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

Yoneda, Ryoma; Suzuki, Shiho; Mashima, Tsukasa; Kondo, Kengo; Nagata, Takashi; Katahira, Masato; Kurokawa, Riki

doi:10.1186/s13578-016-0068-8

Cited by 19 publications

(44 citation statements)

References 26 publications

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“…The RNase inhibitor (SIN-201) was purchased from Toyobo Life Science. HeLa cell NE was prepared as previously described protocol [29][30][31].…”

Section: Antibodies and Reagentsmentioning

confidence: 99%

“…Most certain criterion for RBPs is just to detect binding ability to RNA. Recently, we have established a novel assay to detect binding of RNA-binding proteins, using biotinylated RNA oligos to capture RBPs with Western blot of specific antibodies against RBPs [28,29]. The assay more confidently detects RNA binding than the traditional gel shift assay.…”

Section: Introductionmentioning

confidence: 99%

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Affinity Profiles Categorize RNA-Binding Proteins into Distinctive Groups

Ueda¹

2018

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Recently, we have established a novel assay to detect interaction of RNA-binding proteins (RBPs) with RNA, using biotinylated RNA oligos to capture RBPs with Western blot of specific antibodies against RBPs. The assay detects RNA binding more confidently than the traditional gel shift assay. Starting with completely randomized RNA oligos from 5mer through 12mer length, their binding was examined with HeLa cell nuclear extract (NE). Coomassie brilliant blue-based (CBB) staining did not detect any strong signal. Western blot analysis of typical six RBP antibodies showed four RBPs bound with the random RNA oligos. hnRNPUL2 bound to all from 5mer to 12mer of RNA oligos, while no TLS and hnRNPH1 signal was detected in the random RNA oligo samples. Next, base specificity was examined using sets of oligos of RNA fixed at G, A, U, and C (GAUC RNA oliogs). The RNA oligos fixed at "G" (G RNA oligos) have the most prominent protein bands. A, U, and C of RNA oligos were shown to bind less numbers of protein. Western blot indicated that hnRNPUL2 and hnRNPU bind all four oligos of GAUC at the 10mer length. Contrarily, TLS and hnRNPH1 have no binding with these oligos of GAUC. Then, poly G, A, U and C of RNA at the length of 100mer were tested to see binding profile of RBPs. The CBB staining of the fractions bound with these four polymers of RNA showed that more bands were bound than GAUC RNA oligos. hnRNPU bound well to poly G, A, and, U, but slightly less to poly C. Intriguingly, TLS and hnRNPH1 have binding only to poly G, and also to their common specific sites consisting of GGUG motifs. These data demonstrate that RNA binding is regulated with three factors, length, base composition, and sequence. Furthermore, hnRNPU and hnRNPUL2 have low specificity binding to RNAs, while TLS and hnRNPH1 exert high specific binding. These different propensities in bindings of RBPs are supposed to support specific biological roles in living cells.

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“…The RNase inhibitor (SIN-201) was purchased from Toyobo Life Science. HeLa cell NE was prepared as previously described protocol [29][30][31].…”

Section: Antibodies and Reagentsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Affinity Profiles Categorize RNA-Binding Proteins into Distinctive Groups

Ueda¹

2018

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“…SDS-polyacrylamide gel electrophoresis was performed with 10% gels following coomassie brilliant blue staining [28]. Western blotting was done with anti-TLS monoclonal antibody from the BD bioscience, 611385 with the dilution ratio 1:2000 using standard protocol shown previously [28].…”

Section: Protein Analysismentioning

confidence: 99%

“…Western blotting was done with anti-TLS monoclonal antibody from the BD bioscience, 611385 with the dilution ratio 1:2000 using standard protocol shown previously [28].…”

Section: Protein Analysismentioning

confidence: 99%

“…Thus, their sequences are in full diversity and unlikely to have a common molecular mechanism behind of their biological activity. We have, however, noticed that most of RNAs should have their own RNA-binding protein partners in living cells [28]. Then, we have set RNA-binding proteins as a criterion for classification of biological activities for each lncRNA.…”

Section: Introductionmentioning

confidence: 99%

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Biotin-Lys-His Blocks Aggregation of RNA-binding Protein TLS, a Cause of Amyotrophic Lateral Sclerosis

Ueda¹

2017

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RNA-binding protein TLS/FUS is a causative gene for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TLS mutations induce the propensity of TLS to form aggregates in motor neurons causing neuronal degenerative lesions to their necrosis. TLS is prone to be precipitated in high concentration around 10 mg/ml, while mutated TLS is suspected to be precipitated even lower or physiological concentration in the motor neurons. An unidentified agent from infections would cause formation of the precipitation of TLS through their surface antigens. The precipitation driven with agents might be attribute to a small compound appeared on the surface. Biotinylated isoxazole (BISOX) has been reported to be precipitated with divergent RNA-binding proteins including TLS in nuclear extracts of cultured mammalian cells. We have published a molecular model for crystal formation of BISOX with TLS providing a plat form for searching novel regulators to precipitate TLS in neuronal disorders. Because BISOX is an artificial compound, we have further explored to obtain naturally occurring agents to induce the TLS precipitation and generated a conceivable biological compound, biotin-Lys-His (BLH). We have examined the precipitation of BLH with HeLa cell nuclear extract, but did not detect any TLS signal. Then, we add BLH to reaction of BISOX with TLS, and serendipitously observed a robust inhibitory effect of BLH on the formation of crystal of BISOX with TLS. We employed in silico analysis to show how BLH blocks the crystal formation of BISOX with TLS. The computational analysis of the events presented a model that BLH should be incorporated into the crystal formation of BISOX but some steric hindrance placed by BLH blocks growing of the crystal of BISOX and TLS. These results provide the potentiality that BLH should block the aggregate formation of TLS in ALS, leading to a seed for drug discovery against ALS, although it needs future endeavor to find more compounds to have effect on the TLS aggregation.

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FUS Aggregation by Shear Stress on Pipetting and Its Suppression by Non-coding RNA

Katahira

2023

Phase Separation in Living Cells

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

Cited by 19 publications

References 26 publications

Affinity Profiles Categorize RNA-Binding Proteins into Distinctive Groups

Affinity Profiles Categorize RNA-Binding Proteins into Distinctive Groups

Biotin-Lys-His Blocks Aggregation of RNA-binding Protein TLS, a Cause of Amyotrophic Lateral Sclerosis

FUS Aggregation by Shear Stress on Pipetting and Its Suppression by Non-coding RNA

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