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. 2020 Feb 14;10(1):2629.
doi: 10.1038/s41598-020-59496-0.

RNA Sequence and Length Contribute to RNA-induced Conformational Change of TLS/FUS

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Free PMC article

RNA Sequence and Length Contribute to RNA-induced Conformational Change of TLS/FUS

Nesreen Hamad et al. Sci Rep. .
Free PMC article

Abstract

Translocated in liposarcoma (TLS)/fused in sarcoma (FUS) is a multitasking DNA/RNA binding protein implicated in cancer and neurodegenerative diseases. Upon DNA damage, TLS is recruited to the upstream region of the cyclin D1 gene (CCND1) through binding to the promotor associated non-coding RNA (pncRNA) that is transcribed from and tethered at the upstream region. Binding to pncRNA is hypothesized to cause the conformational change of TLS that enables its inhibitive interaction with histone acetyltransferases and resultant repression of CCND1 expression, although no experimental proof has been obtained. Here, the closed-to-open conformational change of TLS on binding pncRNA was implied by fluorescence resonance energy transfer. A small fragment (31 nucleotides) of the full-length pncRNA (602 nucleotides) was shown to be sufficient for the conformational change of TLS. Dissection of pncRNA identified the G-rich RNA sequence that is critical for the conformational change. The length of RNA was also revealed to be critical for the conformational change. Furthermore, it was demonstrated that the conformational change of TLS is caused by another target DNA and RNA, telomeric DNA and telomeric repeat-containing RNA. The conformational change of TLS on binding target RNA/DNA is suggested to be essential for biological functions.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
The hypothesized closed-to-open conformational change of TLS on binding to pncRNA for the repression of CCND1 and the strategy for the detection of the conformational change by FRET. (a), The mechanism of the repression of CCND1 by TLS on DNA damage. A closed-to-open conformational change of TLS is hypothesized on binding pncRNA, which enables TLS to interact with CBP/p300 and repress their histone acetyltransferase activities, resulting in the repression of CCND1. (b), Schematic illustration of the protein used in this study, MBP-BFP-TLS-GFP-6xHis. Residue numbers of TLS are indicated. Positions of ALS-linked mutations, K510R and P525L, are also indicated. (c), The strategy for the detection of the closed-to-open conformational change of TLS by FRET. (d), 10% SDS-PAGE of MBP-BFP-TLS-GFP-6xHis, arrowhead (right), and the molecular mass markers (left). These are cropped gels. The uncropped full-length gel is presented in Supplementary Fig. S2 online.
Figure 2
Figure 2
Detection of the closed-to-open conformational change of TLS on binding of pncRNA by FRET. (a), The localization, length, sequence and secondary structure of pncRNA. (b), Fluorescence spectrum of TLS (100 nM), excited at 402 nm, in either the absence (blue) or presence (red) of an equimolar amount of full-length pncRNA of 602 nt. (c), The names and sequences of fragments of R31 of pncRNA. (d), Fluorescence spectrum of TLS in either the absence (blue) or presence (red) of an equimolar amount of R31 of pncRNA. (e), Absolute values of the change in the relative FRET efficiency, ∆E, where E = IGFP/ (IGFP + IBFP), on the addition of each fragment of R31 of pncRNA. Three independent results were averaged for each fragment. |∆E| is presented as a mean ± standard deviation. *indicates p value < 0.05. (f), |∆ E| of R6, GAGGGU, and its mutants.
Figure 3
Figure 3
The conformational change of TLS caused by either DNA counterparts of the fragments of pncRNA or oligomers of U residues of various lengths. (a), |∆E| of DNA counterparts, D13, D19 and D31. Three independent results were averaged for each fragment. |∆E| is presented as a mean ± standard deviation. (b), |∆E| of oligomers of U residues of various lengths, U13, U19 and U31.
Figure 4
Figure 4
The difference in affinity toward TLS between a specific RNA, R31 of pncRNA, and non-specific RNA, U31. (a), Fluorescence anisotropy of TAMRA-labeled R31 (100 nM) (red) and TAMRA-labeled U31 (100 nM) (blue) in the course of the addition of TLS. (b), Fluorescence anisotropy of TAMRA-labeled R31 (100 nM) (red) in the presence of a 1.5 molar ratio of TLS in the course of the addition of non-labeled U31 and that of TAMRA-labeled U31 (100 nM) (blue) in the presence of a 1.5 molar ratio of TLS in the course of the addition of non-labeled R31.
Figure 5
Figure 5
The conformational change of TLS caused by TERRA and telomeric DNA. Fluorescence spectrum of TLS in either the absence (blue) or presence (red) of TERRA (a), telomeric DNA (b), mutant TERRA (c), or mutant telomeric DNA (d). (e), |∆E| of each RNA or DNA.

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