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

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.

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.

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.