Intramolecular structure in yeast introns aids the early steps of in vitro spliceosome assembly.
rp5l B pre-mRNA, like many Saccharomyces cerevisiae primary transcripts, contains a secondary structure within its intron sequence. The structure is required for optimal in vivo splicing efficiency and includes two complementary regions near the 5' splice site and the branchpoint (UB1 and DB1, respectively). An intron-containing RNA was probed in vitro with RNase T1 and dimethyl sulfate (DMS), and is folded as expected. We have also examined in vitro splicing of rp5l B pre-mRNA, by analyzing the formation of splicing complexes as well as splicing products. Similar analyses were done with mutant pre-mRNAs containing shortened or lengthened UB1/DB1 base pairing regions. Our experiments indicate that the secondary structure acts at an early step of spliceosome assembly to aid the formation of U1 snRNP-containing commitment complexes. pre-mRNAs were probed with DMS in vivo and the folding takes place inside cells. The effect of the different UB1/DB1 interactions on in vivo splicing efficiency was also analyzed. The results are consistent with the idea that the intramolecular interaction takes place prior to or at the beginning of spliceosome assembly.
Charpentier
B
B
Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02254, USA.
Rosbash
M
M
D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D007438:Introns; D015335:Molecular Probes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry* / Q000378:metabolism; D012326:RNA Splicing; D018075:RNA, Complementary / Q000737:chemistry / Q000235:genetics; D012342:RNA, Small Nuclear / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D006163:Ribonuclease T1 / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D017421:Sequence Analysis; D017381:Spliceosomes / Q000378:metabolism*; D013463:Sulfuric Acid Esters / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D015335:Molecular Probes; D012322:RNA Precursors; D018075:RNA, Complementary; D012342:RNA, Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D013463:Sulfuric Acid Esters; D016914:Ribonuclease H; D006163:Ribonuclease T1; C007482:dimethyl sulfate