Abstract
Many cell biological pathways exhibit overall polarity (net movement of molecules in one direction) even though individual molecular interactions in the pathway are freely reversible. The A2 RNA trafficking pathway exhibits polarity in moving specific RNA molecules from the nucleus to localization sites in the myelin compartment of oligodendrocytes or dendritic spines in neurons. The A2 pathway is mediated by a ubiquitously expressed trans-acting trafficking factor (hnRNP A2) that interacts with a specific 11 nucleotide cis-acting trafficking sequence termed the A2 response element (A2RE) found in several localized RNAs. Five different molecular partners for hnRNP A2 have been identified in the A2 pathway: hnRNP A2 itself, transportin, A2RE RNA, TOG (tumor overexpressed gene) and hnRNP E1, each playing a key role in one particular step of the A2 pathway. Sequential interactions of hnRNP A2 with different molecular partners at each step mediate directed movement of trafficking intermediates along the pathway.Specific "rules of engagement" (both and, either or, only if) govern sequential interactions of hnRNP A2 with each of its molecular partners. Rules of engagement are defined experimentally using three component binding assays to measure differential binding of hnRNP A2 to one partner in the presence of each of the other partners in the pathway. Here we describe rules of engagement for hnRNP A2 binding to each of its molecular partners and discuss how these rules of engagement promote polarity in the A2 RNA trafficking pathway.For molecules with multiple binding partners, specific rules of engagement govern different molecular interactions. Rules of engagement are ultimately determined by structural relationships between binding sites on individual molecules. In the A2 RNA trafficking pathway rules of engagement governing interactions of hnRNP A2 with different binding partners provide the basis for polarity of movement of intermediates along the pathway.
Highlights
Cell biological processes are mediated by pathways of interacting macromolecules
Results and Discussion hnRNP A2 binding to hnRNP A2 HnRNP A2 is a 36 kD RNA binding protein that shuttles between nucleus and cytoplasm [14], interacting with different partners at each step in the A2 RNA trafficking pathway [4]
Fluorescence correlation spectroscopy (FCS) studies indicate that hnRNP A2 forms oligomers in solution, which implies that each hnRNP A2 molecule contains at least two separate homotypic binding sites, one mediating association of monomers to form dimers and one mediating association of dimers to form higher order aggregates
Summary
Comprehensive understanding of a particular cell biological pathway requires: inventory of all molecules involved in the pathway (including concentrations and diffusion coefficients); structures of all macromolecules in the pathway; interactions among molecules in the pathway (including kinetic constants); effects of perturbations of individual components of the pathway; and computational modeling of pathway behaviour [1] Many of these requirements can be met using large scale systematic approaches. If the unlabeled protein increases the amount of cross correlation, this indicates that the target protein binds the labelled partner only if the unlabeled partner binds In this manuscript we will summarize rules of engagement for molecules in the A2 RNA trafficking pathway [4]. We show that polarity in the A2 pathway is determined by rules of engagement governing specific interactions of hnRNP A2 with individual components of the A2 pathway
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