PMC

A. U1 snRNP bound to the proximal strong 5′ splice site (hexagon) adopts a conformation which can efficiently engage U2 snRNP. In this situation, U1 snRNP bound to the strong site out competes U1 snRNP bound at the weak site (pentagon) resulting in the use of the proximal site. Relative efficiencies of engagement of U1 and U2 snRNPs are depicted schematically by the thickness of the lines connecting them; the line from the unused site is dashed. B. In the presence of a splicing silencer adjacent to the proximal site, the interaction of U1 snRNP (square) is altered such that it less efficiently interacts with U2 snRNP. Accordingly, use of the distal site is observed. When both splice sites are “silenced” simultaneously, the proximal site regains its competitive advantage; in both illustrations, the line from the unused site is dashed.

(A)The 106 intronic and 52 exonic sequences which demonstrated strong silencing activity (see text) were grouped into six classes (A, B, C, D intronic, E, F, exonic) based on hierarchical clustering for sequence similarity and are presented as logos created using RNA structure logo (; ). All individual unique sequences including those that did not fall into the six classes are listed in with repeated sequences deleted.
(B) Pre-mRNAs, each containing a specific representative of one of the six classes (Motif D, GGGCCACTTGGA, lane 1; Motif B, CGCTGGTCATTC, lane 2; Motif C, GAGGATCAGCTT, lane 3; Motif A, CGTTAGAGTAGC, lane 4; Motif F, CTTAATTTTAGT, lane 6; Motif E, TAGTTTAGTTAG, lane 7) of silencer was spliced in vitro. Lane 5 is a splicing reaction with a pre-mRNA that does not contain a splicing silencer. The positions of splicing intermediates (free 5′ exon and lariat-3′ exon) are indicated.

(A) A Schematic representation of the base construct used for functional SELEX; the distal 5′ splice site (AUG/GUAAAC) is weak (W) relative to the proximal strong (S) 5′ splice site (AUG/GUAAGU). Only the proximal 5′ splice is used when this pre-mRNA is spliced in vitro (lane 1) whereas the distal site is activated when the proximal 5′ splice site is inactivated by a block mutation, (AUC/CAUUCAUA, lane 2).
(B) In vitro splicing of the same pre-mRNA as in (A) except that the 3′ splice site was changed from AG/G to UC/C to arrest splicing after the first catalytic step. Lane 1, splicing of the pre-mRNA with a wild type proximal 5′ splice site; lane 2, splicing when the proximal 5′ splice site was inactivated by mutation; lane 3, splicing when the proximal 5′ splice site was wild type but a randomized 12 nucleotide sequence was inserted downstream from positions +11 to +22 (see text).
(C) Schematic of the functional SELEX strategy; for a detailed description of individual steps, see the text and Experimental Procedures.
(D) Using the strategy illustrated in (C), the pool of body-labeled pre-mRNAs randomized at positions +11 to +22 relative to the proximal 5′ splice site were spliced in vitro and analyzed after 0 (lane 0), 1 (lane 1), 2, (lane 2), 3 (lane 3) or 4 (lane 4) rounds of selection for lariat-3′ exon intermediates resulting from use of the distal 5′ splice site. The positions of splicing intermediates, free 5′ exon and lariat-3′ exon are indicated.

(A) Schematic representation of pre-mRNAs used to measure kinetic effects on splicing of individual silencer motifs. The strong and weak 5′ splice sites are the same as in . The strong 3′ splice site construct has an unaltered polypyrimidine tract while the weak 3′ splice site contains purine substitutions in the polypyrimidine tract (). For each panel of four constructs, individual silencing motifs as described in the legend to were inserted upstream or downstream of the 5′ splice site as appropriate.
(B) In vitro splicing of the panel of four constructs either lacking any silencer element (no silencer, lanes 1–5) or containing either intronic silencing Motif D (lanes 6–10) or exonic silencing Motif E (lanes 11–15). Autoradiograms of the 60 minute time points of each reaction are shown.
(C,D,E,F) Time courses of splicing of the indicated pre-mRNAs containing or lacking the indicated silencing motif. Each data point is the average of three independent experiments. The percentage of splicing of indicated pre-mRNAs lacking any silencer motif was set to 100%. Values were determined by quantitating the extent of splicing [product/(product + precursor)] for each reaction and are expressed as percent of the extent of splicing of a pre-mRNA lacking any silencing elements.
(G) Quantitation of the extent of splicing at 40 minute time points from three independent time courses for each panel of pre-mRNAs containing the indicated silencing motif. Values expressed as percent were calculated as in C–F. Error bars represent standard deviations of quantified values.

(A) The nuclease protection experimental strategy is shown schematically. RNA molecules containing a uniquely labeled phosphate are incubated with protein(s). After binding, the RNAs are digested with micrococcal nuclease and nuclease-resistant fragments are visualized after fractionation on denaturing gels ()
(B) RNA transcripts spanning −45 to +45 relative to the 5′ splice site were site specifically labeled at the phosphate between the G*U of the 5′ splice site. Each transcript contained a specific silencing motif as indicated or lacked any motif (lanes none). After incubation in HeLa cell nuclear extract, reactions were diluted and digested with micrococcal nuclease (see Experimental Procedures). Following deproteinization, resistant fragments were visualized after gel fractionation. NE is nuclear extract and anti-U1 is a 2′Ome oligonucleotide which hybridizes to the 5′ end of U1 snRNA. The positions of markers of known size are indicated.
(C) Nuclease protection of control or Motif D containing RNAs using purified U1 snRNP. The site specifically labeled RNAs in (B) corresponding to none and Motif D were either incubated in nuclear extract, NE (lanes 1, 2 and 5, 6) or with purified U1 snRNP (lanes 3, 4 and 7, 8) in the absence (lanes 1, 3 and 5, 7) or presence (lanes 2, 4 and 6, 8) of the anti-U1 2′Ome oligonucleotide. Following incubation, reactions were processed as in (B) with some modifications (see ).

(A) HeLa cells were transfected with plasmids expressing the same pre-mRNAs as those assayed in except that the 3′ splice site was wild type. Splicing was assayed by semi-quantitative RT-PCR; products generated from use of the proximal or distal 5′ splice sites are indicated. Lane 1, Motif D; lane 2, Motif B; lane 3, Motif C; lane 4, Motif A; lane 5, no silencer; lane 6, Motif F; lane 7, Motif E; Lane 8, pre-mRNA lacking a silencer in which the proximal 5′ splice site was inactivated by mutation as in .
(B) In vivo splicing of pre-mRNAs identical to those in (A) except that the silencing motif was inserted at positions +11 to +22 (intronic) or −18 to −7 (exonic) relative to the distal 5′ splice site as appropriate; thus each silencing motif was present twice in each pre-mRNA at the same position relative to both the proximal and distal 5′ splice sites. Lane designations are as in (A).
(C) In vivo splicing of pre-mRNAs with duplicated weakened 5′ splice sites (see text). As in (A) each pre-mRNA contained one specific silencing motif upstream or downstream of the proximal 5′ splice site. Lane designations are as in (A).
(D) In vivo splicing of pre-mRNAs with duplicated weakened 5′ splice sites with duplicated silencing motifs. As in (B), the specific silencer motifs were inserted upstream or downstream as appropriate of the distal 5′ splice site when the same motif was upstream or downstream of the proximal 5′ splice site. Lane designations are as in (A).
(E) Body-labeled dual 5′ splice site pre-mRNAs identical to those assayed for splicing in vivo in (A) and (B) except that the 3′ splice site was inactivated by mutation, were spliced in vitro. Each pre-mRNA contained the specific silencing motifs described in the Legend to either upstream or downstream of the proximal 5′ splice site. Lane 1, Motif D; lane 2, Motif B; lane 3, Motif C; lane 4, Motif A; lane 5, no silencer motif; lane 6, Motif F; lane 7, Motif E. For lanes 8–14, specific silencer motifs were inserted either upstream or downstream of the distal 5′ splice site as appropriate such that each pre-mRNA contained identical silencer motifs at the same position relative to both 5′ splice sites. Lane 8, Motifs D; lane 9, Motifs B; lane 10, Motifs C; lane 11, Motifs A; lane 12, no silencers; lane 13, Motifs F; lane 14, Motifs E.
(F) In vitro splicing of pre-mRNAs lacking the distal 5′ splice site in the presence or absence of silencing elements. The same pre-mRNAs as in (E), lanes 1–7, lacking the distal 5′ splice site with a wild type 3′ splice site and with the specific silencer elements present upstream or downstream of the remaining 5′ splice site were spliced in vitro. Lane 1, Motif D; lane 2, Motif B; lane 3, Motif A; lane 5, no silencer; lane 6, Motif F; lane 7, Motif E.

(A) GST-hnRNP A1 pull down of U1 snRNA only in the presence of silencer Motif E. Unlabeled RNA transcripts comprised of regions 45 to +45 relative to the 5′ splice site either lacking a silencing motif (lanes 2, 3) or containing silencing Motif E (lanes 4, 5) were incubated in HeLa cell nuclear extract (lanes 2–7) supplemented with 500 ng GST-hnRNP A1 fusion protein () as indicated (lanes 2–6) either in the absence (lanes 2, 4, 6, 7, 8) or presence (lanes 3, 5) of a 2′Ome oligonucleotide complementary to the 5′ end of U1 snRNA. In lanes 6, 7, 8, no RNA was added. Following incubation, reactions were diluted then bound and eluted from glutathione beads. The eluates were deproteinized and primer extension was performed () with an oligonucleotide complementary to bases 62 to 78 of U1 snRNA. Input (lane 1) denotes a primer extension reaction on RNA extracted from 25% of an aliquot of nuclear extract equivalent to that used in lanes 2–8.
(B) Exactly as in (A) except that after elution from the glutathione beads, proteins were fractionated on a denaturing polyacrylamide gel, blotted and probed with anti-U1A antibody.
(C) The same transcripts described in (A), either lacking a silencer (control, lanes 1, 2, 3, 4) or containing exonic silencer Motif E, were site specifically labeled at position −14 relative to the splice site. Transcripts were then incubated with nuclear extract (GST-hnRNP A1 (lanes 1,5) or pre-supplemented with increasing amounts of recombinant GST-hnRNP A1 proteins (lane 2,3,4 and 6,7,8) and UV cross-linked to detect interacting proteins. Proteins were selected with glutathione beads, digested with RNases, and resolved by SDS-PAGE and visualized by autoradiography (see for the total input proteins before selection). The amount of GST-hnRNP A1 added: 125ng (lanes 2,6), 250ng (lanes 3, 7), 500ng (lanes 4, 8).
(D) Aliquots of the UV cross-linking reactions (lanes 4 and 8 from ) were immunoprecipitated with either anti-U1A antiserum or normal rabbit serum. Bound complexes were then digested with RNases, passed through glutathione sepharose and eluted with reduced glutathione before fractionation by SDS-PAGE. Labeled proteins were visualized by autoradiography.