The abundance of the spliceosomal snRNPs is not limiting the splicing of U12-type introns.
The rate of excision of U12-type introns has been reported to be slower than that of U2-type introns, suggesting a rate-limiting bottleneck that could down-regulate genes containing U12-type introns. The mechanistic reasons for this slower rate of intron excision are not known, but lower abundance of the U12-type snRNPs and slower rate of assembly or catalytic activity have been suggested. To investigate snRNP abundance we concentrated on the U4atac snRNA, which is the least abundant of the U12-type snRNAs and is limiting the formation of U4atac/U6atac complex. We identified mouse NIH-3T3 cell line isolates in which the level of both U4atac snRNA and U4atac/U6atac complexes is reduced to 10%-20% of the normal level. We used these cell lines to investigate splicing efficiency by transient transfection of a reporter gene containing a U12-type intron and by quantitative PCR analysis of endogenous genes. The splicing of the reporter U12-type intron was very inefficient, but the activity could be restored by overexpression of U4atac snRNA. Using these U4atac-deficient NIH-3T3 cells, we confirmed the results of previous studies showing that U12-type introns of endogenous genes are, indeed, excised more slowly than U2-type introns, but we found that the rate did not differ from that measured in cells displaying normal levels of U4atac snRNA. Thus our results suggest that the cellular abundance of the snRNPs does not limit U12-type intron splicing under normal conditions.
Pessa
Heli K J
HK
Institute of Biotechnology, Program on Developmental Biology, PL56 (Viikinkaari 9), 00014 University of Helsinki, Finland.
Ruokolainen
Annukka
A
Frilander
Mikko J
MJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D007438:Introns; D051379:Mice; D041681:NIH 3T3 Cells; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000235:genetics* / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*
D012342:RNA, Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C096986:U12 small nuclear RNA; C405482:U4 small nuclear RNA