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A BBP-Mud2p heterodimer mediates branchpoint recognition and influences splicing substrate abundance in budding yeast.

Wang Q, Zhang L, Lynn B, Rymond BC - Nucleic Acids Res. (2008)

Bottom Line: Unexpectedly, rather than exacerbating bbpDelta56, the mud2::KAN mutation partially suppresses a pre-mRNA accumulation defect observed with bbpDelta56.We propose that a BBP-Mud2p heterodimer binds as a unit to the branchpoint in vivo and serves as a target for the Sub2p-DExD/H-box ATPase and for other splicing factors during spliceosome assembly.In addition, our results suggest the possibility that the Mud2p may enhance the turnover of pre-mRNA with impaired BBP-branchpoint association.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Department of Chemistry, University of Kentucky, Lexington, KY 40506-0225, USA.

ABSTRACT
The 3' end of mammalian introns is marked by the branchpoint binding protein, SF1, and the U2AF65-U2AF35 heterodimer bound at an adjacent sequence. Baker's yeast has equivalent proteins, branchpoint binding protein (BBP) (SF1) and Mud2p (U2AF65), but lacks an obvious U2AF35 homolog, leaving open the question of whether another protein substitutes during spliceosome assembly. Gel filtration, affinity selection and mass spectrometry were used to show that rather than a U2AF65/U2AF35-like heterodimer, Mud2p forms a complex with BBP without a third (U2AF35-like) factor. Using mutants of MUD2 and BBP, we show that the BBP-Mud2p complex bridges partner-specific Prp39p, Mer1p, Clf1p and Smy2p two-hybrid interactions. In addition to inhibiting Mud2p association, the bbpDelta56 mutation impairs splicing, enhances pre-mRNA release from the nucleus, and similar to a mud2::KAN knockout, suppresses a lethal sub2::KAN mutation. Unexpectedly, rather than exacerbating bbpDelta56, the mud2::KAN mutation partially suppresses a pre-mRNA accumulation defect observed with bbpDelta56. We propose that a BBP-Mud2p heterodimer binds as a unit to the branchpoint in vivo and serves as a target for the Sub2p-DExD/H-box ATPase and for other splicing factors during spliceosome assembly. In addition, our results suggest the possibility that the Mud2p may enhance the turnover of pre-mRNA with impaired BBP-branchpoint association.

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Splicing inhibition by the mud2::KAN and bbΔ56 mutations. (A) Northern analysis of RNA extracted from wild-type yeast (WT), the ts splicing mutant, prp38-1, and yeast with the bbpΔ56 and mud2::KAN single mutations and with the combined bbpΔ56 plus mud2::KAN double mutant background. RNA harvested from yeast grown continuously at 23°C (−) and after a 2 h shift to 37°C (+) was hybridized with radiolabeled probes to detect the intronless ADE3 mRNA and the RPS17A pre-mRNA and mRNA. The relative abundance of mRNA and pre-mRNA (M/P ratio) is presented below the image. (B) Northern analysis as in panel A with added samples smy2::KAN and smy2::KAN combined with bbpΔ56 or mud2::KAN. (C) Primer extension analysis of RNA isolated from the indicated yeast backgrounds that express RPS17A reporter gene constructs with the wild-type intron (WT), branchpoint mutants (HZ8, HZ3, HZ10) and 5′ splice site mutant (HZ12). (D) Primer extension of RNA from the bbpΔ56, mud2::KAN double mutant before (left) and after (right) add back of MUD2.
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Figure 5: Splicing inhibition by the mud2::KAN and bbΔ56 mutations. (A) Northern analysis of RNA extracted from wild-type yeast (WT), the ts splicing mutant, prp38-1, and yeast with the bbpΔ56 and mud2::KAN single mutations and with the combined bbpΔ56 plus mud2::KAN double mutant background. RNA harvested from yeast grown continuously at 23°C (−) and after a 2 h shift to 37°C (+) was hybridized with radiolabeled probes to detect the intronless ADE3 mRNA and the RPS17A pre-mRNA and mRNA. The relative abundance of mRNA and pre-mRNA (M/P ratio) is presented below the image. (B) Northern analysis as in panel A with added samples smy2::KAN and smy2::KAN combined with bbpΔ56 or mud2::KAN. (C) Primer extension analysis of RNA isolated from the indicated yeast backgrounds that express RPS17A reporter gene constructs with the wild-type intron (WT), branchpoint mutants (HZ8, HZ3, HZ10) and 5′ splice site mutant (HZ12). (D) Primer extension of RNA from the bbpΔ56, mud2::KAN double mutant before (left) and after (right) add back of MUD2.

Mentions: Since BBP and Mud2p are believed to act cooperatively in branchpoint binding, yeast with the mud2::KAN or bbpΔ56 mutations might be expected to show similar growth characteristics if the only consequence of bbpΔ56 was the loss of Mud2p interaction. However, the bbpΔ56 mutant and the mud2::KAN, bbpΔ56 double mutant grow more slowly than the mud2::KAN strain, suggesting that some additional function of BBP is impaired by the bbpΔ56 lesion (Figure 3B). When pre-mRNA is assayed at 23°C, the mRNA to pre-mRNA ratio (a measure of splicing efficiency) of the RPS17A transcript appears almost equivalent in the wild-type, single and double mutant backgrounds (Figure 5A; the intronless ADE3 mRNA is presented as a sample loading control). At 37°C, splicing is more impaired in the mud2::KAN mutant when compared to the wild-type strain although it remains better than what is observed after inactivation of the essential U4/U6.U5 tri-snRNP protein, Prp38-1p (30). The bbpΔ56 mutant has a somewhat stronger splicing defect than that seen with mud2p::KAN mutant at 37°C, consistent with the lower vitality of this strain. We see no exacerbation of this splicing defect in the mud2::KAN, bbpΔ56 double mutant background (Figure 5A and see below), however. While there is some experimental variability in splicing efficiency with temperature shift, we see no clear splicing defect with the smy2::KAN knockout mutant and no reproducible exacerbation of the mud2::KAN or bbpΔ56 splicing defects by this mutation (Figure 5B).Figure 5.


A BBP-Mud2p heterodimer mediates branchpoint recognition and influences splicing substrate abundance in budding yeast.

Wang Q, Zhang L, Lynn B, Rymond BC - Nucleic Acids Res. (2008)

Splicing inhibition by the mud2::KAN and bbΔ56 mutations. (A) Northern analysis of RNA extracted from wild-type yeast (WT), the ts splicing mutant, prp38-1, and yeast with the bbpΔ56 and mud2::KAN single mutations and with the combined bbpΔ56 plus mud2::KAN double mutant background. RNA harvested from yeast grown continuously at 23°C (−) and after a 2 h shift to 37°C (+) was hybridized with radiolabeled probes to detect the intronless ADE3 mRNA and the RPS17A pre-mRNA and mRNA. The relative abundance of mRNA and pre-mRNA (M/P ratio) is presented below the image. (B) Northern analysis as in panel A with added samples smy2::KAN and smy2::KAN combined with bbpΔ56 or mud2::KAN. (C) Primer extension analysis of RNA isolated from the indicated yeast backgrounds that express RPS17A reporter gene constructs with the wild-type intron (WT), branchpoint mutants (HZ8, HZ3, HZ10) and 5′ splice site mutant (HZ12). (D) Primer extension of RNA from the bbpΔ56, mud2::KAN double mutant before (left) and after (right) add back of MUD2.
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Figure 5: Splicing inhibition by the mud2::KAN and bbΔ56 mutations. (A) Northern analysis of RNA extracted from wild-type yeast (WT), the ts splicing mutant, prp38-1, and yeast with the bbpΔ56 and mud2::KAN single mutations and with the combined bbpΔ56 plus mud2::KAN double mutant background. RNA harvested from yeast grown continuously at 23°C (−) and after a 2 h shift to 37°C (+) was hybridized with radiolabeled probes to detect the intronless ADE3 mRNA and the RPS17A pre-mRNA and mRNA. The relative abundance of mRNA and pre-mRNA (M/P ratio) is presented below the image. (B) Northern analysis as in panel A with added samples smy2::KAN and smy2::KAN combined with bbpΔ56 or mud2::KAN. (C) Primer extension analysis of RNA isolated from the indicated yeast backgrounds that express RPS17A reporter gene constructs with the wild-type intron (WT), branchpoint mutants (HZ8, HZ3, HZ10) and 5′ splice site mutant (HZ12). (D) Primer extension of RNA from the bbpΔ56, mud2::KAN double mutant before (left) and after (right) add back of MUD2.
Mentions: Since BBP and Mud2p are believed to act cooperatively in branchpoint binding, yeast with the mud2::KAN or bbpΔ56 mutations might be expected to show similar growth characteristics if the only consequence of bbpΔ56 was the loss of Mud2p interaction. However, the bbpΔ56 mutant and the mud2::KAN, bbpΔ56 double mutant grow more slowly than the mud2::KAN strain, suggesting that some additional function of BBP is impaired by the bbpΔ56 lesion (Figure 3B). When pre-mRNA is assayed at 23°C, the mRNA to pre-mRNA ratio (a measure of splicing efficiency) of the RPS17A transcript appears almost equivalent in the wild-type, single and double mutant backgrounds (Figure 5A; the intronless ADE3 mRNA is presented as a sample loading control). At 37°C, splicing is more impaired in the mud2::KAN mutant when compared to the wild-type strain although it remains better than what is observed after inactivation of the essential U4/U6.U5 tri-snRNP protein, Prp38-1p (30). The bbpΔ56 mutant has a somewhat stronger splicing defect than that seen with mud2p::KAN mutant at 37°C, consistent with the lower vitality of this strain. We see no exacerbation of this splicing defect in the mud2::KAN, bbpΔ56 double mutant background (Figure 5A and see below), however. While there is some experimental variability in splicing efficiency with temperature shift, we see no clear splicing defect with the smy2::KAN knockout mutant and no reproducible exacerbation of the mud2::KAN or bbpΔ56 splicing defects by this mutation (Figure 5B).Figure 5.

Bottom Line: Unexpectedly, rather than exacerbating bbpDelta56, the mud2::KAN mutation partially suppresses a pre-mRNA accumulation defect observed with bbpDelta56.We propose that a BBP-Mud2p heterodimer binds as a unit to the branchpoint in vivo and serves as a target for the Sub2p-DExD/H-box ATPase and for other splicing factors during spliceosome assembly.In addition, our results suggest the possibility that the Mud2p may enhance the turnover of pre-mRNA with impaired BBP-branchpoint association.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Department of Chemistry, University of Kentucky, Lexington, KY 40506-0225, USA.

ABSTRACT
The 3' end of mammalian introns is marked by the branchpoint binding protein, SF1, and the U2AF65-U2AF35 heterodimer bound at an adjacent sequence. Baker's yeast has equivalent proteins, branchpoint binding protein (BBP) (SF1) and Mud2p (U2AF65), but lacks an obvious U2AF35 homolog, leaving open the question of whether another protein substitutes during spliceosome assembly. Gel filtration, affinity selection and mass spectrometry were used to show that rather than a U2AF65/U2AF35-like heterodimer, Mud2p forms a complex with BBP without a third (U2AF35-like) factor. Using mutants of MUD2 and BBP, we show that the BBP-Mud2p complex bridges partner-specific Prp39p, Mer1p, Clf1p and Smy2p two-hybrid interactions. In addition to inhibiting Mud2p association, the bbpDelta56 mutation impairs splicing, enhances pre-mRNA release from the nucleus, and similar to a mud2::KAN knockout, suppresses a lethal sub2::KAN mutation. Unexpectedly, rather than exacerbating bbpDelta56, the mud2::KAN mutation partially suppresses a pre-mRNA accumulation defect observed with bbpDelta56. We propose that a BBP-Mud2p heterodimer binds as a unit to the branchpoint in vivo and serves as a target for the Sub2p-DExD/H-box ATPase and for other splicing factors during spliceosome assembly. In addition, our results suggest the possibility that the Mud2p may enhance the turnover of pre-mRNA with impaired BBP-branchpoint association.

Show MeSH