Terminal Adenosyl Transferase Activity of Posttranscriptional Regulator HuR Revealed by Confocal On-Bead Screening

Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR

“…In contrast, the binding of HuR to target mRNA is structurally attributed to three highly conserved RNA-recognition motifs (RRMs) with the N-terminal tandem RRM domains RRM1 and RRM2 representing the main ARE-binding domains of HuR [25]. By contrast, the less conserved C-terminal RRM3 of HuR is thought to be mainly relevant for stabilization of RNA-protein complexes and is furthermore characterized by its terminal adenosyl transferase activity [26]. According to the many signaling events which are known to modulate nucleo-cytoplasmic HuR shuttling, previous data from our and other laboratories implicate that posttranslational modifications, namely methylation [27] and phosphorylation of HuR represent critical features in the control of different HuR functions.…”

The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets

“…34,[44][45][46] RRM3 is involved in binding long poly-A tails of mRNAs [47][48][49] and in catalyzing the 3 0 -terminal adenosyl modification of non-polyadenylated RNA substrates. 50 Besides its role in RNA recognition, RRM3 is also responsible for the formation of HuR multimers, 34 as occurs with the homologous Drosophila ELAV protein. 51 In fact, mutations in the RRM3 domain of Drosophila ELAV protein lead to a temperature-sensitive phenotype by impairing HuR oligomerization, highlighting the crucial role of this RRM module.…”

Combinatorial/Library Peptide Synthesis

“…After completion of the scan, the hits are ranked by their intensities, and the best are picked by an actuated capillary and placed into individual containers for structural evaluation. The bead picking process is slower and can retrieve about 1 bead/min [280][281][282]. Resynthesis and retesting of the active compounds has shown that surface inhomogeneity prevents precise ranking of the ligand quality based on the solid support fluorescence reading (Figure 12.10).…”