Measurement and Analysis of Yeast Pre-mRNA Sequence Contribution to Splicing Efficiency

B. C. Rymond; C. Pikielny; B. Seraphin; P. Legrain; M. Rosbash

doi:10.1016/0076-6879(90)81116-C

Measurement and Analysis of Yeast Pre-mRNA Sequence Contribution to Splicing Efficiency

B. C. Rymond, C. Pikielny, B. Seraphin, P. Legrain, M. Rosbash

Biology

Research output: Contribution to journal › Article › peer-review

12 Scopus citations

Abstract

This chapter examines yeast pre-mRNA sequence contribution to splicing efficiency. A primer complementary to intron sequences, RB27 can be used to determine the relative abundance of premRNA and lariat molecules. On addition of a small, splicing-competent substrate and ATP to a yeast whole cell extract, the time-dependent appearance of three splicing complexes could be visualized. Presumably the carrier RNA eliminates nonspecific and adventitious binding of proteins to the spliceosome complexes, thereby increasing the electrophoretic mobility of the complexes and improving resolution. The modified pre-mRNA is then added to an in vitro assembly reaction, and the splicing complexes are resolved on native polyacrylamide gels. The expectation is that the assembly process should be sensitive to chemical modification at nucleotide positions required for the formation of spliceosomes. If a particular modification is extremely deleterious to assembly it should be absent from the complex-derived RNA. The DNA oligonucleotides should be size-selected by HPLC chromatography or polyacrylamide gel electrophoresis prior to labeling. This precaution removes the shorter, incompletely synthesized molecules that compete for labeling and may lead to background problems during primer extension.

Original language	English
Pages (from-to)	122-147
Number of pages	26
Journal	Methods in Enzymology
Volume	181
Issue number	C
DOIs	https://doi.org/10.1016/0076-6879(90)81116-C
State	Published - Jan 1 1990

Bibliographical note

Funding Information:
We would hke to thank former members of our laboratory, Jose Rodriguez, Leo Kretzner, and John Teem, for significantc ontributions to this work and current members of our laboratory, Nadja Abovich, and HildurC olotfor assistance in preparing the manuscript. This work was supported by National Institutes of Health Grant GM23549 to M.R., an NIH postdoctoral fellowshipt o B.R.C., and a Fogarty InternationalF ellowship to P.L.

ASJC Scopus subject areas

Biochemistry
Molecular Biology

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title = "Measurement and Analysis of Yeast Pre-mRNA Sequence Contribution to Splicing Efficiency",

abstract = "This chapter examines yeast pre-mRNA sequence contribution to splicing efficiency. A primer complementary to intron sequences, RB27 can be used to determine the relative abundance of premRNA and lariat molecules. On addition of a small, splicing-competent substrate and ATP to a yeast whole cell extract, the time-dependent appearance of three splicing complexes could be visualized. Presumably the carrier RNA eliminates nonspecific and adventitious binding of proteins to the spliceosome complexes, thereby increasing the electrophoretic mobility of the complexes and improving resolution. The modified pre-mRNA is then added to an in vitro assembly reaction, and the splicing complexes are resolved on native polyacrylamide gels. The expectation is that the assembly process should be sensitive to chemical modification at nucleotide positions required for the formation of spliceosomes. If a particular modification is extremely deleterious to assembly it should be absent from the complex-derived RNA. The DNA oligonucleotides should be size-selected by HPLC chromatography or polyacrylamide gel electrophoresis prior to labeling. This precaution removes the shorter, incompletely synthesized molecules that compete for labeling and may lead to background problems during primer extension.",

author = "Rymond, {B. C.} and C. Pikielny and B. Seraphin and P. Legrain and M. Rosbash",

note = "Funding Information: We would hke to thank former members of our laboratory, Jose Rodriguez, Leo Kretzner, and John Teem, for significantc ontributions to this work and current members of our laboratory, Nadja Abovich, and HildurC olotfor assistance in preparing the manuscript. This work was supported by National Institutes of Health Grant GM23549 to M.R., an NIH postdoctoral fellowshipt o B.R.C., and a Fogarty InternationalF ellowship to P.L.",

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AU - Pikielny, C.

AU - Seraphin, B.

AU - Legrain, P.

AU - Rosbash, M.

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PY - 1990/1/1

Y1 - 1990/1/1

N2 - This chapter examines yeast pre-mRNA sequence contribution to splicing efficiency. A primer complementary to intron sequences, RB27 can be used to determine the relative abundance of premRNA and lariat molecules. On addition of a small, splicing-competent substrate and ATP to a yeast whole cell extract, the time-dependent appearance of three splicing complexes could be visualized. Presumably the carrier RNA eliminates nonspecific and adventitious binding of proteins to the spliceosome complexes, thereby increasing the electrophoretic mobility of the complexes and improving resolution. The modified pre-mRNA is then added to an in vitro assembly reaction, and the splicing complexes are resolved on native polyacrylamide gels. The expectation is that the assembly process should be sensitive to chemical modification at nucleotide positions required for the formation of spliceosomes. If a particular modification is extremely deleterious to assembly it should be absent from the complex-derived RNA. The DNA oligonucleotides should be size-selected by HPLC chromatography or polyacrylamide gel electrophoresis prior to labeling. This precaution removes the shorter, incompletely synthesized molecules that compete for labeling and may lead to background problems during primer extension.

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Measurement and Analysis of Yeast Pre-mRNA Sequence Contribution to Splicing Efficiency

Abstract

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ASJC Scopus subject areas

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