2008
DOI: 10.1074/jbc.m806297200
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Solution Conformation and Thermodynamic Characteristics of RNA Binding by the Splicing Factor U2AF65

Abstract: The U2 auxiliary factor large subunit (U2AF 65 ) is an essential pre-mRNA splicing factor for the initial stages of spliceosome assembly. Tandem Pre-mRNA splicing is an essential source of transcript diversity in multicellular eukaryotes (reviewed in Refs. 1 and 2), as reflected by the significant number of cancers and hereditary diseases associated with mutations in pre-mRNA splice site signals or splicing factors (3-5). The splicing machinery (spliceosome) is faced with the task of recognizing relatively s… Show more

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Cited by 33 publications
(49 citation statements)
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“…To investigate the involvement of U2AF 65 inhibition, we examined the effects on U2AF 65 association of mutations in two representative Py tracts (Table 1): (i) a U→A transversion in the Py tract of the neurofibromin 1 gene [NF1(U3 > A)], which creates a new 3′ splice site junction (consensus AG) and leads to familial neurofibromatosis type I (29), and (ii) a U→A transversion in the Py tract of the retinitis pigmentosa 2 (RP2) gene [RP2(U4 > A)] that also introduces an AG dinucleotide and induces exon skipping and, consequently, X-linked retinitis pigmentosa (30). We titrated the Py tract recognition domain of U2AF 65 comprising RRM1 and RRM2 and bordering residues into either the WT or mutated fluoresceinlabeled Py tract RNAs, and then fit the apparent equilibrium dissociation constant (K D ) values to anisotropy changes as described previously (31) (Fig. S1).…”
Section: Resultsmentioning
confidence: 99%
“…To investigate the involvement of U2AF 65 inhibition, we examined the effects on U2AF 65 association of mutations in two representative Py tracts (Table 1): (i) a U→A transversion in the Py tract of the neurofibromin 1 gene [NF1(U3 > A)], which creates a new 3′ splice site junction (consensus AG) and leads to familial neurofibromatosis type I (29), and (ii) a U→A transversion in the Py tract of the retinitis pigmentosa 2 (RP2) gene [RP2(U4 > A)] that also introduces an AG dinucleotide and induces exon skipping and, consequently, X-linked retinitis pigmentosa (30). We titrated the Py tract recognition domain of U2AF 65 comprising RRM1 and RRM2 and bordering residues into either the WT or mutated fluoresceinlabeled Py tract RNAs, and then fit the apparent equilibrium dissociation constant (K D ) values to anisotropy changes as described previously (31) (Fig. S1).…”
Section: Resultsmentioning
confidence: 99%
“…If differentially populated species exist in a dynamic equilibrium, SAS and NMR data represent a weighted average over all coexisting conformations sampled in solution. If SAS data obtained from dynamic systems is mis-interpreted as arising from a single rigid structure, resulting models will be quite far from reality (Jenkins et al, 2008). Although there are software tools available for generating an ensemble of structures (typically in the order of 10.000) that fit the experimental scattering data , it is often challenging to obtain the ''true'' conformational ensemble using SAS data alone.…”
Section: Considering Dynamic Conformational Ensemblesmentioning
confidence: 98%
“…Enthalpy changes may still be determined from isotherms with higher c-values (51). C using a FluoroMax-3 spectrophotometer (HORIBA Jobin Yvon, Kyoto, Japan) were measured and analyzed in a manner similar to that described in (52,53). Both macromolecules were diluted >50-fold in 150 mM NaCl, 25 mM HEPES (pH 7.4), 0.2 mM TCEP, and 0.1 U mL À1 Superase-In (Ambion, Life Technologies, Carlsbad, CA).…”
Section: Itc Methodsmentioning
confidence: 99%