RNA sequence and length contribute to RNA-induced conformational change of TLS/FUS

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 fulllength 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. Translocated in liposarcoma (TLS), also known as fused in sarcoma (FUS), belongs to the TET or FET family (TLS/FUS, Ewing's sarcoma, and TATA-binding protein-associated factor), whose members are multitasking RNA/DNA binding proteins that regulate gene expression, and are implicated in cancer and neurodegenerative diseases. TLS was first identified in a human adipose tissue tumor, a myxoid liposarcoma, as a fusion oncogenic protein that results from chromosomal translocation t(12;16)(q13.3;p11.2) 1. Later, TLS was found to be involved in various devastating neurodegenerative diseases 2-6 , as well as in a wide variety of biological processes through regulation of gene expression 7-15. TLS reportedly binds to a wide variety of RNA and DNA sequences, both structured and unstructured, that are involved in transcription, splicing, and so on 9. Previously, systematic evolution of ligands through exponential enrichment (SELEX) showed that TLS recognizes the GGUG motif 16 , while a photoactivatable ribonucleoside-enhanced crosslinking immunoprecipitation (PAR-CLIP) study and RNA-binding competition study identified an AU-rich stem-loop structure and CGCGC sequence, respectively, as additional targets for TLS 17,18. More recently, normalization of CLIP-seq (CLIP combined with deep sequencing) data by means of Nascent-seq data showed a combination of six motifs (UGUG, CUGG, UGGU, GCUG, GUGG, and UUGG) are also targeted by TLS 19. TLS comprises from the N-to C-terminal ends, a low complexity domain (LC), the first Arginine-Glycine-Glycine rich motif (RGG1), an RNA recognition motif (RRM), the second RGG (RGG2), a Zinc finger domain (ZnF), and the third RGG (RGG3) (Fig. 1b). The LC and RGGs are known to be...

Non-coding RNA suppresses FUS aggregation caused by mechanistic shear stress on pipetting in a sequence-dependent manner

“…4 a). We had already revealed that this spectrum change reflects the compact-to-extended conformational change of FUS with pncRNA on the basis of FRET and HS-AFM analyses 23 , 24 . When further strokes of pipetting were applied, however, the spectrum rarely changed, if any (Fig.…”

“…In our previous studies, we showed that full-length (602 nucleotide residues) and fragments of pncRNA (Supplementary Fig. S1 ) can induce a conformational change of FUS 23 , 24 . Interestingly, this time we found that the reduction in the fluorescence intensity caused by shear stress on pipetting was not observed when full-length pncRNA was added to the FUS fusion protein (Fig.…”

“…We have been studying the mechanism of the transcription regulation of a cell cycle activator, the cyclin D1 gene ( CCND1 ), by FUS in response to DNA damage 1 , 23 – 25 . We showed that a long non-coding RNA that is transcribed from the promoter region of CCND1 , which was named promoter-associated non-coding RNA (pncRNA), can induce a conformational change of FUS.…”

“…This leads to CCND1 transcription suppression and subsequent cell cycle arrest, which may provide the time needed for DNA damage repair. We used FRET assaying to detect the effect of pncRNA on the conformational change of FUS 23 . FUS fusion protein with blue fluorescence protein (BFP) and green fluorescence protein (GFP) attached to its N- and C-termini, respectively, was used for the FRET assays.…”

“…Only the RRM and ZnF domains are structured, the others being regarded as intrinsically disordered regions (IDRs). Previously, we showed by fluorescence resonance energy transfer (FRET) and high-speed atomic force microscopy (HS-AFM) analyses that FUS takes on a compact conformation in its free-form but becomes extended upon binding to RNA/DNA 23 , 24 .…”