PROTEIN SYNTHESIS:

"Believe It or Not--Translation in the Nucleus".

Matthias W. Hentze

Gene Expression Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1,
D-69117 Heidelberg, Germany.

E-mail:   hentze@embl-heidelberg.de

The concept of nuclear translation is not entirely new. Earlier studies showed that a small fraction of amino acids are incorporated into polypeptides in the nucleus. However, many attributed this finding to cytoplasmic contamination (2). More recently, structures with the biochemical and pharmacological characteristics of
 polyribosomes have been described in the nuclei of the slime mould Dictyostelium (3). This work, however, did not indicate whether these structures were unambiguously localized in the nucleus or whether they carried out protein synthesis. Although these earlier reports of nuclear translation were greeted with skepticism, it is now accepted that most components of the translation machinery are present in the nucleus. For example, the two ribosomal subunits are assembled in the nucleolus, translation initiation and elongation factors reside in the nucleus, and even the addition of amino acids to transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases can take place in the nucleus (4-6). But the question is, can these separate components unite and orchestrate protein synthesis in the nucleus?

In their study, Iborra et al. adopted a strategy that would enable them to visualize nuclear protein synthesis. They pinpointed nuclear sites of translation by labeling permeabilized mammalian cells or purified nuclei with fluorescent lysine. The accumulation of nuclear fluorescence was time dependent and sensitive to inhibitors of eukaryotic protein synthesis (cycloheximide and puromycin), but not to the bacterial translation inhibitor chloramphenicol (see the figure). The authors estimate that nuclear translation accounts for about 10 to 15% of protein synthesis in the cell. 

Evidence for nuclear translation.
(1) New protein synthesis in the nucleus (green dots) and the cytoplasm--as indicated by nuclear and cytoplasmic fluorescence after incorporation of fluorescent lysine--is equally sensitive to
inhibitors of eukaryotic translation.
(2) Isolated nuclei show no detectable extranuclear or perinuclear fluorescence, indicating that proteins made in the cytoplasm are not being imported into the nucleus.
(3) Purified nuclei display undiminished intranuclear fluorescence.
(4) Nuclear translation sites are not randomly distributed, but overlap with sites of gene transcription as indicated by immunogold labeling (yellow dots).
(5) Stimulation of transcription by increasing the concentration of nucleotides enhances nuclear but not cytoplasmic fluorescence.
(6) Nuclear fluorescence is not affected by blocking the import of proteins into the nucleus by thapsigargin.

In principle, exclusion of a single vital component of the translation apparatus from the nucleus of a living cell should result in translation being restricted to the cytoplasm. How can we be sure that no such cytoplasmic translation factor leaked into the nucleus during cell permeabilization or nuclear isolation? To address this concern, the authors pretreated cells with thapsigargin--which inhibits the import of proteins into the nucleus and diffusion of proteins through nuclear pores--and then permeabilized them in the presence of the drug. This treatment effectively prevented a 40-kD fluorescein-dextran marker molecule from entering the nucleus, but did not affect nuclear translation. Although one cannot completely exclude the possibility that translation factors leaked into the nucleus, the results of this experiment are reassuring. There is no doubt that Iborra and colleagues have mounted a case of unprecedented strength in support of nuclear translation.

The Iborra et al. paper is sure to spur intense discussion between "believers" and "converts" on the one hand, and "nonbelievers" on the other. Doubtless, nonbelievers will demand to see nuclear translation in intact cells rather than in permeabilized cells or purified nuclei. As was the case with local translation at synapses in the central nervous system, we need more evidence to confirm that local translation products are not transported. The persuasive power of electron micrographs illustrating puromycin-sensitive nuclear ribosomes at work will win some converts. Harnessing the power of genetics and RNA interference to produce mutants that carry out either nuclear or cytoplasmic translation but not both would garner additional converts.

Few readers will fail to be fascinated by what nuclear translation can tell us about, for example, the origin of eukaryotic cells (7). Perhaps nuclear translation serves the same purpose as the purported restriction of translation to the cytoplasm does: namely, to prevent synthesis of faulty proteins. Nuclear translation provides the cell with an additional opportunity to assess the integrity of mRNAs before they are exported to the cytoplasm. If detected in the nucleus, "faulty" mRNAs may be subjected to intranuclear degradation or altered splicing (to avoid the production of mRNAs with premature stop codons). Such processes complement the more conventional pathway that degrades mRNAs with premature stop codons after translation by ribosomes in the cytoplasm (8-10). How will mRNAs that need to decode UGA stop codons for selenoprotein synthesis pass the nuclear translation test? Are proteins produced by nuclear translation functional, or are they all destined for degradation by the proteasome? What is the interplay between nuclear translation and export of mature mRNAs out of the nucleus? A recent study reports that premature translation termination codons induce the accumulation of unspliced precursor mRNAs at the site of transcription (11). Is this discovery a smoking gun, highlighting a consequence of linking nuclear transcription and translation? For players and spectators alike, future research on translation, whether in the nucleus or the cytoplasm, is likely to be full of suspense and surprises.

References

1. F. J. Iborra, D. A. Jackson, P. R. Cook, Science 293, 1139 (2001).

2. J. A. Goidl, W. R. Allen, Trends Biochem. Sci. 3, N225 (1978).

3. G. Mangiarotti, Biochemistry 38, 3996 (1999).

4. E. Lund, J. E. Dahlberg, Science 282, 2082 (1998).

5. G. J. Arts et al., EMBO J. 17, 7430 (1998).

6. J. Dostie, F. Lejbkowicz, N. Sonenberg, J. Cell Biol. 148, 239 (2000).

7. J. A. Lake, M. C. Rivera, Proc. Natl. Acad. Sci. U.S.A. 91, 2880 (1994).

8. M. W. Hentze, A. E. Kulozik, Cell 96, 307 (1999).

9. S. Li, M. F. Wilkinson, Immunity 8, 135 (1998).

10. L. E. Maquat, G. G. Carmichael, Cell 104, 173 (2001).

11. O. Mühlemann et al., Mol. Cell, in press.

1. Wells WA, "Translation in the Nucleus", J. Cell Biol. vol. 154, no. 1, p. 12 (July 9, 2001).

2. Brachet J, "Biochemical Cytology", Academic Press Inc., New York, 1957, pp. 97-105 and 133-135.

3. Crick F, "Central Dogma of Molecular Biology", Nature 227: 561-563 (August 8, 1970).

4. "Metabolism and Morphology of Ribonucleoprotein Particles from the Cell Nucleus of Lymphocytes".

5. "In Vitro Incorporation of Amino Acids into the Proteins of Isolated Nuclear Ribosomes",

6. "Nuclear Ribosomes and RNA-RNA Duplexes".