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-tai l structure, with the conserved motifs H and ACA located in the hinge and t ail, respectively. Synthetic RNA transcripts and extracts from HeLa cells w ere used to reconstitute human U17 and other H/ACA ribonucleoproteins (RNPs ) in vitro. Competition and UV crosslinking experiments showed that protein s of about 60, 29, 23, and 14 kDa interact specifically with U17 RNA. Excep t 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 suff icient to form an RNP, and U17/3'st could compete other full-length H/ACA s noRNAs for assembly. The H/ACA-like domain that constitutes the 3' moiety o f human telomerase RNA (hTR), and its 3'-terminal stem-loop (hTR/3'st), als o 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 (h GAR1) 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 con tain hGAR1 and that binding of hGAR1 does not appear to be a prerequisite f or the assembly of the other H/ACA proteins.