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. 2019 Dec 10;48(3):1508–1530. doi: 10.1093/nar/gkz1148

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Processing activity of purified semi-recombinant holo U7 snRNP. (A) Recombinant core U7 snRNP containing full length Lsm11 and SmB/Δ1–95 was assembled on synthetic U7–5′pcB snRNA tagged at the 5′ end with biotin and a photo-cleavable linker. (B) The assembled core was bound to Δ51 FLASH, immobilized on SA beads and incubated with mouse nuclear extract to recruit the HCC. The reconstituted semi-recombinant holo U7 snRNP was eluted to solution by UV irradiation and a fraction (10%) was analyzed by silver staining (lane 1). Silver staining of UV-eluted core U7 snRNP/Δ51 FLASH complex (no incubation with the nuclear extract) is shown in lane 2. Silver staining of UV-eluted U7–5′pcB snRNA (no Sm ring and FLASH) incubated with the same nuclear extract is shown in lane 3. (C) The same fraction (10%) of the three UV-eluted samples was analyzed by mass spectrometry. The table lists top scoring components (scores > 500) of semi-recombinant holo U7 snRNP (lane 1 in panel B). (D) Top scoring proteins identified in the UV-eluted sample of U7–5′pcB snRNA incubated with the nuclear extract (lane 3 in panel B). (E) A fraction (5%) of the UV-eluted samples shown in panel B were tested for the ability to support processing of mH2a/2m-3′B pre-mRNA either in the absence or in the presence of SLBP and/or an oligonucleotide blocking mouse U7 snRNA (αU7), as indicated at the top of each lane. (F) Processing of mH2a/2m-3′B pre-mRNA in a mouse nuclear extract alone (lane 1) or in the presence of SLBP, stem–loop RNA (SL) or αU7 oligonucleotide, as indicated at the top of each lane. (G) Processing of mH2a/2m-3′B pre-mRNA by a fraction (0.5%) of the UV-eluted semi-recombinant holo U7 snRNP alone (lane 2) or in the presence of WT SLBP (lane 3), or its mutant versions unable to support processing (lanes 4–5).