Protein composition of catalytically active U7-dependent processing complexes assembled on histone pre-mRNA containing biotin and a photo-cleavable linker
| Autor: | Xiao Cui Yang, Zbigniew Dominski, William F. Marzluff, Aleksandra Skrajna, Michal Dadlez |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Streptavidin Ultraviolet Rays Biotin Cleavage (embryo) Mass Spectrometry Histones 03 medical and health sciences chemistry.chemical_compound Mice Genetics RNA and RNA-protein complexes RNA Precursors Tumor Cells Cultured Animals Drosophila Proteins snRNP RNA Messenger Ribonucleoprotein SLBP RNA Cleavage biology Nucleotides Ribonucleoprotein U7 Small Nuclear Cell biology 030104 developmental biology Histone chemistry biology.protein Biocatalysis RNA 3' End Processing Precursor mRNA |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | 3′ end cleavage of metazoan replication-dependent histone pre-mRNAs requires the multi-subunit holo-U7 snRNP and the stem–loop binding protein (SLBP). The exact composition of the U7 snRNP and details of SLBP function in processing remain unclear. To identify components of the U7 snRNP in an unbiased manner, we developed a novel approach for purifying processing complexes from Drosophila and mouse nuclear extracts. In this method, catalytically active processing complexes are assembled in vitro on a cleavage-resistant histone pre-mRNA containing biotin and a photo-sensitive linker, and eluted from streptavidin beads by UV irradiation for direct analysis by mass spectrometry. In the purified processing complexes, Drosophila and mouse U7 snRNP have a remarkably similar composition, always being associated with CPSF73, CPSF100, symplekin and CstF64. Many other proteins previously implicated in the U7-dependent processing are not present. Drosophila U7 snRNP bound to histone pre-mRNA in the absence of SLBP contains the same subset of polyadenylation factors but is catalytically inactive and addition of recombinant SLBP is sufficient to trigger cleavage. This result suggests that Drosophila SLBP promotes a structural rearrangement of the processing complex, resulting in juxtaposition of the CPSF73 endonuclease with the cleavage site in the pre-mRNA substrate. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|