Cullin 4B Is Recruited to Tristetraprolin-Containing Messenger Ribonucleoproteins and Regulates TNF-α mRNA Polysome Loading

TNF-alpha is a central mediator of inflammation and critical for host response to infection and injury. TNF-alpha biosynthesis is controlled by transcriptional and posttranscriptional mechanisms allowing for rapid, transient production. Tristetraprolin (TTP) is an AU-rich element binding protein that regulates the stability of the TNF-alpha mRNA. Using a screen to identify TTP-interacting proteins, we identified Cullin 4B (Cul4B), a scaffolding component of the Cullin ring finger ligase family of ubiquitin E3 ligases. Short hairpin RNA knockdown of Cul4B results in a significant reduction in TNF-alpha protein and mRNA in LPS-stimulated mouse macrophage RAW264.7 cells as well as a reduction in TTP protein. TNF-alpha message t(1/2) was reduced from 69 to 33 min in LPS-stimulated cells. TNF-3' untranslated region luciferase assays utilizing wild-type and mutant TTP-AA (S52A, S178A) indicate that TTP function is enhanced in Cul4B short hairpin RNA cells. Importantly, the fold induction of TNF-alpha mRNA polysome loading in response to LPS stimulation is reduced by Cul4B knockdown. Cul4B is present on the polysomes and colocalizes with TTP to exosomes and processing bodies, which are sites of mRNA decay. We conclude that Cul4B licenses the TTP-containing TNF-alpha messenger ribonucleoprotein for loading onto polysomes, and reduction of Cul4B expression shunts the messenger ribonucleoproteins into the degradative pathway. The Journal of Immunology, 2012, 188: 1828-1839.