RESUMO
Rhabdomyosarcoma (RMS) is an aggressive pediatric tumor with a poor prognosis for metastasis and recurrent disease. Large-scale sequencing endeavors demonstrate that Rhabdomyosarcomas have a dearth of precisely targetable driver mutations. However, IGF-2 signaling is known to be grossly altered in RMS. The insulin receptor (IR) exists in two alternatively spliced isoforms, IR-A and IR-B. The IGF-2 signaling molecule binds both its innate IGF-1 receptor as well as the insulin receptor variant A (IR-A) with high affinity. Mitogenic and proliferative signaling via the canonical IGF-2 pathway is, therefore, augmented by IR-A. This study shows that RMS patients express increased IR-A levels compared to control tissues that predominantly express the IR-B isoform. We also found that Hif-1α is significantly increased in RMS tumors, portraying their hypoxic phenotype. Concordantly, the alternative splicing of IR adapts to produce more IR-A in response to hypoxic stress. Upon examining the pre-mRNA structure of the gene, we identified a potential hypoxia-responsive element, which is also the binding site for the RNA-binding protein CUG-BP1 (CELF1). We designed Splice Switching Oligonucleotides (SSO) against this binding site to decrease IR-A levels in RMS cell lines and, consequently, rescue the IR-B expression levels. SSO treatment resulted in a significant reduction in cell proliferation, migration, and angiogenesis. Our data shows promising insight into how impeding the IGF-2 pathway by reducing IR-A expression mitigates tumor growth. It is evident that Rhabdomyosarcomas use IR alternative splicing as yet another survival strategy that can be exploited as a therapeutic intervention in conjunction with already established anti-IGF-1 receptor therapies.
RESUMO
Aminoacyl-tRNA synthetases are multidomain proteins that catalyze the covalent attachment of amino acids to their cognate transfer RNA. Various domains of an aminoacyl-tRNA synthetase perform their specific functions in a highly coordinated manner to maintain high accuracy in protein synthesis in cells. The coordination of their function, therefore, requires communication between domains. In this study we explored the relevance of enzyme motion in domain-domain communications. Specifically, we attempted to probe whether the communication between distantly located domains of a multidomain protein is accomplished through a coordinated movement of structural elements. We investigated the collective motion in Thermus thermophilus leucyl-tRNA synthetase by studying the low frequency normal modes. We identified the mode that best described the experimentally observed conformational changes of T. thermophilus leucyl-tRNA synthetase upon substrate binding and analyzed the correlated and anticorrelated motions between different domains. Furthermore, we used statistical coupling analysis to explore if the amino acid pairs and/or clusters whose motions are thermally coupled have also coevolved. Our study demonstrates that a small number of residues belong to the category whose coupled thermal motions correspond to evolutionary coupling as well. These residue clusters constitute a distinguished set of interacting networks that are sparsely distributed in the domain interface. Residues of these networking clusters are within van der Waals contact, and we suggest that they are critical in the propagation of long range mechanochemical motions in T. thermophilus leucyl-tRNA synthetase.
