Identification of Essential Components of RNA Binding Domain of TLS/FUS

Abstract

TLS/FUS is RNA-binding protein having multiple functions of regulations of genes, homeostasis, and cellular growth. Recent studies show that TLS is involved in phase separation and occasionally forms precipitation related to neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). RNA has been reported to suppress phase separation, droplet formation, and concomitant precipitation of TLS, suggesting that RNA is a possible candidate for ALS drug discovery. Our experiments demonstrated that a long noncoding RNA, promoter-associated noncoding RNA (pncRNA-D), specifically binds TLS and represses its phase separation and precipitation. To obtain competent drug seeds, it is essential to reveal mechanism of action of lncRNAs with specificity to TLS and inhibitory activity on phase separation and related precipitation. For this purpose, several lncRNAs (lncRNAs 1 to 6) were selected upon assays with GST-TLS binding and inhibition on the precipitation. With criteria of binding specificity for TLS, lncRNA3 has been selected for further analysis for RNA-binding ability. Initially, RNA-binding region at TLS amino acid sequence was identified from four fragments of TLS. RNA binding assay with biotinylated lncRNA3 precipitated with avidin magnetic beads indicated clearly that TLS binds the fragment 4 (373-526 aa), C-terminus end of TLS. Then, dissecting fragment 4 presents four regions, RGG2, zinc finger, RGG3, and the nuclear localization signal (NLS) region in this order. Experiments with extensive deletion mutants indicated that just one deletion out of the four regions irs not enough to delete the TLS binding, although combinatorial deletion of zinc finger with other three regions almost wiped off the lncRNA3 binding. Remarkably, each of four regions alone has no binding to TLS, either. Collectively, RGG2, zinc finger, RGG3, and NLS all are essential for binding to lncRNA3, but are required to work synergistically for full binding. These data indicate that dynamic assembly of RNA-binding domain works for action of lncRNAs and possibly has allosteric effect on intrinsically disordered region (IDR) of N-terminus of TLS, implying relation of RNA-binding with phase separation and the resultant precipitation.