title: Reconstitution of the human U snRNP assembly machinery reveals stepwise Sm protein organization.
abstract: The assembly of spliceosomal U snRNPs depends on the coordinated action of PRMT5 and SMN complexes in vivo. These trans-acting factors enable the faithful delivery of seven Sm proteins onto snRNA and the formation of the common core of snRNPs. To gain mechanistic insight into their mode of action, we reconstituted the assembly machinery from recombinant sources. We uncover a stepwise and ordered formation of distinct Sm protein complexes on the PRMT5 complex, which is facilitated by the assembly chaperone pICln. Upon completion, the formed pICln-Sm units are displaced by new pICln-Sm protein substrates and transferred onto the SMN complex. The latter acts as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to prevent mis-assembly and to ensure the transfer of Sm proteins to cognate RNA. Investigation of mutant SMN complexes provided insight into the contribution of individual proteins to these activities. The biochemical reconstitution presented here provides a basis for a detailed molecular dissection of the U snRNP assembly reaction.
authors.affiliation: Department of Biochemistry, Biocenter, University of Würzburg, Würzburg, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Neuenkirchen
authors.affiliation: Department of Biochemistry, Biocenter, University of Würzburg, Würzburg, Germany.
authors.forename: Clemens
authors.initials: C
authors.lastname: Englbrecht
authors.affiliation: Department of Biochemistry, Biocenter, University of Würzburg, Würzburg, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Ohmer
authors.affiliation: Department of Biochemistry, Biocenter, University of Würzburg, Würzburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Ziegenhals
authors.affiliation: Research Group of 3D Electron Cryomicroscopy, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany utz.fischer@biozentrum.uni-wuerzburg.de ashwin.chari@mpibpc.mpg.de.
authors.forename: Ashwin
authors.initials: A
authors.lastname: Chari
authors.affiliation: Department of Biochemistry, Biocenter, University of Würzburg, Würzburg, Germany Department of Radiation Medicine and Applied Sciences, University of California, San Diego, San Diego, CA, USA utz.fischer@biozentrum.uni-wuerzburg.de ashwin.chari@mpibpc.mpg.de.
authors.forename: Utz
authors.initials: U
authors.lastname: Fischer
mesh_terms: D000818:Animals; D055541:DEAD Box Protein 20 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015778:Minor Histocompatibility Antigens; D009134:Muscular Atrophy, Spinal / Q000235:genetics; D009154:Mutation; D011484:Protein-Arginine N-Methyltransferases / Q000235:genetics / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D055532:SMN Complex Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C406815:GEMIN4 protein, human; C449808:GEMIN5 protein, human; D015778:Minor Histocompatibility Antigens; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; D017411:Ribonucleoproteins, Small Nuclear; D055532:SMN Complex Proteins; C405483:U1 small nuclear RNA; C400529:PRMT5 protein, human; D011484:Protein-Arginine N-Methyltransferases; D055541:DEAD Box Protein 20
keywords: PRMT5; SMN; assembly; pICln; snRNP