An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway.
Previous studies have identified five distinct mammalian splicing complexes that assemble on pre-mRNA in vitro. Of these complexes, which include H, E, A, B, and C, only the B and C complexes have been isolated and shown directly to be functional intermediates in the splicing pathway. In this report we carried out a systematic analysis of the temporal and functional relationships among the H, E, A, and B complexes. Using gel filtration to isolate each complex, we show that H complex, which consists primarily of hnRNP proteins, assembles first in either the presence or absence of ATP. Subsequently, E complex, which contains stably bound U1 snRNP, is detected in reactions lacking ATP, whereas A complex, which contains stably bound U1 and U2 snRNPs, is detected in reactions containing ATP. We show that E complex can be chased into A and B complexes and that A complex can be chased into B complex. Both E and A complexes can also be chased into spliced products. In contrast, H complex cannot be chased into A or B complexes or spliced products under the same conditions. We conclude that in addition to the two spliceosome complexes (B and C), two distinct pre-splicesome complexes (E and A) are functional intermediates in the splicing pathway. Comparison of the efficiency of splicesome assembly on different pre-mRNAs has revealed dramatic differences. We show that these differences are first apparent at the time of E complex assembly. Thus, we conclude that E complex commits pre-mRNA to the splicing pathway and that this step is critical in determining the efficiency of mammalian spliceosome assembly.
Michaud
S
S
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115.
Reed
R
R
D000255:Adenosine Triphosphate / Q000502:physiology*; D000260:Adenoviruses, Human / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D046911:Macromolecular Substances; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear; D012441:Saccharomyces cerevisiae / Q000235:genetics
D046911:Macromolecular Substances; D012322:RNA Precursors; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D000255:Adenosine Triphosphate