TNIP2 is a Hub Protein in the NF-κB Network with Both Protein and RNA Mediated Interactions

The NF-κB family of transcription factors is pivotal in controlling cellular responses to environmental stresses; abnormal nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling features in many autoimmune diseases and cancers. Several components of the NF-κB signaling pathway have been reported to interact with the protein TNIP2 (also known as ABIN2), and TNIP2 can both positively and negatively regulate NF-κB- dependent transcription of target genes. However, the function of TNIP2 remains elusive and the cellular machinery associating with TNIP2 has not been systematically defined. Here we first used a broad MudPIT/Halo Affinity Purification Mass Spectrometry (AP-MS) approach to map the network of proteins associated with the NF-κB transcription factors, and establish TNIP2 as an NF-κB network hub protein. We then combined AP-MS with biochemical approaches in a more focused study of truncated and mutated forms of TNIP2 to map protein associations with distinct regions of TNIP2. NF-κB interacted with the N-terminal region of TNIP2. A central region of TNIP2 interacted with the endosomal sorting complex ESCRT-I via its TSG101 subunit, a protein essential for HIV-1 budding, and a single point mutant in TNIP2 disrupted this interaction. The major gene ontology category for TNIP2 associated proteins was mRNA metabolism, and several of these associations, like KHDRBS1, were lost upon depletion of RNA. Given the major association of TNIP2 with mRNA metabolism proteins, we analyzed the RNA content of affinity purified TNIP2 using RNA-Seq. Surprisingly, a specific limited number of mRNAs was associated with TNIP2. These RNAs were enriched for transcription factor binding, transcription factor cofactor activity, and transcription regulator activity. They included mRNAs of genes in the Sin3A complex, the Mediator complex, JUN, HOXC6, and GATA2. Taken together, our findings suggest an expanded role for TNIP2, establishing a link between TNIP2, cellular transport machinery, and RNA transcript processing.