The binding specificity of Translocated in LipoSarcoma/FUsed in Sarcoma with lncRNA transcribed from the promoter region of cyclin D1

Abstract

Background: Translocated in LipoSarcoma (TLS, also known as FUsed in Sarcoma) is an RNA/DNA binding protein whose mutation cause amyotrophic lateral sclerosis. In previous study, we demonstrated that TLS binds to long noncoding RNA, promoter-associated ncRNA-D (pncRNA-D), transcribed from the 5' upstream region of cyclin D1 (CCND1), and inhibits the expression of CCND1.

Results: In order to elucidate the binding specificity between TLS and pncRNA-D, we divided pncRNA-D into seven fragments and examined the binding with full-length TLS, TLS-RGG2-zinc finger-RGG3, and TLS-RGG3 by RNA pull down assay. As a result, TLS was able to bind to all the seven fragments, but the fragments containing reported recognition motifs (GGUG and GGU) tend to bind more solidly. The full-length TLS and TLS-RGG2-zinc finger-RGG3 showed a similar interaction with pncRNA-D, but the binding specificity of TLS-RGG3 was lower compared to the full-length TLS and TLS-RGG2-zinc finger-RGG3. Mutation in GGUG and GGU motifs dramatically decreased the binding, and unexpectedly, we could only detect weak interaction with the RNA sequence with stem loop structure.

Conclusion: The binding of TLS and pncRNA-D was affected by the presence of GGUG and GGU sequences, and the C terminal domains of TLS function in the interaction with pncRNA-D.

Keywords: Long noncoding RNA; TLS/FUS; pncRNA.

Fig. 1

The binding between full-length TLS and fragmented pncRNA-Ds. a The position of pncRNA-D and CCND1. The fragmented pncRNA-Ds are shown at the bottom. Black and white boxes indicate GGUG and GGU sequence, respectively. Since fragment 3 and 4 did not contain any GGUG or GGU motifs, we considered them as a negative control. b and c Western blot analysis were conducted with HeLa nuclear extract (NE). Seven fragmented pncRNA-Ds (b) and shortened fragment 1 and 7 (c) were incubated with HeLa NE, and the affinity between TLS and each fragment was examined by RNA pull down assay. Five and ten percent of the protein used for RNA pull down assays were loaded as input N = 5

Fig. 2

The binding between full-length TLS and the fragment 1–1. a Computational analysis predicting the secondary structure of pncRNA-D by CentroidFold (http://www.ncrna.org/centroidfold/). The position of the fragment 1–1 is shown in red box. b Imino-imino (left) and imino-amino/base (right) proton regions of a NOESY spectrum with a mixing time of 300 ms, the assignments of imino protons being indicated. Cross peaks to two amino protons and H2 are boxed, respectively. c 1D imino proton spectra of the fragment 1–1, the 3′ end of the fragment 1–1, G49A mutant of the 3′ end of the fragment 1–1 and G46A mutant of the 3′ end of the fragment 1–1, respectively, from top to bottom. d The two possible secondary structures of the fragment 1–1, the upper one being concluded as a right one. e Western blot analysis were performed to detect the binding between 5′ and 3′ ends of the fragment 1–1. Ten percent of the protein used for RNA pull down assay was loaded as input N = 5

Fig. 3

The binding between truncated TLS and pncRNA-D. a The domain structure of TLS. GST tag was attached to the 5′ end of the truncated TLS-1 to -5. RGG RGG repeat domain, RRM RNA recognition motif, ZF zinc finger domain. b The interaction between TLS-1 and -5 and pncRNA-D with different length was examined by RNA pull down assay. N = 3. c Binding of TLS-4 (top) and -5 (bottom) with fragmented pncRNA-D were examined by RNA pull down assay followed by western blot analysis. Purified TLS-4 and TLS-5 were incubated with pncRNA-D fragments, and the signals were detected with anti-GST antibody. N = 5. d The binding between the fragments 1–1, 7–1, the 5′ end and the 3′ end of fragment 1–1 with TLS-4 or TLS-5 were examined by RNA pull down assay as in (b) and (c). N = 4. In all the experiments, 10 % of the protein used for RNA pull down assay was loaded as input

Fig. 4

NMR titration experiments for TLS-5 with the fragment 1–1, the 5′ end of the fragment 1–1, the 3′ end of the fragment 1–1 and U13. a and b ¹H–¹⁵N HSQC spectra of TLS-5 with either the fragment 1–1 (a), the 5′ end of the fragment 1–1 (b), the 3′ end of the fragment 1–1 (c) or U13 (d), with the molar ratios of 1:0, 1:0.5, 1:1.0, 1:1.5 and 1:2.0, respectively

Fig. 5

The effect of the mutation (GGUG to CCUC; GGU to CCU) on the binding of the fragment 1–1 and TLS. a The position of GGU and GGUG where mutation was induced in the fragment 1–1 is shown in black box (left), and the RNA sequences are listed in the table (right). Sequence of the 5′ end of the fragment 1–1 is underlined. b The binding between mutated fragments and full-length TLS, TLS-4, and TLS-5 was examined by RNA pull down assay N = 3. Ten percent of the protein used for RNA pull down assay was loaded as input