tRNA-like structures will provide a critical piece of evidence to support the notion that the 3′UTRs of these plant viruses are directly involved in the translation process and may be considered a conceptual step toward understanding the general mechanisms of 3′UTR involvement in the regulation of gene expression. The tRNA-like structure of a 3′UTR would also provide a critical piece of evidence supporting the notion that the function of these 3′UTRs is related to their ability to form tRNA-like structures. The involvement of the 3′UTR in translation may not be limited to plant viruses. Two other examples found in mammalian cells are the 3′UTR of mRNAs coding for the ornithine decarboxylase (ODC) and the vascular endothelial growth factor (VEGF). In mammalian cells, the polyamines putrescine, spermidine, and spermine are essential for proliferation and differentiation. The mammalian ODC catalyzes the first and rate-limiting steps in the polyamine biosynthetic pathway. VEGF plays an important role in tumor angiogenesis and cancer progression. Both of these essential proteins are regulated at multiple levels, including the mRNA level, via 3′UTRs. It has been proposed by Dr. Bruce Shapiros laboratory, of the CCR, that both RNAs share a secondary structure similar to that of the turnip crinkle virus 3′UTR fragment (tcvRNA), and therefore, possibly the tertiary structure. We have applied the G2G method to determine the global structure of the tcvRNA. A manuscript that describes the structure determination of the tcvRNA is currently under consideration of a journal. 3. The gene expression of VEGF is regulated at multiple stages, including but not limited to post-transcription and translational levels. The down-regulation of the VEGF expression has been mapped to specific interactions between RNA125 and one of proteins in the GAIT complex. An earlier study by Dr. Fox's laboratory showed that the down-regulation involves specific interactions between the linker domain of the glutamyl-prolyl tRNA synthetase (EPRS), which is one of components of the GAIT complex, and a 29-nt hairpin (H29) that is entirely composed of A or U nucleotides and is the part of the 125-nt RNA . The structural basis for this important interaction is unknown. The EPRS linker domain (ELD) consists of three dual-helical bundles and the interaction site for H29 has been mapped to the first two helical bundles, r1r2, by Dr. Fox's group using biochemical methods. r1r2 contains 114 amino acids and the three-dimensional structure of the first dual-helical bundle has been reported. The helical structure of r1r2 differs from known RNA-binding structural motifs and may represent a new RNA-binding motif. We have completed assigning the backbone signals of r1r2 and are in the process of mapping the RNA/protein interaction surface. We are currently investigating the structural basis of down- and up-regulation of the VEGF expression using combination of NMR and SAXS. We have established a collaboration with Professor Paul Fox's group of the Cleveland Clinic to corroborate our structural results with biological functions.
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