RESUMO
Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is a multitasking RNA/DNA binding protein. FUS aggregation is implicated in various neurodegenerative diseases. RNA was suggested to modulate phase transition of FUS. Here, we found that FUS transforms into the amorphous aggregation state as an instant response to the shear stress caused by usual pipetting even at a low FUS concentration, 100 nM. It was revealed that non-coding RNA can suppress the transformation of FUS into aggregates. The suppressive effect of RNA on FUS aggregation is sequence-dependent. These results suggested that the non-coding RNA could be a prospective suppressor of FUS aggregation caused by mechanistic stress in cells. Our finding might pave the way for more research on the role of RNAs as aggregation inhibitors, which could facilitate the development of therapies for neurodegenerative diseases.
Assuntos
RNA não Traduzido/genética , Proteína FUS de Ligação a RNA/genética , Proteínas de Ligação a DNA/genética , Agregados Proteicos/genética , Proteínas de Ligação a RNA/genética , Resistência ao Cisalhamento/fisiologiaRESUMO
High-speed AFM revealed the conformational change of fused in sarcoma (FUS) from a compact to an extended structure upon binding of non-coding RNA, which is supposed to allow FUS to bind to CBP/p300 for transcriptional interference. Thus, a mechanistic insight into transcription regulation by FUS and non-coding RNA is provided.
Assuntos
RNA não Traduzido/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Ligantes de Maltose/química , Microscopia de Força Atômica , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Proteína FUS de Ligação a RNA/químicaRESUMO
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.
Assuntos
RNA não Traduzido/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Sequência de Bases , Sítios de Ligação , Transferência Ressonante de Energia de Fluorescência , Humanos , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , RNA não Traduzido/química , Proteína FUS de Ligação a RNA/químicaRESUMO
Somatic hypermutation (SHM) requires not only the expression of activation-induced cytidine deaminase, but also transcription in the target regions. However, how transcription guides activation-induced cytidine deaminase in targeting SHM to the Ig genes is not fully understood. Here, we found that the "facilitates chromatin transcription" (FACT) complex promotes SHM by RNAi screening of transcription elongation factors. Furthermore, FACT and histone H3.3, a hallmark of transcription-coupled histone turnover, are enriched at the V(D)J region, 5' flanking sequence of the Sµ switch region and the light chain Jκ 5 segment region in the Ig loci. The regions with the most abundant deposition of FACT and H3.3 were also the most efficient targets of SHM. These results demonstrate the importance of histone-exchanging dynamics at the chromatin of SHM targets, especially in Ig genes.