Alternatively Expressed Domains of AU-rich Element RNA-binding Protein 1 (AUF1) Regulate RNA-binding Affinity, RNA-induced Protein Oligomerization, and the Local Conformation of Bound RNA Ligands

Abstract

AU-rich element RNA-binding protein 1 (AUF1) binding to AU-rich elements (AREs) in the 3-untranslated regions of mRNAs encoding many cytokines and other regulatory proteins modulates mRNA stability, thereby influencing protein expression. AUF1-mRNA association is a dynamic paradigm directed by various cellular signals, but many features of its function remain poorly described. There are four isoforms of AUF1 that result from alternative splicing of exons 2 and 7 from a common pre-mRNA. Preliminary evidence suggests that the different isoforms have varied functional characteristics, but no detailed quantitative analysis of the properties of each isoform has been reported despite their differential expression and regulation. Using purified recombinant forms of each AUF1 protein variant, we used chemical cross-linking and gel filtration chromatography to show that each exists as a dimer in solution. We then defined the association mechanisms of each AUF1 isoform for ARE-containing RNA substrates and quantified relevant binding affinities using electrophoretic mobility shift and fluorescence anisotropy assays. Although all AUF1 isoforms generated oligomeric complexes on ARE substrates by sequential dimer association, sequences encoded by exon 2 inhibited RNA-binding affinity. By contrast, the exon 7-encoded domain enhanced RNA-dependent protein oligomerization, even permitting cooperative RNA-binding activity in some contexts. Finally, fluorescence resonance energy transfer-based assays showed that the different AUF1 isoforms remodel bound RNA substrates into divergent structures as a function of protein:RNA stoichiometry. Together, these data describe isoform-specific characteristics among AUF1 ribonucleoprotein complexes, which likely constitute a mechanistic basis for differential functions and regulation among members of this protein family.

Crystal structure of the YTH domain of YTHDF2 reveals mechanism for recognition of N6-methyladenosine

“…Figure 1C). A similar two-step binding mode was observed in other RNA-binding proteins [13]. It is likely that binding to RNA facilitates the further recognition of m 6 A.…”

Cleavage and polyadenylation specificity factor 30: An RNA-binding zinc-finger protein with an unexpected 2Fe–2S cluster

“…Other RNA-binding proteins that recognize their targets in a cooperative manner are the human zipcode-binding protein IMP-1, HIV-1 Rev protein, hordeiviral γb protein, human La protein, and tomato bushy stunt virus p33 protein (35)(36)(37)(38). There are also other AUrich RNA-binding proteins such as AUF-1 (p42), Hsp70, and HuR, which bind to their respective RNA partners in a cooperative manner (39)(40)(41). Moreover, a two-domain construct of CPSF30 has been crystallized bound to the NS1 influenza A virus protein, where it is present as a dimer (21).…”

Direct Binding of Specific AUF1 Isoforms to Tandem Zinc Finger Domains of Tristetraprolin (TTP) Family Proteins

“…The creation of plasmids His-AUF1p45 and AUF1p37 have been described (30). Purification of these recombinant proteins has also been described (30,31).…”

“…The creation of plasmids His-AUF1p45 and AUF1p37 have been described (30). Purification of these recombinant proteins has also been described (30,31). GFPHis-TZF (hTTP) was purified using the HisPur Ni-NTA Resin kit from Thermo Scientific, Rockford, IL, as per the manufacturer's instructions.…”