Biophysical and structural investigation into the RNA-binding properties of TIAR

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The RNA-binding protein (RBP) TIAR [related to TIA (T-cell-restricted intracellular antigen)-1] plays a multifunctional role in the regulation of gene expression through binding target mRNA via its RNA recognition motifs (RRMs). TIAR binds to mRNA at AU-rich elements (AREs) in their 3’ untranslated regions (UTRs) and is involved in translational repression via the formation of stress granules, particularly under conditions of cellular stress. TIAR has also been shown to bind to single-stranded DNA (ssDNA) and be involved in splicing. This study aims to better understand the biophysical and structural basis for TIAR binding to its target oligonucleotides. TIAR has been reported previously to preferentially bind to U-rich sequences. However, a surprising discovery by our collaborators of a cytosine-rich motif targeted by TIAR initiated our investigation of whether TIAR was really capable of binding this C-rich motif. Firstly, we describe the development of a surface plasmon resonance (SPR) protocol for accurate measurements of TIAR-RNA interactions in vitro. The ability of constructs of TIAR, comprising all or some of its 3 RRMs, to bind to the C-rich consensus motif was then verified using the optimized protocol. Through this analysis, TIAR12 and TIAR123 showed low but significant binding to the C-rich sequence which ultimately led to the elucidation of the C-rich motif as a novel TIAR target. Similar to TIAR, HuR (Hu antigen R) is an RRM-containing ARE-binding protein that is involved in stabilization of the mRNA transcript. It binds to AREs via its RRMs and with seemingly overlapping specificity with TIAR. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nM range, with higher overall affinity for U-rich RNA. However, both proteins show slower dissociation from AU-rich RNA, indicating what may be a truer measure of their binding preference. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nM affinity, whereas HuR affinity is reduced to a µM level. SAXS data for TIAR12/RNA complex are more consistent with a flexible, elongated shape and not the compact shape of HuR12/RNA suggesting that these proteins interact with their targets in fundamentally different ways. We show using SPR, specific roles of individual TIAR domains for its high affinity binding to oligonucleotide targets. We not only confirm RRM2 as the major binding domain, but also show that the strong affinity binding to U-rich RNA and T-rich DNA only occurs in the presence of RRM1 and the extension region C-terminal to RRM2. On its own, RRM1 shows preferred binding to DNA over RNA. RRM3 makes little contribution to the overall binding affinities to both RNA and DNA targets. We further characterize the interaction between RRM2 with the C-terminal extension and an ARE target using NMR spectroscopy. 1H-15N HSQC titration experiments reveal specific residues involved in RNA binding including those in RNP1, RNP2, beta sheets, and the extension region. Lastly, we report our attempts at crystallizing TIAR-oligonucleotide complexes. Although crystals for the complex were not obtained from these trials, the efforts serve as a useful guideline for future trials. In summary, the work presented here advances our understanding of biophysical basis for protein-RNA interactions in post-transcriptional gene regulation and provides insight into the mechanism underlying the complex interplay of their interactions leading to different outcomes for the mRNA, and its encoded protein in the cell.