Arginine methylation of translocated in liposarcoma (TLS) inhibits its binding to long noncoding RNA, abrogating TLS-mediated repression of CBP/p300 activity

Abstract

Translocated in liposarcoma (TLS) is an RNA-binding protein and a transcription-regulatory sensor of DNA damage. TLS binds promoter-associated noncoding RNA (pncRNA) and inhibits histone acetyltransferase (HAT) activity of CREB-binding protein (CBP)/E1A-binding protein P300 (p300) on the cyclin D1 () gene. Although post-translational modifications of TLS, such as arginine methylation, are known to regulate TLS's nucleocytoplasmic shuttling and assembly in stress granules, its interactions with RNAs remain poorly characterized. Herein, using various biochemical assays, we confirmed the earlier observations that TLS is methylated by protein arginine methyltransferase 1 (PRMT1) The arginine methylation of TLS disrupted binding to pncRNA and also prevented binding of TLS to and inhibition of CBP/p300. This result indicated that arginine methylation of TLS abrogates both binding to pncRNA and TLS-mediated inhibition of CBP/p300 HAT activities. We also report that an arginine residue within the Arg-Gly-Gly domain of TLS, Arg-476, serves as the major determinant for binding to pncRNA. Either methylation or mutation of Arg-476 of TLS significantly decreased pncRNA binding and thereby prevented a pncRNA-induced allosteric alteration in TLS that is required for its interaction with CBP/p300. Moreover, unlike WT TLS, an R476A TLS mutant did not inhibit promoter activity in luciferase reporter assays. Taken together, we propose the hypothesis that arginine methylation of TLS regulates both TLS-nucleic acid and TLS-protein interactions and thereby participates in transcriptional regulation.

Affinity Profiles Categorize RNA-Binding Proteins into Distinctive Groups

“…At the third category, we found hnRNPH1 and TLS which recognize a binding site, 5'(1-1), located in pncRNA-D transcribed from the cyclin D1 gene promoter. Intriguingly, 5'(1-1) has been identified as specific site for TLS [28][29][30]. Our experiments demonstrated that hnRNPH1 also binds the site specific to TLS.…”

“…Our binding assays have effectively demonstrated that RBPs have distinctive subgroups classified with criteria of their binding affinity [28,29]. The experiments using random RNA oligos unpredictably indicated that hnRNPUL2 and hnRNPU bind to these RNA oligos which are typically used for negative control of RNA binding assays [28,29,47]. It has been reported that hnRNPUL2 is a part of RNA polymerase II general transcription factor complex [48] and also works on DNA repair processes [49,50], indicating roles in forming nuclear structures.…”

“…Our binding assays have effectively demonstrated that RBPs have distinctive subgroups classified with criteria of their binding affinity [28,29]. The experiments using random RNA oligos unpredictably indicated that hnRNPUL2 and hnRNPU bind to these RNA oligos which are typically used for negative control of RNA binding assays [28,29,47].…”

“…Most certain criterion for RBPs is just to detect binding ability to RNA. Recently, we have established a novel assay to detect binding of RNA-binding proteins, using biotinylated RNA oligos to capture RBPs with Western blot of specific antibodies against RBPs [28,29]. The assay more confidently detects RNA binding than the traditional gel shift assay.…”

Post-translational epigenetics: PRMT7 regulates RNA-binding capacity and protein stability to controlLeishmaniaparasite virulence

“…Although arginine methylation has been mostly associated with changes in protein-protein interactions (50,51), there are previous examples of direct modulation of RNA-protein binding in mammalian cells (52)(53)(54). PRMTs have previously been shown to regulate the RNA affinity of target RBPs in other systems (55,56). In T. brucei, the RNA-binding proteins DRBD18 and PRMT1 have been shown to modulate the fate of mRNAs according to methylation state, impacting both protein and mRNA binding (45,50).…”