Different modes of interaction by TIAR and HuR with target RNA and DNA

Abstract

TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U–rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2′-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways.

Identification of novel proteins binding the AU-rich element of α-prothymosin mRNA through the selection of open reading frames (RIDome)

“…The value we observed with ELAVL1 is in a good agreement with K D values previously reported. 39 In contrast to previous reports, full-length ELAVL1 and GST-ORF ELAVL1 did not show a significant difference in affinity for the ARE PTMA target. 39 Similarly, we did not observe a significant difference in binding properties between full-length RBM38 and GST-ORF RBM38 (0.4 nM vs. 1 nM, respectively).…”

“…To directly compare the RNA-binding properties of ELAVL1-, RBM38-, RALY-and R3HDM2-derived GST-fusion products, we used surface plasmon resonance (SPR) to measure 39,40 we wanted to compare these to the properties obtained with the GST-ORF ELAVL1 product. Thus, we cloned the full-length coding sequence of ELAVL1 from HEK293 cells, expressed it in E. coli and performed SPR analyses as above.…”

“…39 The oligonucleotide used in the analyses was the ARE PTMA . The chemical biotinylation of the oligonucleotide allowed the immobilization of the RNA onto streptavidin-coated sensor chips (Series S Sensor Chip SA, Biacore).…”

Translational coregulation of 5′TOP mRNAs by TIA-1 and TIAR

“…Based on previous studies implicating TIA-1 and TIAR in translational repression (Piecyk et al 2000 Kim et al 2007) and in binding to pyrimidine-rich sequences (Dember et al 1996;Lopez de Silanes et al 2005;Mazan-Mamczarz et al 2006;Kim et al 2007Kim et al , 2011, we speculated that TIA-1 and TIAR may play a role in the control of 59TOP mRNA translation. To first determine whether TIA-1 and TIAR exist in complex with 59TOP mRNAs, we established human HEK293 cell lines stably expressing Flagtagged TIA-1 or TIAR under control of tetracycline-inducible promoters.…”

Modulation of Gene Expression by RNA Binding Proteins: mRNA Stability and Translation

“…While HuR is ubiquitously expressed, HuB, HuC and HuD are primarily neuronal [10]. HuR, also known as embryonic lethal, abnormal vision, Drosophila-like 1 (ELAV L1), binds mRNAs bearing AU-and U-rich sequences, which are considered binding signatures, or RNA-recognition motifs (RRMs) TIA-1 and TIAR RBPs bind AU/U-rich sequences in the 3'UTR of target transcripts and suppress mRNA translation [25]. However, they can also modulate translation through 5' terminal oligopyrimidine tracts (5'TOP) in response to changes in the cellular environment [26].…”