A Mechanism for the Regulation of Pre-mRNA 3′ Processing by Human Cleavage Factor Im  Kirk M Brown, Gregory M Gilmartin  Molecular Cell  Volume 12, Issue 6, Pages 1467-1476 (December 2003) DOI: 10.1016/S1097-2765(03)00453-2

Figure 1 SELEX Determination of the RNA Binding Specificity of CFIm (A) Recombinant human CFIm 68/25 kDa heterodimer purified from Sf9 cells, resolved on a 10% SDS-polyacrylamide gel, and stained with Coomassie brilliant blue R. (B) Sequence of the 33 clones analyzed from the final CFIm selection pool. The frequency with which each sequence was identified is listed at the right. Spaces have been inserted in the sequence to emphasize the common elements: (1) UGUAA, (2) UGUA(C/G/U), and (3) GC-rich segment. Uppercase letters denote sequences within the randomized region of the SELEX RNA; lowercase letters denote primer binding site sequences. (C) Gel mobility shift analysis of the initial SELEX RNA pool, SELEX RNAs 1 and 2 (as listed in panel [B]), and the final SELEX RNA pool. The 32P-labeled RNAs were incubated with 2 pmol of CFIm, and the complexes were resolved on a 6% (29:1) nondenaturing polyacrylamide gel. Molecular Cell 2003 12, 1467-1476DOI: (10.1016/S1097-2765(03)00453-2)

Figure 2 CFIm RNA Aptamers Inhibit Poly(A) Site Cleavage and Poly(A) Addition In Vitro (A) Poly(A) site cleavage. Uniformly 32P-labeled HIV-1 and Ad-L3 poly(A) site-containing RNA substrates were incubated in HeLa cell nuclear extract along with 3′dATP in the absence (lanes 1 and 8) or presence of the indicated amount of unlabeled SELEX RNA 1 (lanes 2, 3, 9, and 10), SELEX RNA 2 (lanes 6, 7, 13, and 14), or a negative control RNA (sequence described in Experimental Procedures) (lanes 4, 5, 11, and 12). The incubations were carried out for 30 min at 30°C, and the RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. (B) Poly(A) addition. Uniformly 32P-labeled precleaved HIV-1 and Ad-L3 poly(A) site-containing RNA substrates were incubated in HeLa cell nuclear extract along with ATP in the absence (lanes 1 and 8) or presence of the indicated amount of unlabeled SELEX RNA 1 (lanes 2, 3, 9, and 10), SELEX RNA 2 (lanes 4, 5, 11, and 12), or negative control RNA (lanes 6, 7, 13, and 14). The incubations were carried out for 15 min at 30°C, and the RNA products were isolated and resolved on a denaturing 5% polyacrylamide gel. Molecular Cell 2003 12, 1467-1476DOI: (10.1016/S1097-2765(03)00453-2)

Figure 3 A CFIm RNA Aptamer Inhibits 3′ Processing Complex Formation at Both the HIV-1 and Ad-L3 Poly(A) Sites In Vitro (A) Uniformly 32P-labeled HIV-1 and Ad-L3 poly(A) site-containing RNAs were incubated in HeLa cell nuclear extract alone (lanes 1, 4, 6, and 9) or in the presence of the indicated amount of SELEX RNA 1 (lanes 2, 3, 7, and 8) or negative control RNA (lanes 5 and 10). The incubations were carried out for 10 min at 30°C, and the RNA:protein complexes were resolved on a nondenaturing 3% (80:1) polyacrylamide gel. (B) Uniformly 32P-labeled SELEX RNA 1 and negative control RNA were incubated in the presence of purified HeLa cell CPSF (lanes 1 and 4), CFIm (lanes 2 and 5), or both CPSF and CFIm (lanes 3 and 6). The incubations were carried out for 10 min at 30°C, and the RNA:protein complexes were resolved on a nondenaturing 4% (80:1) polyacrylamide gel. Molecular Cell 2003 12, 1467-1476DOI: (10.1016/S1097-2765(03)00453-2)

Figure 4 The Inhibition of 3′ Processing Complex Formation and Poly(A) Addition by a CFIm RNA Aptamer Is Reversible (A) A uniformly 32P-labeled HIV-1 poly(A) site-containing RNA was incubated in HeLa cell nuclear extract in the absence (lane 1) or presence (lanes 2–7) of the indicated amount of SELEX RNA 1 for 10 min at 30°C. The reactions were then placed on ice, the indicated amount of an antisense (lanes 4 and 5) or sense (lanes 6 and 7) DNA oligonucleotide was added, and the reactions were returned to 30°C for an additional 10 min. The RNA:protein complexes were resolved on a nondenaturing 3% (80:1) polyacrylamide gel. (B) A uniformly 32P-labeled precleaved HIV-1 poly(A) site-containing RNA was incubated in HeLa cell nuclear extract in the absence (lanes 1–4) or presence (lanes 5–8) of 25 pmol of SELEX RNA 1 for 10 min at 30°C. The reactions were placed on ice, 25 pmol of antisense (lanes 2 and 6) or sense (lanes 4 and 8) DNA oligonucleotide was added along with ATP, and the reactions were returned to 30°C for an additional 20 min. The RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. Molecular Cell 2003 12, 1467-1476DOI: (10.1016/S1097-2765(03)00453-2)

Figure 5 CFIm Binding and Regulation of Cleavage at the Human CFIm 68 kDa Subunit Poly(A) Site In Vitro (A) The sequence encompassing the primary poly(A) site of the human CFIm 68 kDa subunit pre-mRNA. An asterisk denotes the primary site of endonucleolytic cleavage (ascertained by EST frequency). (B) Uniformly 32P-labeled RNAs containing either the CFIm 68 kDa subunit poly(A) site sequence depicted in (A) (lanes 1–5) or the Ad-L3 poly(A) site (lanes 6–10) were incubated with the indicated amount of CFIm. Incubations were carried out for 10 min at 30°C, and the RNA:protein complexes were resolved on a nondenaturing 4% (80:1) polyacrylamide gel. (C) Uniformly 32P-labeled RNAs containing the Ad-L3 and CFIm 68 kDa subunit poly(A) sites were preincubated in the presence of the indicated amount of CFIm for 10 min at 30°C. The reactions were then placed on ice, and HeLa cell nuclear extract and 3′ dATP were added. The reactions were then returned to 30°C for an additional 30 min. The RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. Molecular Cell 2003 12, 1467-1476DOI: (10.1016/S1097-2765(03)00453-2)

Figure 6 Sequence-Dependent Regulation of Poly(A) Site Cleavage by CFIm In Vitro Uniformly 32P-labeled RNAs containing the following poly(A) sites were preincubated in the presence of the indicated amount of CFIm for 10 min at 30°C: (1) Ad-L3 (lanes 1–5), (2) wild-type CFIm 68 kDa subunit (lanes 6–10 and 21–25), (3) 68 kDa Δ−22 (CFIm 68 kDa subunit poly(A) site containing a UGUAAUAAA to ACAAAUAAA mutation) (lanes 11–15), (4) 68 kDa Δ+7 (CFIm 68 kDa subunit poly(A) site containing a UGUAA to ACAAA mutation seven bases downstream of the cleavage site) (lanes 16–20), (5) 68 kDa Δ−55 (CFIm 68 kDa subunit poly(A) site containing a UGUAA to ACAAA mutation 55 nt upstream of the cleavage site) (lanes 26–30), and (6) 68 kDa Δ−55/−22/+7 (CFIm 68 kDa subunit poly(A) site containing all three UGUAA to ACAAA mutations) (lanes 31–35). The reactions were then placed on ice, and HeLa cell nuclear extract and 3′ dATP were added. The reactions were then returned to 30°C for an additional 30 min. The RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. Molecular Cell 2003 12, 1467-1476DOI: (10.1016/S1097-2765(03)00453-2)

Figure 7 Sequence-Specific Enhancement of Poly(A) Addition by CFIm In Vitro (A) Uniformly 32P-labeled precleaved RNAs containing the following poly(A) sites were preincubated in the presence of the indicated amount of CFIm for 10 min at 30°C: (1) wild-type CFIm 68 kDa subunit (lanes 1–5), (2) 68 kDa Δ−55 (lanes 6–10), (3) 68 kDa Δ−22 (lanes 11–15), and (4) 68 kDa Δ−55/−22 (lanes 16–20). The reactions were then placed on ice, and purified HeLa cell CPSF, recombinant poly(A) polymerase, and ATP were added. The reactions were then returned to 30°C for an additional 15 min. The RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. (B) Uniformly 32P-labeled precleaved RNAs containing the following poly(A) sites were preincubated in the presence of the indicated amount of CFIm for 10 min at 30°C: (1) wild-type CFIm 68 kDa subunit (lanes 1–5), (2) 68 kDa Δ−55 (lanes 6–10), (3) 68 kDa Δ−22 (lanes 11–15), and (4) 68 kDa Δ−55/−22 (lanes 16–20). The RNA:protein complexes were resolved on nondenaturing 4% (80:1) polyacrylamide gels. Molecular Cell 2003 12, 1467-1476DOI: (10.1016/S1097-2765(03)00453-2)