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Title: The molecular basis for the regulation of the cap-binding complex by the importins

Authors:
; ; ; ;  [1]
  1. (Cornell)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1171230
Resource Type:
Journal Article
Resource Relation:
Journal Name: Nat. Struct. Mol. Biol.; Journal Volume: 16; Journal Issue: (9) ; 09, 2009
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Dias, Sandra M.G., Wilson, Kristin F., Rojas, Katherine S., Ambrosio, Andre L.B., and Cerione, Richard A.. The molecular basis for the regulation of the cap-binding complex by the importins. United States: N. p., 2015. Web. doi:10.1038/nsmb.1649.
Dias, Sandra M.G., Wilson, Kristin F., Rojas, Katherine S., Ambrosio, Andre L.B., & Cerione, Richard A.. The molecular basis for the regulation of the cap-binding complex by the importins. United States. doi:10.1038/nsmb.1649.
Dias, Sandra M.G., Wilson, Kristin F., Rojas, Katherine S., Ambrosio, Andre L.B., and Cerione, Richard A.. Thu . "The molecular basis for the regulation of the cap-binding complex by the importins". United States. doi:10.1038/nsmb.1649.
@article{osti_1171230,
title = {The molecular basis for the regulation of the cap-binding complex by the importins},
author = {Dias, Sandra M.G. and Wilson, Kristin F. and Rojas, Katherine S. and Ambrosio, Andre L.B. and Cerione, Richard A.},
abstractNote = {},
doi = {10.1038/nsmb.1649},
journal = {Nat. Struct. Mol. Biol.},
number = (9) ; 09, 2009,
volume = 16,
place = {United States},
year = {Thu Feb 19 00:00:00 EST 2015},
month = {Thu Feb 19 00:00:00 EST 2015}
}
  • The binding of capped RNAs to the cap-binding complex (CBC) in the nucleus, and their dissociation from the CBC in the cytosol, represent essential steps in RNA processing. Here we show how the nucleocytoplasmic transport proteins importin-{alpha} and importin-{beta} have key roles in regulating these events. As a first step toward understanding the molecular basis for this regulation, we determined a 2.2-{angstrom} resolution X-ray structure for a CBC-importin-{alpha} complex that provides a detailed picture for how importin-{alpha} binds to the CBP80 subunit of the CBC. Through a combination of biochemical studies, X-ray crystallographic information and small-angle scattering experiments, we thenmore » determined how importin-{beta} binds to the CBC through its CBP20 subunit. Together, these studies enable us to propose a model describing how importin-{beta} stimulates the dissociation of capped RNA from the CBC in the cytosol following its nuclear export.« less
  • Patients who have pseudohypoparathyroidism type I associated with Albright hereditary osteodystrophy commonly have a genetic deficiency of the ..cap alpha.. subunit of the G protein that stimulated adenylyl cyclase ..cap alpha..G/sub s/. To discover the molecular mechanism that causes ..cap alpha..G/sub s/ deficiency in these patients, the authors examined eight kindreds with one or more members affected with Albright hereditary osteodystrophy or pseudohypoparathyroidism and ..cap alpha..G/sub s/ deficiency. In these families, ..cap alpha..G/sub s/, deficiency and the Albright hereditary osteodystrophy phenotype were transmitted together in a dominant inheritance pattern. Using a cDNA hybridization probe for ..cap alpha..G/sub s/, restriction analysismore » with several analysis with several endonucleases showed no abnormalities of restriction fragments or gene dosage. RNA blot and dot blot analysis of total RNA from cultured fibroblasts obtained from the patients revealed approx. 50% reduced mRNA levels for ..cap alpha..G/sub s/ in affected members of six of the pedigrees but normal levels in affected members of the two other pedigrees, compared to mRNA levels in fibroblasts from unaffected individuals. By contrast, mRNA levels encoding the ..cap alpha.. subunit of the G protein that inhibits adenylyl cyclase were not altered. These findings suggest that several molecular mechanisms produce ..cap alpha..G/sub s/ deficiency in patients with pseudohypoparathyroidism type Ia and that major gene rearrangements or deletions are not a common cause for ..cap alpha..G/sub s/ deficiency in pseudohypoparathyroidism type I.« less
  • It has been suggested that the cap-binding protein complex is involved in ATP-mediated melting of 5'-mRNA secondary structure to facilitate ribosome binding during initiation of translation in eukaryotic cells. Consequently, we have studied the interaction of dATP/ATP with the eukaryotic cap-binding protein complex by UV photoaffinity labeling. UV irradiation of the cap-binding protein complex in the presence of (alpha-TSP)dATP/ATP resulted in the cross-linking of this compound to the 50-kDa polypeptide of the complex. This polypeptide is almost identical to the previously characterized eukaryotic initiation factor (eIF) 4A. We examined the ability of dATP/ATP to cross-link to eIF-4A and found thatmore » it cross-links less efficiently (approximately 60-fold on a molar basis) compared to the cross-linking obtained for the eIF-4A component of the cap-binding protein complex. Irradiation of purified eIF-4A together with the cap-binding protein complex in the presence of (alpha-TSP)dATP resulted in greater than additive labeling of the eIF-4A component of the cap-binding protein complex and purified eIF-4A, suggesting a synergistic interaction between purified eIF-4A, the cap-binding protein complex, and dATP/ATP. We also report that photoaffinity labeling of eIF-4A and the eIF-4A component in the cap-binding protein complex is stimulated by eIF-4B, but not by other initiation factors or mRNA.« less
  • Hemocyanin (Hc) is an oxygen carrier protein in which oxygen binding is regulated by allosteric effectors such as H{sup +} and L-lactate. Isothermal titration calorimetric measurements showed that L-lactate binds to dodecameric and heterohexameric Hc and to the CaeSS3 homohexamer but not to the CaeSS2 monomer. The binding of lactate caused no change in the optical absorption and x-ray absorption spectra of either oxy- or deoxy-Hc, suggesting that no structural rearrangement of the active site occurred. At pH 6.5, the oxygen binding rate constant k{sub obs} obtained by flash photolysis showed a significant increase upon addition of L-lactate, whereas L-lactatemore » addition had little effect at pH 8.3. Lactate binding caused a concentration-dependent shift in the interhexameric distances at pH 6.5 based on small angle x-ray scattering measurements. These results show that L-lactate affects oxygen affinity at pH 6.5 by modulating the global structure of Hc without affecting its binuclear copper center (the active site). In contrast to this, the active site structure of deoxy-Hc is affected by changes in pH (Hirota, S., Kawahara, T., Beltramini, M., Di Muro, P., Magliozzo, R. S., Peisach, J., Powers, L. S., Tanaka, N., Nagao, S., and Bubacco, L. (2008) J. Biol. Chem. 283, 31941-31948). Upon addiction of lactate, the kinetic behavior of oxygen rebinding for Hc was heterogeneous under low oxygen concentrations at pH 6.5 due to changes in the T and R state populations, and the equilibrium was found to shift from the T toward the R state with addition of lactate.« less