Human Pre-mRNA Cleavage Factor Im Is Related to Spliceosomal SR Proteins and Can Be Reconstituted In Vitro from Recombinant Subunits  Ursula Rüegsegger, Diana Blank, Walter Keller  Molecular Cell  Volume 1, Issue 2, Pages 243-253 (January 1998) DOI: 10.1016/S1097-2765(00)80025-8

Figure 1 Predicted Amino Acid Sequences of the 25 kDa and the 68 kDa Subunits of CF Im (A) The 227 aa ORF of the 25 kDa subunit. Lys-C peptides 25L23b (183–189), 25L23c (57–73), and 25L23d (108–112) are indicated by stippled boxes. (B) The 551 aa ORF of the 68 kDa subunit. Lys-C peptides 68L6 (125–138) and 68L19 (162–168) are indicated by stippled boxes, RNP1 (124–131) and RNP2 (83–88) by closed boxes, and the whole RNA binding domain is framed. Proline residues between amino acids 208 and 398 are indicated by closed boxes, positively charged residues (R and K) between amino acids 489 and 551 by darkly shaded boxes, and negatively charged (D and E) serine and threonine residues in lightly shaded boxes. Molecular Cell 1998 1, 243-253DOI: (10.1016/S1097-2765(00)80025-8)

Figure 5 Chromatography of Reconstituted CF Im25/68 on Phenyl Superose (A) SDS–PAGE of Phenyl Superose fractions. Aliquots of 14.4 μl of the load (L) and the flow-through (2), of 5 μl of the fractions indicated at the bottom, and of 7 μl of CF Im purified from HeLa cell nuclear extract (Mono Q; CF Im) were separated on a 10% gel and stained with Coomassie brillant blue R. The labeling of the figure is as in Figure 2A. (B) Western blot of Phenyl Superose fractions. Aliquots of 3 μl of the load (L) and the flow-through (2), of 1 μl of the fractions indicated at the bottom, and of 1 μl of CF Im purified from HeLa cell nuclear extract (CF Im) were separated on a 10% gel and blotted onto a nitrocellulose membrane, and the blot was detected with affinity-purified α-25 kDa and α-68 kDa serum. (C) Cleavage of SV40 late pre-mRNA. Assays were carried out as described in the Experimental Procedures for 70 min at 30°C with 0.5, 1, and 2 μl of CF Im purified from HeLa cell nuclear extract (CF Im), 1.5 μl of the load (L) and the flow-through (2), and 0.3 μl of the fractions indicated at the bottom. Analysis of the samples and labeling are as in Figure 2B. Molecular Cell 1998 1, 243-253DOI: (10.1016/S1097-2765(00)80025-8)

Figure 2 The Cloned Polypeptides Are Subunits of CF Im (A) Western blot analysis of HeLa cell cytoplasmic and nuclear extract and purified CF Im. Proteins were separated on a 10% SDS–polyacrylamide gel and blotted onto a nitrocellulose membrane. Lanes 1–3 were detected with affinity-purified α-25 kDa, lanes 4–6 with α-68 kDa serum. Lanes 1 and 4: 20 μg of cytoplasmic extract; lanes 2 and 5: 20 μg of nuclear extract; and lanes 3 and 6: 1.5 μl of CF Im (Mono Q). The fraction loaded contains stoichiometric amounts of the 59 and 68 kDa polypeptides. The molecular masses of the size standards in kDa are indicated on the left. Arrowheads on the right indicate the subunits of CF Im. (B and C) Cleavage assays of immunodepletion experiments with a partially purified CF Im fraction. Assays were carried out as described in Experimental Procedures for 90 min at 30°C with SV40 late pre-mRNA as the substrate. Samples were analyzed on denaturing 6% w/v polyacrylamide gels. Sizes (in nt) of DNA standards (lanes M) are indicated on the left. The migration behavior of the SV40 late pre-mRNA substrate and the upstream cleavage product are indicated on the right. Lanes 2: SV40 late pre-mRNA incubated without protein fractions; lanes 3: SV40 late pre-mRNA incubated in the presence of CstF, CPSF, PAP, and partially purified CF IIm; lanes 4–6: 1, 3, and 6 μl of partially purified CF Im used for immunodepletion experiments; lanes 7–9: 1, 3, and 6 μl of CF Im depleted with preimmune serum ([B], p-25 kDa; [C], p-68 kDa); lanes 10–12: 1, 3, 6 μl of CF Im depleted with immune serum ([B], α-25 kDa; [C], α-68 kDa); lanes 13: as lanes 12, but 1.5 μl of highly purified CF Im (Mono Q) was added; lanes 14: 1.5 μl of highly purified CF Im (Mono Q). Molecular Cell 1998 1, 243-253DOI: (10.1016/S1097-2765(00)80025-8)

Figure 3 MAb 16H3 Recognizes the Three Large Subunits of CF Im Western blot analysis of HeLa cell cytoplasmic and nuclear extract, of purified SR proteins, and CF Im was performed as in Figure 2A with MAb 16H3 (hybridoma cell supernatant). Lane 1: 20 μg of cytoplasmic extract; lane 2: 20 μg of nuclear extract; lane 3: 800 ng of SR proteins (Zahler et al. 1992); lane 4: 3 μl of CF Im (Mono Q). The fraction loaded contains stoichiometric amounts of the 59 kDa and 68 kDa polypeptides. The labeling of the figure is as in Figure 2A. Asterisks at the right indicate SRp75, SRp55, SRp40, and SRp20, respectively. Molecular Cell 1998 1, 243-253DOI: (10.1016/S1097-2765(00)80025-8)

Figure 4 Time Course of Cleavage Reactions without Preincubation or after Preincubation of CF Im with L3 Pre-mRNA or without RNA For details, see the Experimental Procedures and the text. 100% cleavage was arbitrarily defined as the cleavage activity obtained after preincubating CF Im with L3 pre-mRNA and a reaction time of 120 min (2.25 U). (B) is an enlargement of part of (A). Molecular Cell 1998 1, 243-253DOI: (10.1016/S1097-2765(00)80025-8)