Coordinated Action of Two Double-Stranded RNA Binding Motifs and an RGG Motif Enables Nuclear Factor 90 To Flexibly Target Different RNA Substrates

Abstract

The mechanisms of how RNA binding proteins (RBP) bind to and distinguish different RNA molecules are yet uncertain. Here, we performed a comprehensive analysis of the RNA binding properties of multidomain RBP nuclear factor 90 (NF90) by investigating specifically the functional activities of two double-stranded RNA binding motifs (dsRBM) and an RGG motif in the protein's unstructured C-terminus. By comparison of the RNA binding affinities of several NF90 variants and their modes of binding to a set of defined RNA molecules, the activities of the motifs turned out to be very different. While dsRBM1 contributes little to RNA binding, dsRBM2 is essential for effective binding of double-stranded RNA. The protein's immediate C-terminus, including the RGG motif, is indispensable for interactions of the protein with single-stranded RNA, and the RGG motif decisively contributes to NF90's overall RNA binding properties. Conformational studies, which compared wild-type NF90 with a variant that contains a pseudophosphorylated residue in the RGG motif, suggest that the NF90 C-terminus is involved in conformational changes in the protein after RNA binding, with the RGG motif acting as a central regulatory element. In summary, our data propose a concerted action of all RNA binding motifs within the frame of the full-length protein, which may be controlled by regulation of the activity of the RGG motif, e.g., by phosphorylation. This multidomain interplay enables the RBP NF90 to discriminate RNA features by dynamic and adaptable interactions.

The properties of the RNA-binding protein NF90 are considerably modulated by complex formation with NF45

“…The NF90 b variants were generated by sitedirected mutagenesis and PCR (Table S1) as described earlier. [45] Expression and purification of proteins. The cDNA sequence encoding NF90 b and NF45 were expressed in E. coli BL21-CodonPlus ® (DE3)-RP.…”

“…Protein synthesis was induced by adding 1 mM isopropyl-β-1-thio-D-galactopyranoside. NF90 b , its variants and NF45 were isolated and purified from inclusion bodies according to Schmidt et al [45] Briefly, the cells were harvested, centrifuged, re-suspended in 0.1 M Tris/HCl pH 7.0, 1 mM EDTA and lysed using a French Press homogenizer. Following DNase I treatment and incubation with Triton X-100, the inclusion bodies (IB) were collected by centrifugation and washed.…”

“…[45][46][47] Recently, we systematically examined the RNA-binding mechanisms of purified NF90, analyzing specifically the activities of the dsRBM and RGG/RG motifs. [45] The two dsRBMs participate very differently in RNA binding. While dsRBM2 is a major determinant in the association of NF90 with dsRNA, the impact of dsRBM1 on RNA binding is minimal.…”

“…In sum, these studies revealed a dynamic, cooperative and coordinated action of the three motifs in the RNA-binding process of NF90, which enables the protein to distinguish different features of target RNA ligands. [45] 6…”

ILF3 contributes to the establishment of the antiviral type I interferon program

“…NF90/110 are involved in regulating different steps of gene expression, including pre-mRNA splicing, miRNA biogenesis, and mRNA stability amongst others, and in controlling the life cycle of several viruses (reviewed in (14)(15)). Both ILF3 isoforms bind RNA through two tandem dsRNA-binding motifs and an RGG-rich domain (16)(17). In agreement with their association with polyribosomes, these factors can negatively regulate the translation of cellular mRNAs, and in particular, mRNAs containing AU-rich motifs (18)(19).…”