A New Method for Large-scale Enrichment and Stepwise Identification of RNA-protein Complexes

doi:10.7503/cjcu20240091

Abstract

Abstract:

RNA-protein complexes（RPC） regulate various physiological processes in organisms and play a crucial role in the synthesis and degradation of RNA. For deep RPC profiling in addition to develop RNA-binding proteins（RBP） enrichment methods， some researchers also turned their attention to RNA-associated proteins（RAP）， which interact with RBP via protein-protein interaction. However， previous methods often enrich all proteins in the RPC as a whole and cannot differentiate RBP from RAP. Therefore， we reported a new method using combination of 254 nm UV crosslinking of RBP and RNA and DSP crosslinking of RBP and RAP. Next， RBP and RAP can be eluted in a stepwise way and separately identified by mass spectrometry. A total of 2007 high confidence RBP and 927 high confidence RAP were identified in HeLa cells， of which 243 RAP were identified for the first time. This method aims to refine the RNA-protein interaction network and provide a favorable foundation for subsequent biological investigations.

Key words: RNA binding protein, Proteomics, Enrichment, Separation, Mass spectrometry

CLC Number:

O657

TrendMD:

DONG Peiying, LIU Tong, QIN Weijie. A New Method for Large-scale Enrichment and Stepwise Identification of RNA-protein Complexes[J]. Chem. J. Chinese Universities, 2024, 45(11): 20240091.

Figures/Tables 3

References 32

1	Müller⁃McNicoll M.， Neugebauer K. M.， Nat. Rev. Genet.， 2013， 14， 275—287
2	Chen Y. Y.， Gui Z.， Hu D.， Chen M. Y.， He J.， Yu S. Y.， Feng Y. Q.， Wang J.， Yuan B. F.， Chinese Chem. Lett.， 2023， 35， 108522
3	Guo C.， Zhang X. X.， Hong X. J.， Hu Y. Q.， Mao L. N.， Jiang K. Z.， Chinese Chem. Lett.， 2023， 35， 108867
4	Wang Q.， Jiang Q.， Li D.， Yang Z.， Gao L.， Liu F.， Li C.， Feng Y.， He Z.， Luo C.， Sun J.， Chinese Chem. Lett.， 2023， 35， 108683
5	Hu B.， Kong S.， Weng Y.， Zhao D.， Hussain A.， Jiao Q.， Zhan S.， Qiu L.， Lin J.， Xie M.， Li B.， Huang Y.， Chinese Chem. Lett.， 2023， 34， 108210
6	Hocq R.， Paternina J.， Alasseur Q.， Genovesio A.， Le Hir H.， Nucleic Acids Res.， 2018， 46， 11553—11565
7	Hudson W. H.， Ortlund E. A.， Nat. Rev. Mol. Cell Biol.， 2014， 15， 749—760
8	Al⁃Husini N.， Tomares D. T.， Pfaffenberger Z. J.， Muthunayake N. S.， Sa⁃mad M. A.， Zuo T.， Bitar O.， Aretakis J. R.， Bharmal M. -H. M.， Gega A.， Mol. Cell， 2020， 78， 670—682
9	Valentin⁃Vega Y. A.， Wang Y. D.， Parker M.， Patmore D. M.， Kanagaraj A.， Moore J.， Rusch M.， Finkelstein D.， Ellison D. W.， Gilbertson R. J.， Sci. Rep.， 2016， 6， 25996
10	Somasekharan S. P.， El⁃Naggar A.， Leprivier G.， Cheng H.， Hajee S.， Grunewald T. G. P.， Zhang F.， Ng T.， Delattre O.， Evdokimova V.， Cell Biol.， 2015， 208， 913—929
11	Brownsword M. J.， Locker N.， Rev. RNA， 2022， 14， e1741
12	Verdile V.， de Paola E.， Paronetto M. P.， Front. Genet.， 2019， 10， 173
13	Ramaswami M.， Taylor J. P.， Parker R.， Cell， 2013， 154， 727—736
14	Castello A.， Fischer B.， Eichelbaum K.， Cell， 2012， 1393—1406
15	He C.， Sidoli S.， Warneford Thomson， Mol. Cell， 2016， 416—430
16	Jao C. Y.， Salic A.， Proc.， Natl. Acad. Sci. USA， 2008， 15779—15784
17	Urdaneta E. C.， Vieira⁃Vieira C. H.， Hick T.， Wessels H.， Figini D.， Moschall R.， Medenbach J.， Ohler U.， Granneman S.， Selbach M.， Beckmann B. M.， Nat. Commun.， 2019， 10（1）， 990
18	Chomczynski P.， Sacchi N.， Anal. Biochem.， 1987， 156—159
19	Chomczynski P.， Sacchi N.， Nat. Protoc.， 2006， l， 581—585
20	Smith T.， Villanueva E.， Queiroz M. L.， Dawson C. S.， Elzek M.， Urdaneta E. C.， Current Opinion in Chem. Bio.， 2020， 54， 70—75
21	Trendl J.， Schwarzl T.， Horos R.， Cell， 2018， 37（2）， 169—178
22	Castello A.， Horos R.， Strein C.， Nat. Protoc.， 2013， 8（3）， 491—500
23	Bae J. W.， Kwon S. C.， Na Y.， Nat. Struct. Mol. Biol.， 2020， 27（7）， 678—682
24	Ryan D. P.， Matthews J. M.， Current Opinion in Structural Bio.， 2005， 15， 441—446
25	Ripin N.， Parker R.， Cell， 2023 ， 186， 4737—4756
26	Caudron⁃Herger M.， Rusin S. F.， Adamo M. E.， Barreau E.， Kettenbach A. N.， Diederichs S.， Mol. Cell， 2019， 75， 184—199
27	Street L.， Rothamel K.， Brannan K.， Jin W.， Bokor B.， Dong K.， Rhine K.， bioRxiv， 2023， 544225
28	Zhang Z.， Liu T.， Dong H.， Li J.， Sun H.， Nucleic Acids Research， 2021，49（11），e65
29	Li Y.， Xia C.， Zhao H.， Xie Y.， Zhang Y.， Zhang W.， Yu Y.， Wang J.， Qin W.， Chinese Chem. Lett.， 2022， 34， 107377
30	Parimalam S. S.， Oguchi Y.， Abdelmoez M. N.， Tsuchida A.， Anal. Chem.， 2018， 90， 21， 12512—12518
31	Xiang C. C.， Mezey E.， Chen M.， Key S.， Ma L.， Brownstein M. J.， Nucleic Acids Research， 2004， 32（22），e185
32	Mugler C. F.， Hondele M.， Heinrich S.， Sachdev R.， Vallotton P.， Koek A. Y.， Chan L. Y.， Weis K.， eLife， 2016， 5， e18746

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