Wilhelm bernhard workshop on the cell nucleus

LOCALIZATION OF COMPONENTS OF THE SIGNAL RECOGNITION PARTICLE IN THE AMPLIFIED NUCLEOLI OF XENOPUS OOCYTES

Sommerville J.¹, Mullin A.¹, Gilchrist K.¹, Lewandowski L.B.², Brumwell C.L.², Politz J.C.² and Pederson T.².

¹School of Biology, Bute Medical Buildings, University of St Andrews, St Andrews, Scotland; ²Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, USA.
The nucleolus is the site of pre-ribosome assembly, but this compartment of the nucleus is now known to have other functions. One additional function came from the discovery that the signal recognition particle (SRP) RNA traffics through the nucleolus. Here we report the use of Xenopus oocytes to track the incorporation of both SRPRNA and a protein component of the SRP into the large, amplified, extrachromosomal nucleoli of germinal vesicles (oocyte nuclei). GFP-tagged SRP19 protein injected into the oocyte cytoplasm was imported into the nucleus and rapidly localized in the nucleoli. The dynamics of incorporation revealed that at 2 h post-injection GFP-SRP19 was located mainly in the dense fibrillar component (DFC) of the nucleoli and by 4 h the signal had spread throughout the granular component (GC). In contrast, Alexa 488-tagged SRPRNA injected into the cytoplasm was not imported into the nucleus, but on injection directly into the nucleus it was targeted to the nucleoli. That SRPRNA is normally present in extrachromosomal nucleoli was confirmed by in situ hybridization with complementary Cy3-labelled probes. Treatment of injected oocytes with leptomycin B, a specific inhibitor of the CRM1 (exportin-1) nuclear export pathway, caused GFP-SRP19 protein to accumulate in the nucleoplasm, suggesting that the SRP uses the same nuclear export pathway as does the 60S ribosomal subunit. These results establish that extrachromosomal nucleoli retain the same affinity for SRP components as is observed with somatic cell nucleoli that are built around the chromosomal nucleolar organizer. This suggests that the developmental strategy of amplifying nucleoli during amphibian oogenesis may be to produce a maternal stockpile not only of ribosomes, as classically envisioned, but also of signal recognition particles.