Dear All,
I am in some sort of a conundrum because of the results that I have got recently. My RNA binding protein, as the name, binds to ssRNA very well but also binds to dsRNA with equal efficiency. Then I tested it with dsDNA and it somewhat binds to dsDNA, however, it does not bind to ssDNA at all. Now the problem is that we want to figure out how the interaction is taking place. Our other experiments suggest that the interaction with ssRNA is very strong, which suggests that the interaction could be hydrophobic and not electrostatic. However, when we add Heparin, it competes for binding. By our ssDNA vs ssRNA experiments, we deduce that our RBP discriminates between de-oxy-Ribose and Ribose. Are we right in assuming that? Why is it that even though we have proof that it binds very strongly, it is still competed by Heparin (which we used because it has similar structure and charge as nucleic acids). Also, mostly it is known that the RBPs exploit secondary structures in RNA to bind, why is it then that our protein doesn't bind to ssDNA because I assume that the ssDNA should form the same secondary structure as it ssRNA (from the same sequence)?
I would really appreciate some help from RBP experts.
Thank you
Ikram
RNA binding proteins (RBPs) are key players in the regulation of gene expression. In this chapter we discuss the main protein-RNA recognition modes used by RBPs in order to regulate multiple steps of RNA processing. We discuss traditional and state-of-the-art technologies that can be used to study RNAs bound by individual RBPs, or vice versa, for both in vitro and in vivo methodologies. To help highlight the biological significance of RBP mediated regulation, online resources on experimentally verified protein-RNA interactions are briefly presented. Finally, we present the major tools to computationally infer RNA binding sites according to the modeling features and to the unsupervised or supervised frameworks that are adopted. Since some RNA binding site search algorithms are derived from DNA binding site search algorithms, we discuss the commonalities and novelties introduced to handle both sequence and structural features uniquely characterizing protein-RNA interactions.
Article RNA–Protein Interactions: An Overview
RBPs often, but not in every case, bear structurally and functionally domains, termed RNA recognition motif (RRM) and hnRNP K homology domain (KH), to mention only two prominent examples. These domains are conserved. They interact with RNA sequence motifs (RRM) or can bind to both RNA and DNA, depending on the functional context. In many RBPs several copies of these domains are present and they contribute differebntially to the interaction with specific target RNAs. Vigilin contains 15 KH domains. Some RNAs are discussed to confer structural changes to specific proteins, which enable them to bind to RNAs and are therefore referred to as enigmatic RBPs without known RNA binding domains.
Dear Dirk
Thanks for that, however, I am aware that there are different RNA binding domains in RBPs and that the domains can vary in number. However, I would appreciate if you could throw some light on the properties that RBPs could exploit to differentially bind ssRNA and ssDNA. I feel that the such RBPs, as the case mentioned in my question, (my protein) could make contact with the Ribose and differentially bind to the Ribose in case of RNA and in case of dsDNA it makes contact with the phosphate-sugar backbone. There the bases are occluded. And may be the reason for it to not bind to ssDNA could be that the protein tries to make contact on the base side as opposed to the phosphate backbone and because of the lack of free 2'OH in ssDNA, it does not bind to ssDNA. Thereby, may be we could say that the binding to RNA is driven by free 2'OH. Do you think, I am right in assuming that? However, most of these interaction are supposed to be weak hydrogen bonds, that contradicts my results, my RBP very strongly binds to these RNAs. What type of interaction could it be?
Thanks
Ikram
This might depend on structural properties of the protein, which in case of unstructured peptide motifs probably result in an induced fold that is more stable. Thereby the specificity for defined sequence motifs might be provided.
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