NMR Studies of the RNA Binding Domain of U1A protein and its Complexes with RNA

Eukaryotic splicing is catalysed by five RNA protein complexes called snRNP's. U1A is one protein component of the U1 snRNP particle. The role of U1A in splicing is unclear, but it does bind a hairpin loop from U1 RNA. U1A also binds two internal bulges found in the 3' untranslated region of the mRNA which encodes the U1A protein. This is a negative feedback mechanism for regulating its own expression. The RNA binding domain of U1A is contained within residues 2-102 of U1A, and it includes a RNP domain - a domain which is found in hundreds of RNA binding proteins. The RNA binding domain of U1A was isotope labelled by over-expression in E. coli and it was then studied by heteronuclear multidimensional NMR. Three sets of assignments were obtained; a near-complete assignment of the domain free in solution: a partial assignment of it complexed to a RNA hairpin derived from the Ul-RNA: and a near-complete assignment of the domain complexed to a RNA internal bulge derived from the mRNA which encodes U1A. This is the first assignment of a RNA binding protein in complex with RNA. The assignment of the free U1A protein revealed a third α-helix at its C-terminus, which had not been discovered previously. The partial assignment of the complex with the RNA hairpin confirmed and extended a model of the U1A/RNA complex. Comparison of NMR spectra of the two complexes showed that U1A protein recognises the two different RNA structures in the same way. The methods used to assign the complex with the internal bulge RNA will be discussed in detail. The assignments are compatible with the crystal structure of the complex between U1A and an hairpin RNA, demonstrating that NMR is a useful method for studying protein RNA complexes.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

1996

Author dc:creator

• Howe, Peter W. A.

Advisor dc:contributor.advisor

• Neuhaus, David

• RNA
• U1A protein
• NMR

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1ed8417b-c1db-4f3b-a8a6-cfa8c8e09563/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

en

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.87881

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/340451