title: Six GU-rich (6GUR) FUS-binding motifs detected by normalization of CLIP-seq by Nascent-seq.
abstract: FUS, an RNA-binding protein (RBP), is mutated or abnormally regulated in neurodegenerative disorders. FUS regulates various aspects of RNA metabolisms. FUS-binding sites are rich in GU contents and are highly degenerative. FUS-binding motifs of GGU, GGUG, GUGGU and CGCGC have been previously reported. These motifs, however, are applicable to a small fraction of FUS-binding sites. As CLIP-seq tags are enriched in genes that are highly expressed, we normalized CLIP-seq tags by Nascent-seq tags or RNA-seq tags of mouse N2a cells. Nascent-seq identifies nascent transcripts before being processed for splicing and polyadenylation. We extracted frequently observed 4-nt motifs from Nascent-seq-normalized CLIP regions, RNA-seq-normalized CLIP regions, and native CLIP regions. Specific GU-rich motifs were best detected in Nascent-seq-normalized CLIP regions. Analysis of structural motifs using Nascent-seq-normalized CLIP regions also predicted GU-rich sequence forming a stem structure. Sensitivity and specificity were calculated by examining whether the extracted motifs were present at the cross-linking-induced mutation sites (CIMS), where FUS was directly bound. We found that a combination of six motifs (UGUG, CUGG, UGGU, GCUG, GUGG, and UUGG), which were extracted from Nascent-seq-normalized CLIP-regions, had a better discriminative power than (i) motifs extracted from RNA-seq-normalized CLIP regions, (ii) motifs extracted from native CLIP regions, (iii) previously reported individual motifs, or (iv) 15 motifs in SpliceAid 2. Validation of the 6 GU-rich (6GUR) motifs using CLIP-seq of the cerebrum and the whole brain showed that the 6GURmotifs were specifically enriched in CIMS. The number of the 6GURmotifs in an uninterrupted region was counted and multiplied by four to calculate the area, which was defined as the 6GUR-Score. The 6GUR-Score of 8 or more best discriminated CIMS from CIMS-flanking regions. We propose that the 6GURmotifs predict FUS-binding sites more efficiently than previously reported individual motifs or 15 motifs in SpliceAid 2.
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Jun-Ichi
authors.initials: JI
authors.lastname: Takeda
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Masuda
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan. Electronic address: ohnok@med.nagoya-u.ac.jp.
authors.forename: Kinji
authors.initials: K
authors.lastname: Ohno
mesh_terms: D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D045744:Cell Line, Tumor; D051379:Mice; D059372:Nucleotide Motifs; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D034702:RNA-Binding Protein FUS / Q000737:chemistry / Q000378:metabolism*; D017423:Sequence Analysis, RNA / Q000379:methods* / Q000592:standards
chemical_list: C000599551:FUS protein, mouse; D034702:RNA-Binding Protein FUS; D012313:RNA
keywords: CLIP-seq; FUS; Nascent-seq; RNA-binding motif; RNA-binding protein