Abstract
BackgroundThe RNA recognition motif (RRM) is one of the largest families of RNA binding domains. The RRM is modulated so that individual proteins containing RRMs can specifically recognize RNA targets with diverse sequences and structures. Understanding the principles governing this specificity will be important for the rational modification and design of RRM-RNA complexes.ResultsIn this paper we have investigated the origins of specificity of the N terminal RRM of the U1A protein for stem loop 2 (SL2) of U1 snRNA by substituting modified bases for essential purines in SL2 RNA. In one series of modified bases, hydrogen bond donors and acceptors were replaced by aliphatic groups to probe the importance of these functional groups to binding. In a second series of modified bases, hydrogen bond donors and acceptors were incorrectly placed on the purine bases to analyze the origins of discrimination between cognate and non-cognate RNA. The results of these experiments show that three different approaches are used by the U1A protein to gain specificity for purines. Specificity for the first base in the loop, A1, is based primarily on discrimination against RNA containing the incorrect base, specificity for the fourth base in the loop, G4, is based largely on recognition of the donors and acceptors of G4, while specificity for the sixth base in the loop, A6, results from a combination of direct recognition of the base and discrimination against incorrectly placed functional groups.ConclusionThese investigations identify different roles that hydrogen bond donors and acceptors on bases in both cognate and non-cognate RNA play in the specific recognition of RNA by the U1A protein. Taken together with investigations of other RNA-RRM complexes, the results contribute to a general understanding of the origins of RNA-RRM specificity and highlight, in particular, the contribution of steric and electrostatic repulsion to binding specificity.
Highlights
The RNA recognition motif (RRM) is one of the largest families of RNA binding domains
Strategy for Probing Specificity To investigate the specificity requirements of the U1A protein, we have measured the affinity of the U1A protein for stem loop 2 (SL2) RNA target sites containing modified purine bases
In a second series of modifications, the purines were substituted with alternative hydrogen bond donors or acceptors to investigate the ability of the U1A protein to discriminate against incorrectly placed functional groups
Summary
The RNA recognition motif (RRM) is one of the largest families of RNA binding domains. The RRM is modulated so that individual proteins containing RRMs can recognize RNA targets with diverse sequences and structures. RRMs make limited contacts with the sugar-phosphate backbone compared to other RNA-binding proteins and large cooperative networks of hydrogen bonds are formed with the (page number not for citation purposes). Individual structures of RRM-RNA complexes have been solved [7,8], it remains unclear how this domain forms a general RNA binding scaffold, while individual proteins containing RRMs achieve high specificity for particular RNA sequences. The Nterminal RRM of U1A binds with high affinity to stem loop 2 (SL2) in U1 snRNA and an internal loop target site of nearly identical sequence in the U1A pre-mRNA [9,26]. Cooperative networks of interactions important for complex formation involving amino acids in loops 1, 3, and 6 and β1, β3, and β4 have been identified experimentally and have been suggested computationally [13,15,18,22,27,28,29,30,31]
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