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.

Keywords

• H/ACA RNP
• snoRNP
• dyskeratosis congenita
• nucleocytoplasmic shuttling
• RNP dynamics
• telomerase
• nuclear localization signal