Diffusion-based Transport of Nascent Ribosomes in the Nucleus

Abstract

Although the complex process of ribosome assembly in the nucleolus is beginning to be understood, little is known about how the ribosomal subunits move from the nucleolus to the nuclear membrane for transport to the cytoplasm. We show here that large ribosomal subunits move out from the nucleolus and into the nucleoplasm in all directions, with no evidence of concentrated movement along directed paths. Mobility was slowed compared with that expected in aqueous solution in a manner consistent with anomalous diffusion. Once nucleoplasmic, the subunits moved in the same random manner and also sometimes visited another nucleolus before leaving the nucleus. INTRODUCTIONIn eukaryotes, rRNA transcription and ribosome assembly take place in the nucleolus. Nascent ribosomes then exit the nucleolus and move into the cytoplasm. Multiple ribosomal proteins assemble with rRNA in the nucleolus and may facilitate proper processing of the pre-rRNA primary transcript (Fatica and Tollervey, 2002). Additionally, a significant number of nonribosomal proteins bind to these formative ribosomes in the nucleolus and also in the nucleoplasm after the nascent ribosomal subunits leave the nucleolus Kuersten et al., 2001;Nissan et al., 2002). The binding of particular proteins in a prescribed order is probably necessary for nucleocytoplasmic transport of the processed ribosomal subunits (e.g., Milkereit et al., 2001). The nuclear export of both the large and small ribosomal subunits has been shown to be dependent, at least indirectly, on the Ran-GTPase cycle and the exportin CRM1, and it is likely that CRM1-mediated export of 60S subunits requires the adaptor protein NMD3 (Moy and Silver, 1999;Ho et al., 2000;Gadal et al., 2001;Thomas and Kutay, 2003;Trotta et al., 2003). In situ hybridization experiments in fission yeast have indicated that rRNA may accumulate along short tracks when near the nuclear pores but nonetheless appears to exit from all the pores, not just those near the nucleolus (Léger-Silvestre et al., 1999). In situ hybridization studies in mammalian cells have primarily addressed the distribution of rRNA within the nucleolus (Huang, 2002) rather than the routes of extranucleolar rRNA traffic, because although high signal representing rRNA is routinely detected in both the nucleolus and the cytoplasm, nucleoplasmic signal which might represent ribosomes moving to the nuclear periphery is not easily detectable by in situ hybridization (PuvionDutilleul et al., 1991;Lazdins et al., 1997; J.C.R. Politz, unpublished observations). Therefore, very little is known about the spatial pattern or mechanism of rRNA movement from the nucleolus into the nucleoplasm and then to the nuclear pores for export (Cullen, 2000;Kuersten et al., 2001;Lei and Silver, 2002), although all three classes of eukaryotic RNAs, rRNA, mRNA, and pol III transcripts have been shown to exit from all the nuclear pores (Dworetzky and Feldherr, 1988;Pante et al., 1997;Mattaj and Englmeier, 1998).In previous studies in live cells (Politz et al., 1998, we investigat...

A mRNA and Cognate MicroRNAs Localize in the Nucleolus

“…33,34 28 S-1: 5 0 -GTTACCGGCACTGGACGCCTCGCGGCG CCCATC-3 0 28 S-2: 5 0 -ATGGCTCTCCGCACCGGACCCCGGTCCC-GACTC-3 0 SRP-1: 5 0 -TTCCCACTACTGATCAGCACGGGAGTTT TGACCTT-3 0 SRP-2: 5 0 -GCTCCCGGGAGGTCACCATATTGATGCC-GAACTT-3 0 Cells on coverslips were fixed with 4% (v/v) formaldehyde and permeabilized with cold (4 C) acetone. In situ hybridization and RNase pre-treatment, when desired, were performed as detailed previously 13 except that after hybridization, cells were washed with 40% formamide/2£ SSC (1£ SSC D 0.15 M NaCl/ 0.015 mM sodium citrate, pH 7.0) and then 20% formamide/ 1£ SSC for 30 min.…”

“…The desired rat micro-RNA sequences were obtained from miRBase (Kozomara and Griffiths-Jones, NAR, 2011) (http://mirbase.org). Searches for targets of the 5 nucleolus-associated microRNAs and IGF2 mRNA and other mRNAs were performed with the Mirza program 34 , as refined in the University of Massachusetts Medical School Bioinformatics Core Facility and RNA Therapeutics Institute.…”

Structures and functions in the crowded nucleus: new biophysical insights

“…Diffusion of macromolecules is slowed in the nucleus ( [64,65], reviewed in [66]) probably due to collosions with chromatin and other large obstacles or to viscoelasticity, but nevertheless most macromolecules and multiprotein complexes can explore the entire nuclear volume [67]. Large particles and macromolecules show anomalous diffusion in the nucleus, a lesss-than-linear increase of mean-square displacement with time like that seen in crowded solutions [68].…”

The nucleolus: a raft adrift in the nuclear sea or the keystone in nuclear structure?

“…The nucleolus emerges from the complex mixture of proteins that associate with the rDNA, such as upstream binding factor (UBF) (23). Creation of the spatial domain of the nucleolus may result from high concentrations of binding sites in a small volume effectively causing retention of these proteins (24) by preventing movement out of the zone, as shown for ribosome movement (25). However, rapid shuttling of proteins between the nucleolus and nucleus has been observed (18, 19), suggesting that the nucleolus is a dynamic structure.…”