In vitro assembly of human H/ACA small nucleolar RNPs reveals unique features of U17 and telomerase RNAs.
The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. They usually fold into a two-domain hairpin-hinge-hairpin-tail structure, with the conserved motifs H and ACA located in the hinge and tail, respectively. Synthetic RNA transcripts and extracts from HeLa cells were used to reconstitute human U17 and other H/ACA ribonucleoproteins (RNPs) in vitro. Competition and UV cross-linking experiments showed that proteins of about 60, 29, 23, and 14 kDa interact specifically with U17 RNA. Except for U17, RNPs could be reconstituted only with full-length H/ACA snoRNAs. For U17, the 3'-terminal stem-loop followed by box ACA (U17/3'st) was sufficient to form an RNP, and U17/3'st could compete other full-length H/ACA snoRNAs for assembly. The H/ACA-like domain that constitutes the 3' moiety of human telomerase RNA (hTR), and its 3'-terminal stem-loop (hTR/3'st), also could form an RNP by binding H/ACA proteins. Hence, the 3'-terminal stem-loops of U17 and hTR have some specific features that distinguish them from other H/ACA RNAs. Antibodies that specifically recognize the human GAR1 (hGAR1) protein could immunoprecipitate H/ACA snoRNAs and hTR from HeLa cell extracts, which demonstrates that hGAR1 is a component of H/ACA snoRNPs and telomerase in vivo. Moreover, we show that in vitro-reconstituted RNPs contain hGAR1 and that binding of hGAR1 does not appear to be a prerequisite for the assembly of the other H/ACA proteins.
Dragon
F
F
Friedrich Miescher-Institut, CH-4058 Basel, Switzerland.
Pogacić
V
V
Filipowicz
W
W
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D005998:Glycine / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D020537:RNA, Small Nucleolar / Q000737:chemistry* / Q000235:genetics*; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics*; D020718:Ribonucleoproteins, Small Nucleolar / Q000378:metabolism*; D019098:Telomerase / Q000235:genetics*; D014158:Transcription, Genetic
C485802:GAR1 protein, human; C407731:GAR1 protein, mammalian; D020537:RNA, Small Nucleolar; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; D001120:Arginine; D019098:Telomerase; D005998:Glycine