Studies on the biogenesis of two nucleolar entities: H /ACA RNPs and the nucleolar channel system

Abstract

H/ACA ribonucleoproteins are involved in ribosome biogenesis and telomere maintenance. To investigate the biogenesis of H/ACA RNPs, which consist of four proteins NAP57, NHP2, NOP10 and GAR1 and one H/ACA RNA, and to dissect the role of the putative assembly factor NAF l, we established a cell line to visualize H/ACA RNA transcription in single human cells using fluorescent probes. We show that NAF1 and all of the core proteins, except GAR1, are recruited to the H/ACA RNA transcription site, indicating co-transcriptional assembly. NAF1 shuttles between the nucleus and cytoplasm and is absent from nucleoli and Cajal bodies, the sites where mature H/ACA RNPs function. Normal levels of both NAF1 and NAP57 are essential for H/ACA RNP biogenesis. These results, together with biochemical data, support the following spatial and temporal sequence for H/ACA RNP assembly: NAF1 binds newly synthesized NAP57 in the cytoplasm, possibly along with NHP2 and NOP10, and escorts it to the nascent H/ACA RNA in the nucleoplasm. Later, NAF1 is replaced by GAR1 to yield mature particles that function in nucleoli and Cajal bodies, where they interact with Nopp140.;Interestingly, exogenous expression of the characteristic repeat domain of Nopp140 induces intranuclear stacks of endoplasmic reticulum (ER), termed R-rings that closely resemble the nucleolar channel system (NCS), a human endometrial organelle implicated in blastocyst implantation. Using histochemical localization of the ER marker enzyme glucose-6-phosphatase, we show that the NCS, like the R-rings, is composed of ER. R-ring formation, and possibly, that of the NCS, is initiated by a calcium-mediated interaction of Nopp140 with the inner nuclear membrane. We validate an anti-nucleoporin antibody as a reliable marker to detect the NCS using light microscopy. Analysis of the NCS by light microscopy shows that NCSs are about 10 times as abundant as originally thought. The NCS is present in a tight window during the secretory phase. Taken together, our data provide the first molecular characterization of the NCS and support a role for the nucleolus in human reproduction. Our findings provide an experimental tool for the investigation of the NCS as a marker for endometrial receptivity.