Biomedical Engineering Reference
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localisation, but contains the KH domains found in many
trans
-acting factors
involved in RNA transport. A key question in the recognition of an RNA by a pro-
tein is how specific recognition is achieved. RNA binding proteins interact with the
RNA through two or more of their RBDs, each binding 4-8 nt, increasing the affinity
and specificity of the binding (Fig.
11.4a
).
11.3.2
Experimental Characterisation of RNA:
Protein Binding Preferences
There are several techniques for the determination of the binding preferences of
specific RNA binding proteins. These vary from in vitro-based techniques such as
Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and NMR to
in vivo characterisation using the cross-linking and immunoprecipitation (CLIP)-
based methods. The Zbp1 protein binds b-actin mRNA through a “zipcode” in the
3¢ UTR and is required for localisation. An electrophoretic mobility shift assay
(EMSA) with the third and fourth KH domains revealed that Zbp1 binds just the
first 28 nt of the zipcode (Chao et al.
2010
). This study also involved X-ray structure
determination discussed below. The binding preferences of five
Drosophila
hnRNP
A/B proteins (hrp36, hrp38, hrp40/Sqd, hrp48) were determined using a SELEX
combined with RIP-Chip (RNA binding protein immunoprecipitation coupled to a
microarray chip) approach (Blanchette et al.
2009
). SELEX revealed the nucleotide
binding preferences of the purified proteins, identifying sequence profiles for each
protein. However, SELEX is not an ideal method for the characterisation of multi-
RBD RNA binding proteins as the recognition is through two or more short 4-8 nt
target sequences which, independently, are too low affinity and specificity to attain
reliable results. An alternative to address this limitation, Scaffold-independent anal-
ysis (SIA) is an NMR technique to determine the binding preference for ssRBDs
(Beuth et al.
2007
). A pool of randomised RNA sequences together with the protein
is investigated through changes in the NMR chemical shifts. The interactions with
individual amino acids in the protein for each of the nucleotides are quantified to
give the degree of specificity. SIA has been used to characterise the individual bind-
ing preferences of different KH RBDs of the KSRP protein (Garcia-Mayoral et al.
2008
). Based on this and other structural information, a general model for KSRP-
RNA binding has been proposed. The KH3 domain was also characterised using
CLIP (Ule et al.
2005a
) and the sequence specificity was confirmed using Scaffold
Independent Analysis (SIA) (Beuth et al.
2007
). In addition, microarray analysis
with brain tissue and the Nova (1 and 2) proteins revealed 41 in vivo RNA targets
(Ule et al.
2005b
) .
Biochemical methods such as CLIP have been used to identify RNAs binding a
protein of interest and also map the binding sites on a transcriptome scale. The RNA
binding protein is cross-linked to a target RNA by the formation of a covalent bond
when exposed to UV light. The cross-linking is performed in vivo and so represents
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