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* Laboratorio di Biologia Cellulare e Neurobiologia, Dipartimento di Biologia Animale, Università di Pavia, Italia; and
Istituto di Genetica Molecolare del CNR, Sezione di Istochimica e Citometria, Università di Pavia, Italia
1Correspondence: Laboratorio di Biologia Cellulare e Neurobiologia, Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 10, Pavia 27100, Italy. E-mail: marcobig{at}unipv.it
SPECIFIC AIMS
The purposes of this paper are to 1) follow the movement of a ribosomal protein entering the nucleus, reaching the nucleolus and being exported as a part of the small ribosomal subunit; and 2) investigate the nature of the preribosome export modality in order to verify the existence of an active, energy-consuming mechanism.
PRINCIPAL FINDINGS
The present paper describes the movements of a ribosomal protein, S6, from the cytoplasm to the nucleolus and of the same protein within the small ribosomal subunit (SSU) when exported toward the pores.
1. Nuclear import
We have shown by immuno electron microscopy (EM) (Fig. 1
A) that S6 molecules enter the nucleus and are thereafter found in some (not all) interchromatin granules (IG) clusters.
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The presence of labeling for S6 within IG would suggest the possibility that SSU itself could be stored in these structures. However, this is unlikely since, to date, only ribosomal proteins have been detected in the IG and not rRNA and, consequently, no ribosomal subunits.
IG clusters represent a temporary storage site; thereafter S6 can be visualized on some Cajal-coiled bodies (CBs) both in the vicinity of IG and approaching the nucleolus.
The CBs are known to interact with the nucleolus, where they sequester/release proteins. We confirmed by EM that CBs contain ribosomal proteins, as already described by Jimenez-Garcia and co-workers.
2. Preribosome release from the nucleolus
Ribosome biogenesis takes place in the nucleolus, where the ribosomal proteins recruited from the cytoplasm are associated with rRNA. In this organelle, rRNA transcription occurs at the level of the dense fibrillar component (DFC), and the pre-rRNA molecules are associated with proteins into processomes.
However, ribosomal proteins, later to be found in the ribosomal subunits, are very early associated with pre-rRNA; some of them have been shown to bind within the DFC but most are present in the granular component (GC).
When associated with other SSU proteins, S6 moves to the periphery of the nucleolus, where S6 is sometimes found to colocalize with nuclear myosin I (NMI).
Therefore, we show for the first time the association of the motor proteins nuclear myosin I (NMI) and actin, and ribosomal subunits.
As a part of the SSU, S6 moves in the nucleoplasm associated with RNA.
The immunolabeling shows that a subset of SSUs, labeled by anti-S6 antibody (Ab) and stained by terbium (a specific stain for RNA at EM), colocalizes with NMI and nuclear actin, reaching the nuclear pores.
We confirmed such an association by performing 1) an immunoprecipitation assay (IP) and 2) an incorporation of anti-NMI or antiactin antibodies into living cells. Both these approaches suggest a possible interaction (direct or indirect) of the ribosomal protein S6 with the nuclear motor proteins.
Moreover, a statistical analysis of the gold labeling of S6 and the motor proteins, as well as a measurement of the difference in RNA export between samples kept at 37°C or 23°C, confirms that 
10% of the SSUs move with an energy-consuming process.
CONCLUSIONS AND SIGNIFICANCE
Our hypothesis is summarized in the schematic diagram (Fig. 2
A). We suggest that:
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1. IG clusters may play a role in the storage or/and in possible modifications of the ribosomal proteins during the nucleoplasmic transit toward the nucleolus
2. CBs are likely to represent a carrier to the nucleolus
3. NMI and actin may have a fundamental role in the nucleoplasmic SSU transport
In our opinion, the last point is intriguing, because it may encourage an enlargement of the spectrum of possible involvements of NMI and actin in different biological areas.
We have shown here for the first time the direct or indirect interaction among S6, NMI, and actin (indicated by our results in IP assay and with the blockade of NMI or actin) and the simultaneous occurrence of these proteins with single, selectively stained RNA molecules present within SSUs. These results support our hypothesis that an active SSU export exists. We suggest that SSUs can be helped by NMI and actin to leave the nucleolus to the nuclear pore. Our findings need not, however, be in conflict with the diffusion mechanism described for the large ribosomal subunit. One could hypothesize that both mechanisms, diffusive and active, may actually coexist for the subunit movements; the diffusive one, however, could guarantee the transport of the majority of the subunits, the active one a subset only. The advantage of the double mechanism is to assure that while most subunits would move without consuming energy, the movement of the remnant could be modulated, that is, increased or decreased according to the cell needs at a specific moment of the cell cycle.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5339fje
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