Signal recognition particle RNA localization within the nucleolus differs from the classical sites of ribosome synthesis

Abstract

The nucleolus is the site of ribosome biosynthesis, but is now known to have other functions as well. In the present study we have investigated how the distribution of signal recognition particle (SRP) RNA within the nucleolus relates to the known sites of ribosomal RNA synthesis, processing, and nascent ribosome assembly (i.e., the fibrillar centers, the dense fibrillar component (DFC), and the granular component). Very little SRP RNA was detected in fibrillar centers or the DFC of the nucleolus, as defined by the RNA polymerase I–specific upstream binding factor and the protein fibrillarin, respectively. Some SRP RNA was present in the granular component, as marked by the protein B23, indicating a possible interaction with ribosomal subunits at a later stage of maturation. However, a substantial portion of SRP RNA was also detected in regions of the nucleolus where neither B23, UBF, or fibrillarin were concentrated. Dual probe in situ hybridization experiments confirmed that a significant fraction of nucleolar SRP RNA was not spatially coincident with 28S ribosomal RNA. These results demonstrate that SRP RNA concentrates in an intranucleolar location other than the classical stations of ribosome biosynthesis, suggesting that there may be nucleolar regions that are specialized for other functions.

Diffusion-based Transport of Nascent Ribosomes in the Nucleus

“…In parallel experiments it was observed that oligo(dT) or oligo(dA), or oligos containing repeating CTG or CAG sequences did not concentrate in the nucleolus, and in fact, appeared to be excluded from the nucleolus ( Figure 2B and our unpublished results). In standard in situ hybridization experiments with fixed cells, the 28S antisense oligos generated signal in the nucleolus and the cytoplasm as expected ( Figure 2C; see also Politz et al, 2002), whereas only background levels of signal were detected with control oligos ( Figure 2D). J.C.R.…”

“…Oligodeoxynucleotides complementary to these five regions of 28S rRNA, each 33 nucleotides in length, were synthesized with four, approximately evenly spaced aminohexyl-modified thymidines (see MATERIALS AND METHODS), and these sites were labeled with fluorescein as described Politz et al, 2002). RNA hybrids formed with oligos labeled in this manner are less susceptible to degradation in vivo, perhaps because the evenly spaced aminohexyl arms interfere with RNase H binding (Ueno et al, 1997;J.C.R.…”

“…Oligodeoxynucleotides complementary to 28S rRNA were as follows (see Gerbi, 1996 andDeRijk et al, 1999; for database and nomenclature information also see Politz et al, 2002): Oligo 1 in loop E11_1 (D7b): G*TACCGGCA-C*GGACGCC*CGCGGCGCCCA*C; Oligo 2 in loop E9_1 (D-7a): C*GAGG-GCAACGGAGGCCA*CGCCCG*CCCT*C; Oligo 3 in loop B13_1 (D1): G*ACGCCACAT*TCCCGCGCC*CGGCGCGCG*C; Oligo 4 in loop C1_1 (D2): C*CGCGCCGCCGGG*TCAATCC*CCGGGCGG*C; and Oligo 5 in loops H1_2, H1_3 (D12): A*GGCTC*CCGCACCGGACCCCGG*CCCGAC*C, where the asterisk indicates positions of aminohexyl-modified thymidine residues coupled during synthesis (Integrated DNA Technologies, Iowa City, IA).…”

“…After 2 h, the medium was replaced with fresh DMEM (with serum) and the cells were incubated for another 30 min to 1 h. Immediately before imaging, the medium was changed to Leibovitz's L15 medium (Life Technologies, Rockville, MD) with 10% serum. Oligodeoxynucleotides complementary to 28S rRNA were as follows (see Gerbi, 1996 and DeRijk et al, 1999; for database and nomenclature information also see Politz et al, 2002): Oligo 1 in loop E11_1 (D7b): G*TACCGGCA-C*GGACGCC*CGCGGCGCCCA*C; Oligo 2 in loop E9_1 (D-7a): C*GAGG-GCAACGGAGGCCA*CGCCCG*CCCT*C; Oligo 3 in loop B13_1 (D1): G*ACGCCACAT*TCCCGCGCC*CGGCGCGCG*C; Oligo 4 in loop C1_1 (D2): C*CGCGCCGCCGGG*TCAATCC*CCGGGCGG*C; and Oligo 5 in loops H1_2, H1_3 (D12): A*GGCTC*CCGCACCGGACCCCGG*CCCGAC*C, where the asterisk indicates positions of aminohexyl-modified thymidine residues coupled during synthesis (Integrated DNA Technologies, Iowa City, IA).HPLC-purified oligos were labeled with either fluorescein or caged-fluorescein (caged-fl; CMNB2AF in Mitchison et al, 1994) as described by . …”

“…Also, incubation with a mixture of antidigoxigenin and antibiotin antibodies (1:250 dilution of each) was at 4°C overnight in a humidified chamber, and 0.5% normal sheep serum and 0.5% normal goat serum instead of 1% BSA was used in the washes before and after antibody binding. In situ hybridization experiments were performed exactly as described (Politz et al, 2002). …”