Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

Abstract

Three different base paired stems form between U2 and U6 snRNA over the course of the mRNA splicing reaction (helices I, II and III). One possible function of U2/U6 helix II is to facilitate subsequent U2/U6 helix I and III interactions, which participate directly in catalysis. Using an in vitro trans-splicing assay, we investigated the function of sequences located just upstream from the branch site (BS). We find that these upstream sequences are essential for stable binding of U2 to the branch region, and for U2/U6 helix II formation, but not for initial U2/BS pairing. We also show that non-functional upstream sequences cause U2 snRNA stem-loop IIa to be exposed to dimethylsulfate modification, perhaps reflecting a U2 snRNA conformational change and/or loss of SF3b proteins. Our data suggest that initial binding of U2 snRNP to the BS region must be stabilized by an interaction with upstream sequences before U2/U6 helix II can form or U2 stem-loop IIa can participate in spliceosome assembly.

Figure 1

Substrates, crosslinks and trans-splicing assay. (A) U2 snRNA interactions with U6 snRNA and the mRNA precursor. U snRNA stem–loops are numbered conventionally (4); U2/U6 helices I, II and III are indicated as hI, hII and hIII; the branch site is denoted BS; and the exons are boxed. Partial, complete or alternative base pairing is denoted by parallel lines, loops and bulges by curved lines. U2 snRNA can in principle pair with the BS or with U6 to extend helix III. Adapted from (88). (B) trans-splicing substrates. The Adeno 5′ SS is a synthetic oligonucleotide with a 3′ terminal deoxythymidine to prevent degradation. The Adeno 3′ RNA contains the anchoring site (AS), the most probable BS (underlined; see , fig. 3), polypyrimidine tract (Py), 3′ splice site (3′ SS) and 3′ exon. (C) In vitro trans-splicing. ³²P-labeled Adeno 3′ RNA was incubated with unlabeled Adeno 5′ SS oligonucleotide in HeLa nuclear extract at 30°C for 120 min. RNA was purified and resolved by denaturing 12% PAGE. The positions of Y-branched splicing intermediate, Adeno 3′ RNA, Y-branched intron and X are shown on the right. X is an exonuclease degradation product of Adeno 3′ RNA resulting from protection in the BS region (–93). Left to right, lanes 1–4. Splicing intermediates are seen most easily on short exposure (lanes 1 and 2), ligated exons on long exposure (lanes 3 and 4).

Figure 1

Substrates, crosslinks and trans-splicing assay. (A) U2 snRNA interactions with U6 snRNA and the mRNA precursor. U snRNA stem–loops are numbered conventionally (4); U2/U6 helices I, II and III are indicated as hI, hII and hIII; the branch site is denoted BS; and the exons are boxed. Partial, complete or alternative base pairing is denoted by parallel lines, loops and bulges by curved lines. U2 snRNA can in principle pair with the BS or with U6 to extend helix III. Adapted from (88). (B) trans-splicing substrates. The Adeno 5′ SS is a synthetic oligonucleotide with a 3′ terminal deoxythymidine to prevent degradation. The Adeno 3′ RNA contains the anchoring site (AS), the most probable BS (underlined; see , fig. 3), polypyrimidine tract (Py), 3′ splice site (3′ SS) and 3′ exon. (C) In vitro trans-splicing. ³²P-labeled Adeno 3′ RNA was incubated with unlabeled Adeno 5′ SS oligonucleotide in HeLa nuclear extract at 30°C for 120 min. RNA was purified and resolved by denaturing 12% PAGE. The positions of Y-branched splicing intermediate, Adeno 3′ RNA, Y-branched intron and X are shown on the right. X is an exonuclease degradation product of Adeno 3′ RNA resulting from protection in the BS region (–93). Left to right, lanes 1–4. Splicing intermediates are seen most easily on short exposure (lanes 1 and 2), ligated exons on long exposure (lanes 3 and 4).

Figure 1

Substrates, crosslinks and trans-splicing assay. (A) U2 snRNA interactions with U6 snRNA and the mRNA precursor. U snRNA stem–loops are numbered conventionally (4); U2/U6 helices I, II and III are indicated as hI, hII and hIII; the branch site is denoted BS; and the exons are boxed. Partial, complete or alternative base pairing is denoted by parallel lines, loops and bulges by curved lines. U2 snRNA can in principle pair with the BS or with U6 to extend helix III. Adapted from (88). (B) trans-splicing substrates. The Adeno 5′ SS is a synthetic oligonucleotide with a 3′ terminal deoxythymidine to prevent degradation. The Adeno 3′ RNA contains the anchoring site (AS), the most probable BS (underlined; see , fig. 3), polypyrimidine tract (Py), 3′ splice site (3′ SS) and 3′ exon. (C) In vitro trans-splicing. ³²P-labeled Adeno 3′ RNA was incubated with unlabeled Adeno 5′ SS oligonucleotide in HeLa nuclear extract at 30°C for 120 min. RNA was purified and resolved by denaturing 12% PAGE. The positions of Y-branched splicing intermediate, Adeno 3′ RNA, Y-branched intron and X are shown on the right. X is an exonuclease degradation product of Adeno 3′ RNA resulting from protection in the BS region (–93). Left to right, lanes 1–4. Splicing intermediates are seen most easily on short exposure (lanes 1 and 2), ligated exons on long exposure (lanes 3 and 4).

Figure 2

Sequences upstream of the branch site are essential for complex A assembly and formation of U2/U6 helix II within the trans-spliceosome. (A) Upstream mutations affect assembly of the Adeno 3′ RNA substrate into complex A. Nuclear extracts were incubated with ³²P-labeled Adeno 3′ RNA, or the UP-GG and UP-GA substrates (10 ng), with (+) or without (–) unlabeled Adeno 5′ SS oligonucleotide (440 ng), under trans-splicing conditions at 30°C for 20 min (G.Ast and A.M.Weiner, submitted for publication). RNP complexes were then resolved by native 4% PAGE. The positions of the H, A and B (U2/U4/U5/U6/3′ RNA) complexes are indicated on the left. Formation of complex A was both ATP- and temperature-dependent (data not shown). Left to right, lanes 1–6. (B) Upstream mutations affect the U2/U6 helix II interaction. After incubation as in (A), reactions were crosslinked with psoralen on ice, and the deproteinized RNAs resolved by denaturing 5% PAGE. The identities of crosslinked products obtained with the Adeno 3′ RNA substrate are indicated on the left (see Fig. 4) (70). The identity of the U2/BS crosslinks obtained with the UP-GG and UP-GA substrates was confirmed by RNase H protection with a DNA oligonucleotide complementary to U2 positions 28–42; cleavage of both complexes with a DNA oligonucleotide complementary to the 3′ SS served as a positive control (Fig. 4 and data not shown). The wild-type and mutant U2/BS crosslinks differ in size because the wild-type Adeno 3′ RNA substrate has 27 additional 5′ terminal nucleotides. Left to right, lanes 1–6.

Figure 2

Sequences upstream of the branch site are essential for complex A assembly and formation of U2/U6 helix II within the trans-spliceosome. (A) Upstream mutations affect assembly of the Adeno 3′ RNA substrate into complex A. Nuclear extracts were incubated with ³²P-labeled Adeno 3′ RNA, or the UP-GG and UP-GA substrates (10 ng), with (+) or without (–) unlabeled Adeno 5′ SS oligonucleotide (440 ng), under trans-splicing conditions at 30°C for 20 min (G.Ast and A.M.Weiner, submitted for publication). RNP complexes were then resolved by native 4% PAGE. The positions of the H, A and B (U2/U4/U5/U6/3′ RNA) complexes are indicated on the left. Formation of complex A was both ATP- and temperature-dependent (data not shown). Left to right, lanes 1–6. (B) Upstream mutations affect the U2/U6 helix II interaction. After incubation as in (A), reactions were crosslinked with psoralen on ice, and the deproteinized RNAs resolved by denaturing 5% PAGE. The identities of crosslinked products obtained with the Adeno 3′ RNA substrate are indicated on the left (see Fig. 4) (70). The identity of the U2/BS crosslinks obtained with the UP-GG and UP-GA substrates was confirmed by RNase H protection with a DNA oligonucleotide complementary to U2 positions 28–42; cleavage of both complexes with a DNA oligonucleotide complementary to the 3′ SS served as a positive control (Fig. 4 and data not shown). The wild-type and mutant U2/BS crosslinks differ in size because the wild-type Adeno 3′ RNA substrate has 27 additional 5′ terminal nucleotides. Left to right, lanes 1–6.

Figure 3

The UP-GG upstream mutant cannot be rescued by additional intron sequences. (A and B) As in Figure 2A and B, but using a UP-GG derivative containing 100 additional 5′ nucleotides. The Adeno 3′ and UP-GG+100 substrates differ in size (see Materials and Methods) causing equivalent crosslinked products to have different gel mobilities, as indicated on left.

Figure 3

The UP-GG upstream mutant cannot be rescued by additional intron sequences. (A and B) As in Figure 2A and B, but using a UP-GG derivative containing 100 additional 5′ nucleotides. The Adeno 3′ and UP-GG+100 substrates differ in size (see Materials and Methods) causing equivalent crosslinked products to have different gel mobilities, as indicated on left.

Figure 4

The U2/U6/BS crosslink contains U2/U6 helix II. (A and B) Unlabeled Adeno 5′ SS and ³²P-labeled Adeno 3′ RNA were incubated in nuclear extract as in Figure 2A, and crosslinked as in Figure 2B. RNA was then purified, subjected to RNase H cleavage targeted by the indicated oligodeoxynucleotides, and the products resolved by denaturing 5% PAGE. The positions of the U2/BS and U2/U6/BS crosslinks, and the 3′ RNA substrate, are indicated. (A) Left to right, lanes 1–7. (B) Left to right, lanes 1–6.

Figure 4

The U2/U6/BS crosslink contains U2/U6 helix II. (A and B) Unlabeled Adeno 5′ SS and ³²P-labeled Adeno 3′ RNA were incubated in nuclear extract as in Figure 2A, and crosslinked as in Figure 2B. RNA was then purified, subjected to RNase H cleavage targeted by the indicated oligodeoxynucleotides, and the products resolved by denaturing 5% PAGE. The positions of the U2/BS and U2/U6/BS crosslinks, and the 3′ RNA substrate, are indicated. (A) Left to right, lanes 1–7. (B) Left to right, lanes 1–6.

Figure 5

U2 snRNP undergoes a conformational change upon binding to the mutant UP-GG substrate containing G-rich sequences upstream of the BS. (A) Primer extension assay for U2 snRNA conformational changes. Nuclear extracts were incubated with wild-type Adeno 3′ RNA or the UP-GG mutant under trans-splicing conditions for 20 min at 30°C, with or without the Adeno 5′ SS. The reactions were treated for 12 min at room temperature with DMS (2 µl of DMS in dioxane, 5% v/v). DMS modification sites were detected by primer extension using purified RNA as template and U2(79-98) as primer. Modification sites were localized by comparison to a sequencing ladder generated with the same primer (lanes 1–6). (B) The DMS reactivity of nucleotides in U2 stem–loop IIa is denoted by arrowheads, with larger arrowheads indicating greater reactivity. For reference, the alternative U2 conformation (39,49) is shown schematically. (C) The U2/AS interaction, possibly involving U2 snRNP component SF3b, stabilizes the initial U2/BS interaction and allows formation of U2/U6 helix II.

Figure 5

U2 snRNP undergoes a conformational change upon binding to the mutant UP-GG substrate containing G-rich sequences upstream of the BS. (A) Primer extension assay for U2 snRNA conformational changes. Nuclear extracts were incubated with wild-type Adeno 3′ RNA or the UP-GG mutant under trans-splicing conditions for 20 min at 30°C, with or without the Adeno 5′ SS. The reactions were treated for 12 min at room temperature with DMS (2 µl of DMS in dioxane, 5% v/v). DMS modification sites were detected by primer extension using purified RNA as template and U2(79-98) as primer. Modification sites were localized by comparison to a sequencing ladder generated with the same primer (lanes 1–6). (B) The DMS reactivity of nucleotides in U2 stem–loop IIa is denoted by arrowheads, with larger arrowheads indicating greater reactivity. For reference, the alternative U2 conformation (39,49) is shown schematically. (C) The U2/AS interaction, possibly involving U2 snRNP component SF3b, stabilizes the initial U2/BS interaction and allows formation of U2/U6 helix II.

Figure 5

U2 snRNP undergoes a conformational change upon binding to the mutant UP-GG substrate containing G-rich sequences upstream of the BS. (A) Primer extension assay for U2 snRNA conformational changes. Nuclear extracts were incubated with wild-type Adeno 3′ RNA or the UP-GG mutant under trans-splicing conditions for 20 min at 30°C, with or without the Adeno 5′ SS. The reactions were treated for 12 min at room temperature with DMS (2 µl of DMS in dioxane, 5% v/v). DMS modification sites were detected by primer extension using purified RNA as template and U2(79-98) as primer. Modification sites were localized by comparison to a sequencing ladder generated with the same primer (lanes 1–6). (B) The DMS reactivity of nucleotides in U2 stem–loop IIa is denoted by arrowheads, with larger arrowheads indicating greater reactivity. For reference, the alternative U2 conformation (39,49) is shown schematically. (C) The U2/AS interaction, possibly involving U2 snRNP component SF3b, stabilizes the initial U2/BS interaction and allows formation of U2/U6 helix II.