We have investigated factors that recognize the splice junctions for mRNA by means of a rapid and sensitive filter binding assay using chemically synthesized single-stranded (ss) DNA (16-21 nucleotides) that includes a splice junction sequence or using RNA transcribed from the DNA. When small nuclear RNA-protein complexes from HeLa cells or rat liver were separated by a DEAE-Sepharose column, U1 RNA-protein complex fractions showed strong binding to ss DNA including a 5' or 3' consensus splice junction sequence. This binding took place in the presence of a large excess of Escherichia coli denatured DNA or RNA, but it was significantly reduced when conserved G-T or A-G within the splice junction was altered. In contrast, the U2 RNA-protein complex fractions did not show significant binding. We also have prepared RNA carrying the splice junction sequence by in vitro transcription of double-stranded splice junction DNA, which was linked to the E. coli lac promoter. By using this RNA, preferential binding to both 5' and 3' splice junction sequences has been confirmed with the partially purified U1 RNA-protein complex fraction described above. When the U1 RNA-protein complex is highly purified, it always retains a strong binding activity for a 5' splice junction. The binding activity for a 3' splice junction is partly or mostly lost during purification. These results strongly suggest that the U1 RNA-protein complex and/or an associated factor participates in the recognition of both 5' and 3' splice junctions.
|ジャーナル||Proceedings of the National Academy of Sciences of the United States of America|
|出版ステータス||Published - 01-12-1984|
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