title: Mapping the Interaction Network of Key Proteins Involved in Histone mRNA Generation: A Hydrogen/Deuterium Exchange Study.
abstract: Histone pre-mRNAs are cleaved at the 3' end by a complex that contains U7 snRNP, the FLICE-associated huge protein (FLASH) and histone pre-mRNA cleavage complex (HCC) consisting of several polyadenylation factors. Within the complex, the N terminus of FLASH interacts with the N terminus of the U7 snRNP protein Lsm11, and together they recruit the HCC. FLASH through its distant C terminus independently interacts with the C-terminal SANT/Myb-like domain of nuclear protein, ataxia-telangiectasia locus (NPAT), a transcriptional co-activator required for expression of histone genes in S phase. To gain structural information on these interactions, we used mass spectrometry to monitor hydrogen/deuterium exchange in various regions of FLASH, Lsm11 and NPAT alone or in the presence of their respective binding partners. Our results indicate that the FLASH-interacting domain in Lsm11 is highly dynamic, while the more downstream region required for recruiting the HCC exchanges deuterium slowly and likely folds into a stable structure. In FLASH, a stable structure is adopted by the domain that interacts with Lsm11 and this domain is further stabilized by binding Lsm11. Notably, both hydrogen/deuterium exchange experiments and in vitro binding assays demonstrate that Lsm11, in addition to interacting with the N-terminal region of FLASH, also contacts the C-terminal SANT/Myb-like domain of FLASH, the same region that binds NPAT. However, while NPAT stabilizes this domain, Lsm11 causes its partial relaxation. These competing reactions may play a role in regulating histone gene expression in vivo.
authors: Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland; Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors: Aleksandra
authors: A
authors: Skrajna
authors: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors: Xiao-Cui
authors: XC
authors: Yang
authors: Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.
authors: Krzysztof
authors: K
authors: Tarnowski
authors: Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.
authors: Kinga
authors: K
authors: Fituch
authors: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors: William F
authors: WF
authors: Marzluff
authors: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: dominski@med.unc.edu.
authors: Zbigniew
authors: Z
authors: Dominski
authors: Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland. Electronic address: michald@ibb.waw.pl.
authors: Michał
authors: M
authors: Dadlez
mesh_terms: D000818:Animals; D004330:Drosophila; D005786:Gene Expression Regulation; D006657:Histones / Q000235:genetics*; D013058:Mass Spectrometry; D008954:Models, Biological; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D014714:Vertebrates
chemical_list: D006657:Histones; D012322:RNA Precursors
keywords: FLASH; Lsm11; Mass spectrometry; NPAT; U7 snRNP