First published online 14 November 2002
doi: 10.1242/jcs.00176
Nuclear RanGTP is not required for targeting small nucleolar RNAs to the nucleolus
Aarthi Narayanan1,
Julia Eifert1,
Kavita A. Marfatia2,
Ian G. Macara3,
Anita H. Corbett2,
Rebecca M. Terns1 and
Michael P. Terns1,*
1 Departments of Biochemistry and Molecular Biology, and Genetics, University of
Georgia, Life Sciences Building, Athens, GA 30602, USA
2 Department of Biochemistry, Emory University, Atlanta, GA 30322, USA
3 Department of Pharmacology, Center for Cell Signaling, University of Virginia
School of Medicine, Charlottesville, VA 22908, USA
View larger version (49K):
[in a new window]
|
Fig. 5. Microinjected U3 and U65 snoRNAs are retained within the nucleus and
localized to nucleoli of Xenopus oocytes after injection of T24N Ran.
(A,C) Recombinant RanT24N in microinjection buffer (bottom panels) or
microinjection buffer alone (top panels) were injected into separate sets of
oocytes. One hour later, one fmole of fluorescently and 32P-labeled
U3 or U65 snoRNA was injected into the same set of oocytes. Nuclear spreads
were made four hours after injection of the RNA, and slides were analyzed by
fluorescence microscopy. Several individual nucleoli are shown in each
differential interference contrast (DIC) panel. The fluorescence signals (RNA)
show that the injected snoRNAs are targeted to a centrally located subregion
of the nucleoli in oocytes, which were injected with the T24N Ran (+T24N).
(B,D) Nuclear (N) and cytoplasmic (C) fractions were obtained from the same
set of injected cells. RNA was extracted from the N and C fractions and
subjected to denaturing PAGE followed by autoradiography. The marker lane (M)
indicate samples prior to injection.
|
|
View larger version (52K):
[in a new window]
|
Fig. 1. Depletion of RCC1 does not affect the nucleolar localization of U3 snoRNA
in tsBN2 cells. Individual cells are observed in the differential interference
contrast images (DIC), and the arrowheads point to distinct nucleoli. Also
shown are the nuclei of these same cells stained with the DNA-specific dye
4,6-diamino-2-phenylindole(DAPI). U3 snoRNA was detected by fluorescence in
situ hybridization (U3 snoRNA) in tsBN2 cells maintained for 6 hours at the
permissive temperature (33°C) or the non-permissive temperature [40°C,
resulting in RCC1 depletion (Ohtsubo et
al., 1987 )]. Immunofluorescence with antibodies against the
trimethyl guanosine cap (TMG) of U snRNAs was also performed simultaneously
and detected with Cy2-tagged anti-rabbit antibodies (TMG panel).
|
|
View larger version (90K):
[in a new window]
|
Fig. 2. U3 and the snR10 (Box C/D and Box H/ACA snoRNAs, respectively) are readily
detected in the yeast nucleolus by fluorescence in situ hybridization (FISH).
(A) U3 and snR10 localize to crescent-shaped nucleoli of S.
cerevisiae as revealed by hybridization with Cy3-labeled, antisense
probes against U3 or snR11 (RNA panel). The merged image shows that the snoRNA
signals (detected by FISH) are directly adjacent to the nucleoplasmic DNA
detected by DAPI staining of the same cells. (B) Colocalization of snoRNAs
with the nucleolar marker protein, Nop1 (fibrillarin). A combination of FISH
(to detect U3 snoRNA) and immunofluorescence (to detect Nop1 protein) was
performed. The merge image indicates an overlay of U3 snoRNA and Nop1 protein.
DIC, differential interference contrast. Arrowheads point to a nucleolus.
|
|
View larger version (86K):
[in a new window]
|
Fig. 3. snoRNAs localize to nucleoli in RanGAP (rna1-1) mutant cells. The
subcellular distribution of U3 and snR10 snoRNAs and polyA+ RNA were
determined by fluorescence in situ hybridization using Cy3-labeled U3, snR10
and oligo d(T) probes as indicated. FISH analysis was performed in a strain
containing a temperature-sensitive mutation in RanGAP (rna1-1) or a
double mutant (rna1-1 rbp1-1) containing also a temperature-sensitive
mutation in the large subunit of RNA polymerase II required for production of
poly(A)+ mRNA. Cells were analyzed both at 25°C (permissive temperature)
and after a shift to the non-permissive temperature of 37°C for three
hours. The merged image shows the position of each RNA relative to the
DAPI-stained nucleoplasmic signal. U3 and snR10 nucleolar signals are observed
in both strains at both permissive and non-permissive temperatures. As
expected, export of poly(A)+ RNA to the cytoplasm is blocked at the
non-permissive temperature.
|
|
View larger version (79K):
[in a new window]
|
Fig. 4. snoRNAs localize to nucleoli in Ran mutant strains. The subcellular
distribution of U3 and snR10 snoRNAs and poly(A)+ RNA were determined by
fluorescence in situ hybridization using Cy3-labeled U3, snR10 and oligo d(T)
probes as indicated. FISH analysis was performed in a strain containing a
temperature-sensitive mutation in Ran (gsp1-1) or a double mutant
(gsp1-1 rbp1-1) containing also a temperature-sensitive mutation in
the large subunit of RNA polymerase II required for production of poly(A)+
mRNA. Cells were analyzed both at 25°C (permissive temperature) and after
a shift to the non-permissive temperature of 37°C for three hours. The
merged image shows the position of each RNA relative to the DAPI-stained
nucleoplasmic signal. U3 and snR10 nucleolar signals are observed in both
strains at both permissive and non-permissive temperatures. As expected,
export of poly(A)+ RNA to the cytoplasm is blocked at the non-permissive
temperature.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2003
