Volume 4, Issue 3, September 2018, Page: 24-31
Affinity Profiles Categorize RNA-Binding Proteins into Distinctive Groups
Naomi Ueda, Division of Gene Structure and Function, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Japan
Riki Kurokawa, Division of Gene Structure and Function, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Japan
Received: Oct. 15, 2018;       Accepted: Oct. 31, 2018;       Published: Nov. 27, 2018
DOI: 10.11648/j.bs.20180403.11      View  28      Downloads  7
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.
TLS, FUS, Random RNA Oligos, hnRNPH1, hnRNPU, hnRNPUL2, DDX21, hnRNPAB
To cite this article
Naomi Ueda, Riki Kurokawa, Affinity Profiles Categorize RNA-Binding Proteins into Distinctive Groups, Biomedical Sciences. Vol. 4, No. 3, 2018, pp. 24-31. doi: 10.11648/j.bs.20180403.11
Copyright © 2018 Authors retain the copyright of this article.
This article is an open access article distributed under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Kurokawa R (2015) Long Noncoding RNAs. In, pp. 257. Springer.
Lipovich L, Tarca AL, Cai J, Jia H, Chugani HT, Sterner KN, Grossman LI, Uddin M, Hof PR, Sherwood CC, et al. (2014) Developmental changes in the transcriptome of human cerebral cortex tissue: long noncoding RNA transcripts. Cereb Cortex 24, 1451-1459, doi: 10.1093/cercor/bhs414.
Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S, Tilgner H, Guernec G, Martin D, Merkel A, Knowles DG, et al. (2012) The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome research 22, 1775-1789, doi: 10.1101/gr.132159.111.
Carninci P & Kasukawa T & Katayama S & Gough J & Frith MC & Maeda N & Oyama R & Ravasi T & Lenhard B & Wells C, et al. (2005) The transcriptional landscape of the mammalian genome. Science 309, 1559-1563, doi: 309/5740/1559 [pii] 10.1126/science.1112014.
Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, Thomas K, Presser A, Bernstein BE, van Oudenaarden A, et al. (2009) Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci U S A 106, 11667-11672, doi: 0904715106 [pii]10.1073/pnas.0904715106.
Necsulea A, Soumillon M, Warnefors M, Liechti A, Daish T, Zeller U, Baker JC, Grutzner F & Kaessmann H (2014) The evolution of lncRNA repertoires and expression patterns in tetrapods. Nature 505, 635-640, doi: 10.1038/nature12943.
Chi KR (2016) Finding function in mystery transcripts. Nature 529, 423-425, doi: 10.1038/529423a.
Kurokawa R (2012) Generation of Functional Long Noncoding RNA Through Transcription and Natural Selection. In Regulatory RNAs, pp. 151-174. Springer.
Djebali S, Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A, Tanzer A, Lagarde J, Lin W, Schlesinger F, et al. (2012) Landscape of transcription in human cells. Nature 489, 101-108, doi: 10.1038/nature11233.
Hon C-C, Ramilowski JA, Harshbarger J, Bertin N, Rackham OJL, Gough J, Denisenko E, Schmeier S, Poulsen TM, Severin J, et al. (2017) An atlas of human long non-coding RNAs with accurate 5′ ends. Nature 543, 199-204, doi: 10.1038/nature21374 http://www.nature.com/nature/journal/v543/n7644/abs/nature21374.html#supplementary-information.
Kurokawa R (2011) Long noncoding RNA as a regulator for transcription. Prog Mol Subcell Biol 51, 29-41, doi: 10.1007/978-3-642-16502-3_2.
Kurokawa R (2011) Promoter-associated long noncoding RNAs repress transcription through a RNA binding protein TLS. Advances in experimental medicine and biology 722, 196-208, doi: 10.1007/978-1-4614-0332-6_12.
Kurokawa R (2015) Initiation of Transcription Generates Divergence of Long Noncoding RNAs. In Long Noncoding RNAs, pp. 69-91. Springer.
Kurokawa R, Rosenfeld MG & Glass CK (2009) Transcriptional regulation through noncoding RNAs and epigenetic modifications. RNA Biol 6, 233-236, doi: 8329 [pii].
Lunde BM, Moore C & Varani G (2007) RNA-binding proteins: modular design for efficient function. Nat Rev Mol Cell Biol 8, 479-490, doi: 10.1038/nrm2178.
Clery A, Blatter M & Allain FH (2008) RNA recognition motifs: boring? Not quite. Current opinion in structural biology 18, 290-298, doi: 10.1016/j.sbi.2008.04.002.
Linder P & Jankowsky E (2011) From unwinding to clamping - the DEAD box RNA helicase family. Nat Rev Mol Cell Biol 12, 505-516, doi: 10.1038/nrm3154.
Ramakrishnan V (2014) The ribosome emerges from a black box. Cell 159, 979-984, doi: 10.1016/j.cell.2014.10.052.
Steitz TA (2008) A structural understanding of the dynamic ribosome machine. Nat Rev Mol Cell Biol 9, 242-253, doi: 10.1038/nrm2352.
Behrmann E, Loerke J, Budkevich TV, Yamamoto K, Schmidt A, Penczek PA, Vos MR, Burger J, Mielke T, Scheerer P, et al. (2015) Structural snapshots of actively translating human ribosomes. Cell 161, 845-857, doi: 10.1016/j.cell.2015.03.052.
Papasaikas P & Valcarcel J (2016) The Spliceosome: The Ultimate RNA Chaperone and Sculptor. Trends in biochemical sciences 41, 33-45, doi: 10.1016/j.tibs.2015.11.003.
Plaschka C, Lin PC & Nagai K (2017) Structure of a pre-catalytic spliceosome. Nature 546, 617-621, doi: 10.1038/nature22799.
Wright PE & Dyson HJ (2015) Intrinsically disordered proteins in cellular signalling and regulation. Nat Rev Mol Cell Biol 16, 18-29, doi: 10.1038/nrm3920.
Castello A, Fischer B, Frese CK, Horos R, Alleaume AM, Foehr S, Curk T, Krijgsveld J & Hentze MW (2016) Comprehensive Identification of RNA-Binding Domains in Human Cells. Molecular Cell 63, 696-710, doi: 10.1016/j.molcel.2016.06.029.
Kwon SC, Yi H, Eichelbaum K, Fohr S, Fischer B, You KT, Castello A, Krijgsveld J, Hentze MW & Kim VN (2013) The RNA-binding protein repertoire of embryonic stem cells. Nature structural & molecular biology 20, 1122-1130, doi: 10.1038/nsmb.2638.
Liepelt A, Naarmann-de Vries IS, Simons N, Eichelbaum K, Fohr S, Archer SK, Castello A, Usadel B, Krijgsveld J, Preiss T, et al. (2016) Identification of RNA-binding Proteins in Macrophages by Interactome Capture. Mol Cell Proteomics 15, 2699-2714, doi: 10.1074/mcp. M115.056564.
Beckmann BM, Horos R, Fischer B, Castello A, Eichelbaum K, Alleaume AM, Schwarzl T, Curk T, Foehr S, Huber W, et al. (2015) The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs. Nat Commun 6, 10127, doi: 10.1038/ncomms10127.
Cui W, Yoneda R, Ueda N & Kurokawa R (2018) Arginine methylation of translocated in liposarcoma (TLS) inhibits its binding to long noncoding RNA, abrogating TLS-mediated repression of CBP/p300 activity. The Journal of biological chemistry, doi: 10.1074/jbc. RA117.000598.
Yoneda R, Suzuki S, Mashima T, Kondo K, Nagata T, Katahira M & Kurokawa R (2016) The binding specificity of Translocated in LipoSarcoma/FUsed in Sarcoma with lncRNA transcribed from the promoter region of cyclin D1. Cell & bioscience 6, 4, doi: 10.1186/s13578-016-0068-8.
Wang X, Arai S, Song X, Reichart D, Du K, Pascual G, Tempst P, Rosenfeld MG, Glass CK & Kurokawa R (2008) Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription. Nature 454, 126-130, doi: nature06992 [pii] 10.1038/nature06992.
Song X, Wang X, Arai S & Kurokawa R (2012) Promoter-associated noncoding RNA from the CCND1 promoter. Methods in molecular biology 809, 609-622, doi: 10.1007/978-1-61779-376-9_39.
Roeder RG (1991) The complexities of eukaryotic transcription initiation: regulation of preinitiation complex assembly. Trends Biochem Sci 16, 402-408.
Roeder RG (2003) Lasker Basic Medical Research Award. The eukaryotic transcriptional machinery: complexities and mechanisms unforeseen. Nature medicine 9, 1239-1244, doi: 10.1038/nm938.
Glass CK & Rosenfeld MG (2000) The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14, 121-141.
Rosenfeld MG, Lunyak VV & Glass CK (2006) Sensors and signals: a coactivator/corepressor/epigenetic code for integrating signal-dependent programs of transcriptional response. Genes & development 20, 1405-1428.
Glass CK, Rosenfeld MG, Rose DW, Kurokawa R, Kamei Y, Xu L, Torchia J, Ogliastro MH & Westin S (1997) Mechanisms of transcriptional activation by retinoic acid receptors. Biochem Soc Trans 25, 602-605.
Kurokawa R, Soderstrom M, Horlein A, Halachmi S, Brown M, Rosenfeld MG & Glass CK (1995) Polarity-specific activities of retinoic acid receptors determined by a co-repressor. Nature 377, 451-454, doi: 10.1038/377451a0.
Kurokawa R, Yu VC, Naar A, Kyakumoto S, Han Z, Silverman S, Rosenfeld MG & Glass CK (1993) Differential orientations of the DNA-binding domain and carboxy-terminal dimerization interface regulate binding site selection by nuclear receptor heterodimers. Genes Dev 7, 1423-1435.
Dreyfuss G, Kim VN & Kataoka N (2002) Messenger-RNA-binding proteins and the messages they carry. Nat Rev Mol Cell Biol 3, 195-205, doi: 10.1038/nrm760.
Hudson WH & Ortlund EA (2014) The structure, function and evolution of proteins that bind DNA and RNA. Nat Rev Mol Cell Biol 15, 749-760, doi: 10.1038/nrm3884.
Cech TR & Steitz JA (2014) The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157, 77-94, doi: 10.1016/j.cell.2014.03.008.
Hentze MW, Castello A, Schwarzl T & Preiss T (2018) A brave new world of RNA-binding proteins. Nat Rev Mol Cell Biol 19, 327-341, doi: 10.1038/nrm.2017.130.
Hasegawa Y, Brockdorff N, Kawano S, Tsutui K & Nakagawa S (2010) The matrix protein hnRNPU is required for chromosomal localization of Xist RNA. Dev Cell 19, 469-476, doi: 10.1016/j.devcel.2010.08.006.
Lambert N, Robertson A, Jangi M, McGeary S, Sharp PA & Burge CB (2014) RNA Bind-n-Seq: quantitative assessment of the sequence and structural binding specificity of RNA binding proteins. Molecular Cell 54, 887-900, doi: 10.1016/j.molcel.2014.04.016.
Zhou Q, Kunder N, De la Paz JA, Lasley AE, Bhat VD, Morcos F & Campbell ZT (2018) Global pairwise RNA interaction landscapes reveal core features of protein recognition. Nat Commun 9, 2511, doi: 10.1038/s41467-018-04729-0.
Jankowsky E & Harris ME (2015) Specificity and nonspecificity in RNA-protein interactions. Nat Rev Mol Cell Biol 16, 533-544, doi: 10.1038/nrm4032.
Khanam T, Muddashetty R, Kahvejian A, Sonenberg N & Brosius J (2006) Poly(A)-Binding Protein Binds to A-Rich Sequences via RNA-Binding Domains 1+2 and 3+4. Vol. 3.
Kim MK & Nikodem VM (1999) hnRNPU Inhibits Carboxy-Terminal Domain Phosphorylation by TFIIH and Represses RNA Polymerase II Elongation. Molecular and Cellular Biology 19, 6833-6844.
Berglund FM & Clarke PR (2009) hnRNP-U is a specific DNA-dependent protein kinase substrate phosphorylated in response to DNA double-strand breaks. Biochemical and biophysical research communications 381, 59-64, doi: 10.1016/j.bbrc.2009.02.019.
Polo SE, Blackford AN, Chapman JR, Baskcomb L, Gravel S, Rusch A, Thomas A, Blundred R, Smith P, Kzhyshkowska J, et al. (2012) Regulation of DNA-end resection by hnRNPU-like proteins promotes DNA double-strand break signaling and repair. Molecular Cell 45, 505-516, doi: 10.1016/j.molcel.2011.12.035.
Song C, Hotz-Wagenblatt A, Voit R & Grummt I (2017) SIRT7 and the DEAD-box helicase DDX21 cooperate to resolve genomic R loops and safeguard genome stability. Genes & development 31, 1370-1381, doi: 10.1101/gad.300624.117.
Calo E, Flynn RA, Martin L, Spitale RC, Chang HY & Wysocka J (2015) RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. Nature 518, 249-253, doi: 10.1038/nature13923.
Khan FA, Jaiswal AK & Szer W (1991) Cloning and sequence analysis of a human type A/B hnRNP protein. FEBS Letters 290, 159-161, doi: doi:10.1016/0014-5793(91)81249-8.
Lane AN, Chaires JB, Gray RD & Trent JO (2008) Stability and kinetics of G-quadruplex structures. Nucleic Acids Research 36, 5482-5515, doi: 10.1093/nar/gkn517.
Booy EP, Meier M, Okun N, Novakowski SK, Xiong S, Stetefeld J & McKenna SA (2012) The RNA helicase RHAU (DHX36) unwinds a G4-quadruplex in human telomerase RNA and promotes the formation of the P1 helix template boundary. Nucleic Acids Research 40, 4110-4124, doi: 10.1093/nar/gkr1306.
Rhodes D & Lipps HJ (2015) G-quadruplexes and their regulatory roles in biology. Nucleic Acids Research 43, 8627-8637, doi: 10.1093/nar/gkv862.
Takahama K & Oyoshi T (2013) Specific binding of modified RGG domain in TLS/FUS to G-quadruplex RNA: tyrosines in RGG domain recognize 2'-OH of the riboses of loops in G-quadruplex. J Am Chem Soc 135, 18016-18019, doi: 10.1021/ja4086929.
Takahama K, Takada A, Tada S, Shimizu M, Sayama K, Kurokawa R & Oyoshi T (2013) Regulation of telomere length by G-quadruplex telomere DNA- and TERRA-binding protein TLS/FUS. Chemistry & biology 20, 341-350, doi: 10.1016/j.chembiol.2013.02.013.
Li W, Notani D & Rosenfeld MG (2016) Enhancers as non-coding RNA transcription units: recent insights and future perspectives. Nature reviews Genetics 17, 207-223, doi: 10.1038/nrg.2016.4.
Hua JT, Ahmed M, Guo H, Zhang Y, Chen S, Soares F, Lu J, Zhou S, Wang M, Li H, et al. (2018) Risk SNP-Mediated Promoter-Enhancer Switching Drives Prostate Cancer through lncRNA PCAT19. Cell 174, 564-575 e518, doi: 10.1016/j.cell.2018.06.014.
Browse journals by subject