PMC

Fig. 6. Schematic illustration of the –20/24 complex deposited on mRNA as a consequence of pre-mRNA splicing. Identified proteins are indicated at arbitrarily chosen positions. The question mark symbolizes potential unidentified component(s).

Fig. 2. A splicing-dependent protection of AdML (A) and β-globin (B) mRNAs from RNase H cleavage in X.laevis oocytes. Control mRNA [(A) lanes 1–9; (B) lanes 1–7] or pre-mRNA [(A) lanes 10–18; (B) lanes 8–14] was injected into oocyte nuclei. Nuclear RNAs were collected immediately [(A) lanes 1 and 10; (B) lanes 1 and 8] or 90 min after injection [(A) lanes 2–9 and 11–18; (B) lanes 2–7 and 9–14]. Where indicated, nuclear RNA samples were incubated with individual cDNA oligos to activate endogenous RNAse H [(A) lanes 3–9 and 12–18; (B) lanes 3–7 and 10–14]. AdML and β-globin RNAs were separated by 15 and 10% denaturing PAGE, respectively. For uncleaved and cleaved samples, one and four oocyte equivalents of RNA, respectively, from pools of 10 oocytes were loaded per lane. Splicing substrates and products are indicated on the right. Black dots indicate specific mRNA cleavage fragments that are markedly reduced with spliced mRNA compared with control mRNA.

Fig. 4. Complete RNase digestion of singly labeled RNAs. (A) Upper panel: splicing time courses for AdML pre-mRNAs containing a single labeled phosphate (star) positioned 20 nt upstream of the 5′ splice site. Splicing substrates, intermediates and products are indicated on the left. RNAs were analyzed as in Figure . Lower panel: complete RNase A (left) or RNase A + T₁ (right) digestion of RNAs at each time point yielded 19 and 8 nt protected fragments (arrows). Protected fragments were separated by 20% denaturing PAGE. (B) Same as (A) with AdML pre-mRNAs containing a single labeled phosphate positioned 24 nt upstream of the 5′ splice site. (C) Upper panels: AdML pre-mRNA (lanes 1, 3 and 4) or control mRNA (lane 2) containing a single labeled phosphate positioned 20 nt upstream of the 5′ splice site was incubated under splicing conditions for the time indicated and separated by 20% (lanes 1 and 2) or 15% (lanes 3 and 4) denaturing PAGE. Lower panels: aliquots of each splicing reaction were subjected to complete RNase A + T₁ digestion with (lane 8) or without (lanes 5–7) prior SDS treatment. Protected fragments were separated as in (A).

Fig. 5. Immunoprecipitation of the protected fragments from spliced mRNA and molecular mass determination of the –20/24 complex. (A) Co-IP of 19 nt (upper panel) or 8 nt (middle panel) protected fragments generated by RNase A or RNase A + T₁ digestion, respectively, of spliced AdML mRNA containing a single labeled phosphate at position –20. Antibodies are as indicated. Lower panel: sequence of AdML mRNA exon 1 between position –31 and the exon–exon junction. Arrows and bold letters indicate –20 and –24 positions and the likely binding site of hnRNP A1, respectively. (B) Superose 6 gel filtration profile of the complex associated with the RNase A-resistant 19 nt fragment of spliced mRNA labeled at position –20 (top). The amount of labeled fragment in each fraction (bottom) was determined by PhosphorImaging (PI units). Elution positions of molecular weight standards and the void volume (v.v.) are indicated.

Fig. 3. Identification of proteins associated with the exon–exon junction region by RNA co-IP. (A) Scheme of co-IP strategy. AdML pre-mRNA was spliced for 2 h, and mRNP was separated from spliceosomes by glycerol gradient fractionation. Spliced mRNA was subsequently cleaved using RNase H and two oligos (–) to produce three fragments: 5′ (white), m (black) and 3′ (shaded). The fragment ‘m’ contains the –20/24 protected region (gray oval). This reaction mixture was then subjected to IP with different antibodies. Control mRNA was analyzed in parallel reactions except that it was not fractionated on a glycerol gradient. (B) Representative RNA co-IP experiments performed with antibodies against Y14, DEK, TAP-C, RNPS1, REF, hnRNPA1 (A1) and ³mCAP. Lanes 1, 6, 11, 14 and 17 correspond to one-fifteenth of input RNA. Lane 18 corresponds to co-IP of spliceosome-associated RNAs. AdML RNAs were analyzed as indicated in Figure . Structures of splicing substrates, intermediates and products as well as mRNA fragments are diagrammed. (C) Co-IP efficiencies of mRNA fragments (5′, m and 3′) and spliceosomes (S, taken as splicing intermediates) with antibodies against the species indicated. Upper and lower rows of histograms correspond to fragments of spliced and control mRNAs, respectively. Co-IP efficiencies are averages of 2–5 independent experiments.

Fig. 1. A splicing-dependent protection of mRNAs from targeted RNase H cleavage. (A) Uniformly labeled TPI control mRNA (lanes 1–15) or TPI pre-mRNA (lanes 16–30) was incubated under splicing conditions in HeLa cell nuclear extract for 0 (lanes 1 and 16), 45 (lanes 2–15) or 90 min (lanes 17–30). Aliquots of the 45 and 90 min reactions were further incubated with the cDNA oligos indicated (short bars underneath the mRNA schematic at the top; lanes 3–15 and 18–30). RNAs were then separated by 14% denaturing PAGE. Each oligo was named (e.g. –96, –24, 0, 48 in top panels) according to its center position relative to the exon–exon junction, which was defined as 0. Splicing substrates, intermediates and products are indicated on the right. Black dots indicate specific mRNA cleavage fragments that are markedly reduced with spliced mRNA compared with control mRNA. Similar RNase H analyses were performed with control and spliced (B) AdML mRNAs, (C) β-globin mRNAs and (D) PIP85.B (left) and PIP85.B+10 (right) mRNAs. The triangle indicates the same cDNA oligo, which was alternatively centered at –12 and –24 for PIP85.B and PIP85.B+10 mRNAs, respectively. Likewise, the asterisk indicates a single cDNA oligo centered at –24 and –36 for each mRNA, respectively. AdML, β-globin and PIP85.B RNAs were separated by 15, 8 and 15% denaturing PAGE, respectively.