Splicing-dependent and -independent modes of assembly for intron-encoded box C/D snoRNPs in mammalian cells.
In mammalian cells, all small nucleolar RNAs (snoRNAs) that guide rRNA modification are encoded within the introns of host genes. An optimal position about 70 nts upstream of the 3' splice site of the host intron is critical for efficient expression of box C/D snoRNAs in vivo, suggesting synergy with splicing. Here, we have used a coupled in vitro splicing-snoRNA processing system to demonstrate that assembly of box C/D snoRNP proteins is the step affected by snoRNA location, and that active splicing is essential for snoRNP assembly. Splicing blockage experiments further reveal that snoRNP proteins bind specifically at the spliceosomal C1 complex stage. In contrast, splicing-independent snoRNP assembly can occur in vitro on snoRNAs that possess stable external stems. In vivo analyses confirm that a stable stem can compensate for the unusual position of those few box C/D snoRNAs located far from the 3' splice site of their host intron.
Hirose
Tetsuro
T
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06536, USA.
Shu
Mei-Di
MD
Steitz
Joan A
JA
D000818:Animals; D002460:Cell Line; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics*; D008322:Mammals / Q000235:genetics*; D015394:Molecular Structure; D011485:Protein Binding / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012342:RNA, Small Nuclear / Q000235:genetics*; D017411:Ribonucleoproteins, Small Nuclear / Q000096:biosynthesis* / Q000235:genetics; D017381:Spliceosomes / Q000235:genetics
D002868:Chromosomal Proteins, Non-Histone; D022821:RNA Splice Sites; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C047458:fibrillarin