Dynamic association and localization of human H/ACA RNP proteins

Abstract

Mammalian H/ACA RNPs are essential for ribosome biogenesis, pre-mRNA splicing, and telomere maintenance. To form mature RNA-protein complexes, one H/ACA RNA associates with four core proteins. In the cell, this process is assisted by at least one nuclear assembly factor, NAF1. Here we report several unanticipated dynamic aspects of H/ACA RNP proteins. First, when overexpressed, NAF1 delocalizes to the cytoplasm. However, its nucleocytoplasmic shuttling properties remain unaffected. These observations demonstrate a subtle equilibrium between NAF1 expression levels and the availability of NAF1 nuclear binding sites. Second, although NAF1 is excluded from mature RNPs in nucleoli and Cajal bodies, NAF1 associates with mature H/ACA RNA in cell lysates. This association occurs post-lysis because it is observed even when NAF1 and the H/ACA RNA are expressed in separate cells. This documents a protein-RNP association in cell lysates that is absent from intact cells. Third, in similar experiments, all H/ACA core proteins, except NAP57, exchange with their exogenous counterparts, portraying an unexpected dynamic picture of H/ACA RNPs. Finally, the irreversible association of only NAP57 with H/ACA RNA and the conundrum that only NAP57 is mutated in X-linked dyskeratosis congenita (even though most core proteins are required for maintaining H/ACA RNAs) may be more than a coincidence.

H/ACA small nucleolar RNA pseudouridylation pockets bind substrate RNA to form three-way junctions that position the target U for modification

“…Together, the structures of the U65hp/S14 complex and the archaeal H/ACA RNP illustrate how this could occur. The H/ACA RNA is believed to remain stably associated with the snoRNP for multiple rounds of pseudouridylation (24), although a very recent study indicates that only Cbf5 is irreversibly associated with the H/ACA RNA (41).…”

SHQ1 is required prior to NAF1 for assembly of H/ACA small nucleolar and telomerase RNPs

“…Therefore, it may depend on those of other proteins, such as the multiple nuclear localization signals of NAP57 (Meier and Blobel 1994;Heiss et al 1999;Youssoufian et al 1999). Third, SHQ1 has to act before cotranscriptional association of NAP57 with H/ACA RNA and other core proteins because SHQ1 only binds NAP57 alone and because, once assembled with the core trimer, H/ACA RNAs will no longer exchange (Wang and Meier 2004;Kittur et al 2006). Therefore, SHQ1 may function as a NAP57 chaperone and/or licensing factor marking it ready for RNP assembly.…”

“…siRNAs were as previously described ) except for siSHQ1, which was a combination of siSHQ1.1, GCU ACGAAAAUUUGUCAAUTT; siSHQ1.2, GGAAGUAGUUGACG AUGAATT; and siSHQ1.3, GGACAGCAAAACCACUUGUTT (Ambion). Cells were maintained, treated, and transfected as we previously described Kittur et al 2006). Similarly, the inducible H/ACA RNA cell line and related analyses ) and the cell line containing integrated Lac operator arrays (Janicki et al 2004) have been described.…”

The Box H/ACA RNP Assembly Factor Naf1p Contains a Domain Homologous to Gar1p Mediating its Interaction with Cbf5p

“…It has recently been shown that overexpressed human Naf1 delocalises to the cytoplasm. 42 This might be due to a concentration effect, shifting the equilibrium between monomeric and dimeric forms of human Naf1. Naf1 most likely assembles along with Cbf5, Nop10 and Nhp2, early during H/ACA snoRNA transcription.…”