Screening protein – Single stranded RNA complexes by NMR spectroscopy for structure determination

Abstract

In the past few years, RNA molecules have been revealed to be at the center of numerous biological processes. Long considered as passive molecules transferring genetic information from DNA to proteins, it is now well established that RNA molecules play important regulatory roles. Associated with that, the number of identified RNA binding proteins (RBPs) has increased considerably and mutations in RNA molecules or RBP have been shown to cause various diseases, such as cancers. It is therefore crucial to understand at the molecular level how these proteins specifically recognise their RNA targets in order to design new generation drug therapies targeting protein–RNA complexes. Nuclear magnetic resonance (NMR) is a particularly well-suited technique to study such protein–RNA complexes at the atomic level and can provide valuable information for new drug discovery programs. In this article, we describe the NMR strategy that we and other laboratories use for screening optimal conditions necessary for structural studies of protein-single stranded RNA complexes, using two proteins, Sam68 and T-STAR, as examples.

Structural basis of RNA recognition and dimerization by the STAR proteins T-STAR and Sam68

“…We have shown previously using NMR spectroscopy that the isolated KH domains of Sam68 and T-STAR are sufficient for binding A/U-rich RNAs 44 . Here, we have determined the X-ray structures of the T-STAR KH domain in its free state, and in complex with AAAUAA; KH-QUA2 in complex with AAUAAU; QUA1-KH in complex with UAAU; and the full STAR domain in complex with AUUAAA ( Fig.…”

Protein–RNA specificity by high-throughput principal component analysis of NMR spectra

“…T-STAR, also referred to as Sam68-like mammalian protein 2 (SLM2) is an important splicing regulator of the Neurexin gene ( 19 ). The protein contains an extended KH domain (STAR domain), which is thought to bind to A-rich sequences, but whose actual specificity is still to be defined ( 20 ). TUT4 is a non-templated poly-uridylase that plays a key role in the biogenesis of the tumour suppressor let-7 miRNA, and that contains three CCHC zinc fingers ( 21 , 22 ).…”

Simple high‐resolution NMR spectroscopy as a tool in molecular biology

“…Additionally, if the protein molecular structure information is available, the annotations and selections can be mirrored to a graphical molecular visualisation, such as PYMOL [12]. We tested the AnalysisAssign CSP module by exploring the binding of Clip2 RNA, AAAUAA, to the testis-signal transduction and activation of RNA (Tstar)-KH domain [13,14]. We imported both the assignments of the free Tstar-KH domain in addition to a series of five 15 N-HSQC spectra of uniformly 15 Nlabelled Tstar-KH at 0.0, 0.5, 1.0, 1.5, and 2.0 eq of Clip2 ligand, directly from the original Sparky data using the inherent data conversion routines of Analy-sisAssign.…”