Extending the Size of Protein–RNA Complexes Studied by Nuclear Magnetic Resonance Spectroscopy

Mackereth, Cameron D.; Simon, Bernd; Sattler, Michael

doi:10.1002/cbic.200500106

Cited by 29 publications

(14 citation statements)

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“…Comparison of NMR spectra of U2AF65, the U2AF heterodimer, and U2AF65-SF1 complexes demonstrates that the conformations and conformational arrangements coupled with RNA binding of U2AF are not affected by the presence of SF1 (43,44). Thus, although 3′ splice site recognition involves cooperative binding of SF1, U2AF65, and U2AF35, the fine-tuning and regulation of the assembly depends on the variability in the Py-tract and its recognition by population shifts involving the RNA binding domains of the U2AF heterodimer.…”

Section: U2af35 Enhances Py-tract Recognition By a Dynamic Populationmentioning

confidence: 99%

Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift

Voithenberg

Sánchez-Rico

Kang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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An essential early step in the assembly of human spliceosomes onto pre-mRNA involves the recognition of regulatory RNA cis elements in the 3′ splice site by the U2 auxiliary factor (U2AF). The large (U2AF65) and small (U2AF35) subunits of the U2AF heterodimer contact the polypyrimidine tract (Py-tract) and the AG-dinucleotide, respectively. The tandem RNA recognition motif domains (RRM1,2) of U2AF65 adopt closed/inactive and open/active conformations in the free form and when bound to bona fide Py-tract RNA ligands. To investigate the molecular mechanism and dynamics of 3′ splice site recognition by U2AF65 and the role of U2AF35 in the U2AF heterodimer, we have combined single-pair FRET and NMR experiments. In the absence of RNA, the RRM1,2 domain arrangement is highly dynamic on a submillisecond time scale, switching between closed and open conformations. The addition of Py-tract RNA ligands with increasing binding affinity (strength) gradually shifts the equilibrium toward an open conformation. Notably, the protein-RNA complex is rigid in the presence of a strong Py-tract but exhibits internal motion with weak Py-tracts. Surprisingly, the presence of U2AF35, whose UHM domain interacts with U2AF65 RRM1, increases the population of the open arrangement of U2AF65 RRM1,2 in the absence and presence of a weak Py-tract. These data indicate that the U2AF heterodimer promotes spliceosome assembly by a dynamic population shift toward the open conformation of U2AF65 to facilitate the recognition of weak Py-tracts at the 3′ splice site. The structure and RNA binding of the heterodimer was unaffected by cancer-linked myelodysplastic syndrome mutants.uring gene expression, the removal of introns is essential for translation of mature mRNA. The splicing process involves a large number of splicing factors for the correct recognition of introns (1). Whereas U1 snRNP contacts the 5′ splice site, recognition of the 3′ splice site involves binding of SF1/BBP to the branch point sequence (BPS) (2-5) and binding of the U2 auxiliary factor (U2AF) heterodimer to the poly-pyrimidine-tract (Py-tract) that precedes the AG dinucleotide at the intron/exon junction. Binding of U2AF to the 3′ splice site during the early steps of spliceosome assembly recruits the U2 snRNP (6-9). The strength, i.e., splicing efficiency, of a 3′ splice site requires recognition of the BPS, Py-tract, and the AG dinucleotide. However, of these three RNA elements, the Py-tract exhibits the largest degree of variability, and thus, weak to strong splice sites are primarily classified depending on the composition of the Py-tract (7, 10).U2AF is a heterodimer consisting of a large (U2AF65) and a small (U2AF35) subunit. U2AF65 harbors two canonical RNA recognition motifs (RRM1,2) and an atypical C-terminal RRM domain, called the U2AF homology motif (UHM). U2AF35 has one RRM (which acts as an UHM), flanked N-and C-terminally by two CCCH-type zinc finger motifs, respectively (Fig. S1A) (9, 11, 12). The U2AF heterodimer is formed by recognition of a peptide motif, cal...

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Section: U2af35 Enhances Py-tract Recognition By a Dynamic Populationmentioning

confidence: 99%

Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift

Voithenberg

Sánchez-Rico

Kang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Furthermore, solution NMR studies allowed to study conformational changes associated with RRM-RNA interactions involving multi-RRM proteins like U2AF (Mackereth et al, 2011) or more recently CPEB (Afroz et al, 2014). Even though X-ray crystallography is still preferentially used to determine structures of large RNA-protein complexes (>30 kDa), their investigation in solution is nevertheless possible using a combination of methods such as paramagnetic relaxation enhancement (Mackereth, Simon, & Sattler, 2005), residual dipolar coupling, and/or electron paramagnetic resonance (EPR) (Duss, Michel, et al, 2014;.…”

Section: Structural Investigations Of Rrms: How Many?mentioning

confidence: 99%

One, Two, Three, Four! How Multiple RRMs Read the Genome Sequence

Afroz

Cieniková

Cléry

et al. 2015

Methods in Enzymology

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“…For large multi-domain proteins the complexity of NMR spectra can be systematically reduced by subunit-selective labeling, i.e., reconstitution of complexes where only one subunit is isotope-labeled and detected in isotope-edited NMR spectra, which select and detect for example only 15 N-bound protons (Mackereth et al, 2005). If the individual protein subunits are already of high-molecular weight, segmental isotope labeling of individual domain is a valuable tool to reduce signal overlap (David et al, 2004;Muralidharan and Muir, 2006;Skrisovska et al, 2010).…”

Section: Examples and A Case Studymentioning

confidence: 99%

“…The U2AF65 RRM1-RRM2/RNA complex is part of a larger multimeric protein complex (Mackereth et al, 2005). For large multi-domain proteins the complexity of NMR spectra can be systematically reduced by subunit-selective labeling, i.e., reconstitution of complexes where only one subunit is isotope-labeled and detected in isotope-edited NMR spectra, which select and detect for example only 15 N-bound protons (Mackereth et al, 2005).…”

Section: Examples and A Case Studymentioning

confidence: 99%

“…We are using the combination of NMR and SAS for structural analysis of a large multi-domain protein complex that plays an essential role in the regulation of pre-mRNA splicing (Mackereth et al, 2005). As a model system derived from this complex we are studying the arrangement of the two RNA Recognition Motif (RRM) domains of the essential splicing factor U2AF65 bound to a short RNA oligonucleotide comprised of nine uridines (U9).…”

Section: Examples and A Case Studymentioning

confidence: 99%

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NMR and small-angle scattering-based structural analysis of protein complexes in solution

Madl

Gabel

Sattler

2011

Journal of Structural Biology

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Extending the Size of Protein–RNA Complexes Studied by Nuclear Magnetic Resonance Spectroscopy

Cited by 29 publications

References 71 publications

Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift

Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift

One, Two, Three, Four! How Multiple RRMs Read the Genome Sequence

NMR and small-angle scattering-based structural analysis of protein complexes in solution

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