ABSTRACT
The vertebrate nuclear pore protein Nup153 contains a novel RNA binding domain. This 150-amino acid region was previously found to bind preferentially to a panel of mRNAs when compared with structured RNAs, such as tRNA, U snRNA, and double-stranded RNA. The ability to broadly recognize mRNA led to the conclusion that the Nup153 RNA binding domain confers a general affinity for single-stranded RNA. Here, we have probed Nup153 RNA recognition to decipher how this unique RNA binding domain discriminates between potential targets. We first mapped the binding determinant within an RNA fragment that associates relatively robustly with the Nup153 RNA binding domain. We next designed synthetic RNA oligonucleotides to systematically delineate the features within this minimal RNA fragment that are key to Nup153 RNA-binding domain binding and demonstrated that the binding preferences of Nup153 do not reflect general preferences of an mRNA/single-stranded RNA-binding protein. We further found that the association between Nup153 and a cellular mRNA can be attributed to an interaction with specific subregions of the RNA. These results indicate that Nup153 can discriminate between mRNA and other classes of RNA transcripts due in part to direct recognition of a loose sequence motif. This information adds a new dimension to the interfaces that can contribute to recognition in mRNA export cargo selection and fate.
Subject(s)
Nuclear Pore Complex Proteins/chemistry , RNA/chemistry , Xenopus Proteins/physiology , Amino Acid Motifs , Animals , Base Sequence , Molecular Sequence Data , Nuclear Pore Complex Proteins/physiology , Nuclear Proteins/chemistry , Protein Binding , Protein Structure, Tertiary , RNA, Messenger/metabolism , Recombinant Proteins/chemistry , Ribonuclease H/chemistry , Transcription, Genetic , Xenopus Proteins/chemistry , Xenopus laevis/metabolismABSTRACT
The vertebrate pore protein Nup153 plays pivotal roles in nuclear pore function. In addition to being important to pore architecture, Nup153 is a key participant in both import and export. The scope of Nup153 function also extends beyond the canonical view of the pore as a trafficking gateway. During the transition into mitosis, Nup153 directs proteins involved in membrane remodeling to the nuclear envelope. As cells exit mitosis, Nup153 is recruited to the chromosomal surface, where nuclear pores are formed anew in a complicated process still under much experimental scrutiny. In addition, Nup153 is targeted for protease cleavage during apoptosis and in response to certain viral infections, providing molecular insight into pore reconfiguration during cell response. Overall, the versatile nature of Nup153 underscores an emerging view of the nuclear pore at the nexus of many key cellular processes.
Subject(s)
Nuclear Pore Complex Proteins/metabolism , Nuclear Pore/chemistry , Nuclear Pore/metabolism , Animals , Apoptosis/physiology , Humans , Nuclear Pore/genetics , Nuclear Pore Complex Proteins/genetics , Protein Transport , RNA-Binding Proteins/metabolismABSTRACT
The nuclear pore protein Nup153 is important for the transport of protein and RNA between the nucleus and cytoplasm. Recently, a novel RNA binding domain (RBD) was mapped within the N-terminal region of Nup153; however, the determinants of RNA association were not characterized. Here we have tested a range of RNAs with different general features to better understand targets recognized by this domain. We have found that the RBD associates with single-stranded RNA with little sequence preference. These results provide new information about a novel RNA binding domain and suggest new models to consider for the contribution of Nup153 to nucleocytoplasmic transport.
