Identification of two cross-linked proteins as SRp20 and REF/Aly. (A) AdML-AG pre-mRNA containing a single 4-thioU and ³²P label at position −20 was incubated under splicing conditions for 60 min prior to UV irradiation. Cross-linked samples were denatured by boiling in the presence of 0.1% SDS, digested with RNase A, and then immunoprecipitated with α-SRp20 antiserum. Similar results were obtained with AdML-AG containing 4-thioU at position −24 (data not shown). (B) Same as A except AdML-AG, AdML-GG, and control mRNA substrates containing a single 4-thioU and ³²P label at position −24 were incubated with α-REF antiserum.

Affinity purification and Western analysis of spliced mRNP. (A) Denaturing gel (15% polyacrylamide) following labeled RNAs during splicing (0 and 60 min, lanes 1,2), RNase H digestion (lane 3), and affinity elution (lane 4). Positions of MS2-tagged pre-mRNA, intermediates, and spliced mRNA are indicated to the left. (B) Western analysis of purified mRNP and C complexes (+/−Mg²⁺). Lane 1 contains 1 μL of HeLa nuclear extract. Lanes 2–4 each contains ∼120 fmole of the indicated complex as estimated from the specific activity of splicing intermediates (C complexes) or mRNA (mRNP).

A model for EJC assembly. EJC assembly is initiated as early as the H complex, when REF and SRp20 become associated with the 5′ exon. Additional EJC components join the complex prior to exon ligation. As the splicing reaction proceeds through exon ligation and mRNP release, at least three additional proteins become closely associated with the RNA, while other factors may remain with the spliceosome. Association of some EJC proteins becomes less stable after mRNP release. Proteins identified by cross-linking are named according to their apparent molecular weights, whereas p32/REF and p25/SRp20 were identified by denaturing immunoprecipitation. Proteins lacking a molecular-mass designation (bottom section) were shown to be present in the C complex or spliced mRNP by mass spectrometry and/or Western analysis.

Splicing and cross-linking of benzophenone-containing AdML RNAs. (Upper panels) Splicing time courses. RNAs were incubated for the times indicated and separated by denaturing PAGE (7%). Substrate and product structures are indicated above and to the left of each panel. Because pre-mRNAs contained only one ³²P label, only a subset of splicing products are detectable. The appearance of doublets for each substrate and product reflects the presence of unligated 3′ RNAs. (Lower panel) Protein cross-linking. After incubation on ice for times indicated, samples were irradiated at 302 nm and digested with RNase A. Molecular mass standards and apparent molecular masses of proteins that cross-linked in a splicing-dependent manner are indicated to the left and right, respectively. Note that lanes 13–16 contained somewhat less input RNA, and thus exhibit reduced cross-linking intensities compared to other samples.

Splicing and cross-linking of 4-thioU-containing RNAs. (A, upper panel) Splicing time courses of AdML-GG and AdML-AG RNAs containing single 4-thioU and ³²P labels at position −24. Splicing precursors, intermediates, and products are indicated to the right. (Lower panel) Pattern of cross-linked proteins for each time point in upper panel. Molecular mass standards and apparent molecular weights of proteins cross-linking in a splicing-specific manner are indicated to the left and right, respectively. (B) Effects of nonspecific competitor RNA on cross-linking pattern for AdML-AG RNA. (Left panel) Following a 60-min incubation under splicing conditions, excess cold competitor RNA was added and reactions incubated for an additional 10 min before UV irradiation and RNase digestion. Open and closed arrows indicate bands that were sensitive or not, respectively, to addition of cold competitor RNA. (Right panel) Denaturing PAGE (15%) of splicing precursors and products in the presence (+) and absence (−) of cold competitor RNA showing that cross-linking differences in the left panel do not simply reflect a difference in the extent of splicing. (C) Glycerol gradient fractionation of splicing reactions containing site-specifically modified AdML-GG (lanes 1–7) or AdML-AG (lanes 9–15) substrates. Substrates were incubated for 90 min (AdML-GG) or 120 min (AdML-AG) and irradiated on ice prior to being loaded onto a 10%–30% glycerol gradient. Following glycerol gradient fractionation, fractions (numbers at top) were divided and either resolved by denaturing PAGE (upper panels) or RNase A digested and separated by SDS-PAGE (lower panels). Lane 8 corresponds to AdML control mRNA incubated for 45 min under splicing conditions, irradiated on ice, and directly resolved by denaturing PAGE (upper panels) or RNase A digested and separated by SDS-PAGE (lower panels), without prior glycerol gradient fractionation. Molecular mass markers and apparent molecular masses of splicing specific cross-linked proteins are indicated to the left and right of each gel, respectively. (D) Same as C except with site-specifically modified PIP.B control mRNA (lanes 1–7) and pre-mRNA (lanes 9–15).

Splicing-dependent protection of 5′ exon intermediates from RNase cleavage. (A) Uniformly labeled AdML-GG pre-mRNA (lanes 1–15) or control 5′ exon RNA (lanes 16–25) was incubated under standard splicing conditions in HeLa cell nuclear extracts for the times indicated. Aliquots of these reactions were further incubated with the cDNA oligonucleotides indicated (lanes 7–15,17–25). RNAs were then separated by 15% denaturing PAGE. Each oligonucleotide was named according to its center position relative to the 5′ splice site, which was defined as 0. Splicing substrates, intermediates, and products are indicated on the left. The asterisk (*) denotes a lariat degradation product often observed in these reactions, which results from 3′–5′ exonucleolytic decay of the accumulated lariat intermediate (Moore and Sharp 1992). Note that in splicing reactions, the RNase H cleavage products migrating below the free 5′ exon derive from both pre-mRNA and free 5′ exons; therefore, 5′ exon intermediate protection is best monitored in splicing reactions by the proportion of intact free 5′ exon remaining after digestion. (B) Similar RNase H analysis using control 5′ exon and spliced α-TM RNAs. (C) Complete RNase digestion of singly labeled AdML RNAs. (Upper panel) Splicing time courses for AdML pre-mRNAs containing a single labeled phosphate at position −24 relative to the 5′ splice site (15% denaturing PAGE). Splicing substrates, intermediates, and products are indicated to the right. (Lower panel) Separation of protected fragments by denaturing PAGE (20%) after RNase A + T₁ digestion. Closed arrows indicate fragments corresponding to the 5′ exon intermediate, whereas the open arrow denotes the fragment corresponding to spliced mRNA. (D) Same as C except using PIP.B+10 pre-mRNA containing a single labeled phosphate at position −24 relative to the 5′ splice site.