The Existence and Localization of Nuclear snoRNAs in Arabidopsis thaliana Revisited

Abstract

Ribosome biogenesis is one cell function-defining process. It depends on efficient transcription of rDNAs in the nucleolus as well as on the cytosolic synthesis of ribosomal proteins. For newly transcribed rRNA modification and ribosomal protein assembly, so-called small nucleolar RNAs (snoRNAs) and ribosome biogenesis factors (RBFs) are required. For both, an inventory was established for model systems like yeast and humans. For plants, many assignments are based on predictions. Here, RNA deep sequencing after nuclei enrichment was combined with single molecule species detection by northern blot and in vivo fluorescence in situ hybridization (FISH)-based localization studies. In addition, the occurrence and abundance of selected snoRNAs in different tissues were determined. These approaches confirm the presence of most of the database-deposited snoRNAs in cell cultures, but some of them are localized in the cytosol rather than in the nucleus. Further, for the explored snoRNA examples, differences in their abundance in different tissues were observed, suggesting a tissue-specific function of some snoRNAs. Thus, based on prediction and experimental confirmation, many plant snoRNAs can be proposed, while it cannot be excluded that some of the proposed snoRNAs perform alternative functions than are involved in rRNA modification.

Keywords: A. thaliana; NGS; cell fractionation; snoRNAs; tissue specificity.

Figure 1

Analysis of the small RNA distribution in Arabidopsis thaliana cells: (a) RNA was isolated from cell lysates (cl) and nuclear fractions (nu) and subjected to agarose gel analysis followed by ethidium bromide staining, and migration of the rRNA precursor is indicated on the left. The presence of the 35S as well as of 27SB rRNA and the absence of 23S rRNA in the nuclear fraction are highlighted by arrowheads. (b) The number of reads (top) and of detected molecules (bottom) is presented. The number of total (column 2) and mapped reads (column 3) is given for each fraction. Subsequently, the number of reads mapped to genes coding for specific RNAs (according to TAIR10) is presented for cell lysate (lysate) and the three replicas of the nuclear fraction (nuc-1 to nuc-3). These ncRNAs are not annotated in TAIR10. On the bottom, the total number of identified RNAs in cell lysate (lysate), the three biological replicas of the nucleus (nuc-1 to nuc-3), accumulated results for nuclear fractions (to. nuc), or all fractions (total) are shown. (c) The codon is indicated as a color code from 5′ (center) to 3′ (outer rim). The total reads per base for the tRNA for each codon is shown (middle light grey background; grey bar: maximal value in one of the nuclear fractions; dark gray: cell lysate; logarithmic scale from 1 to 104). The amino acid occurrence in the proteome of A. thaliana is shown in logarithmic scale between 105 and 106 (outer white rim, black dot). (d) The reads per base found for the rRNA transcript is shown for the four experiments according to the color code on the left. The regions coding for 18S, 5.8S, and 25S are indicated.

Figure 2

The detected snRNAs and snoRNAs in A. thaliana cells. (a,b) The distribution of the snRNAs (a) and snoRNAs (b) annotated in TAIR10 in cell lysate and nuclear fractions is shown. (c,d) The number of identified snoRNAs with C/D box, H/ACA, or unknown fold known present in snOPY or snoRNA DB (c) or additionally identified (d) is presented. For the annotated snoRNAs (Cc), the number for the (predicted) target rRNA is given. (e) The general chromosomal localization of the newly identified putative snoRNAs (d) is shown. The color code indicates the distribution in the two structural groups as in (c). rDNA (grey) and regions with high frequency snoRNAs occurrence (black; [26]) are highlighted.

Figure 3

U3-like snoRNAs of the C/D box family: (a) The structure of the U3 snoRNAs were predicted (see the Materials and Methods section). For comparison, the structure of U3 from yeast [69] is shown. (b,c) A FISH probe (left, Table S4) against the U3-like snoRNA family was used for incubation of root cells of A. thaliana plants before (b) and after RNase treatment (c). DNA was visualized by DAPI staining (second), and the cell shape by recording the bright field image (third). The overlay of all signals is shown for representative cells (right). (d) RNA isolated from nuclear depleted cytoplasm (cy) and nuclear fraction (nu) were subjected to Polyacrylamide Gel Electrophoresis (PAGE)-based separation. The migration of the indicated U25- and the U3-type snoRNAs was probed by northern blotting with specific probes (Table S5). Migration of RNA standards is shown on the left.

Figure 4

snoRNAs of the C/D box family: (a–i) FISH probes (Table S4) against C/D box snoRNAs (Tables S1–S3) were incubated with root cells of A. thaliana plants (second), and DNA was visualized by DAPI staining (third). The overlay between FISH probe signal and DAPI staining is shown for representative cells (first). In (b,e), images for FISH analysis after RNase treatment of the cells are shown exemplarily for snoR29 and U33a. (j) RNA isolated from nuclear depleted cytoplasm (cy) and nuclear fraction (nu) were subjected to acrylamide gel-based separation. The migration of the indicated snoRNAs was probed by northern blotting with specific probes (Table S5). Migration of RNA standards is shown on the left.

Figure 5

snoR100 and snoR160 of the H/ACA family: (a) FISH probes (Table S4) against snoR100 were incubated with root cells of A. thaliana plants and DNA was visualized by DAPI staining. Shown is the overlay between FISH probe signal and DAPI staining, the FISH signal, and the DAPI staining. The arrow points to cytosolic signal. (b) RNA isolated from nuclear depleted (cy) and nuclear fraction (nu) of A. thaliana cell suspension culture was subjected to acrylamide gel-based separation, and migration of indicated snoRNAs was probed by northern blotting (Table S5). Migration of nucleotide standards is shown on the left.

Figure 6

Tissue-specific distribution and localization of snoRNAs: (a) RNA was isolated from roots (R), shoots (S), or flowers (F) of A. thaliana. Ethidium bromide-staining confirmed RNA quality and was used as loading control. (b) Northern blot analysis of the fractions shown in (A) with probes for indicated snoRNAs: Migration of nucleotide standards is shown (left). (c) Density of bands (Figure 6b) was quantified and normalized to 5.8S and 5S density (Figure 6a) and to the abundance in roots. For U24-2, values for the upper (light blue) and lower band (dark blue) were normalized to the density of the lower band. (d) Abundance of all identified snoRNAs expressed as transcripts per million in cell lysate was plotted against the log₂ of the ratio of the average abundance in the nucleus and lysate (SD: error bar). The color code is indicated on the right. The snoRNAs with high abundance in the lysate are named.

Figure 7

Illustration of the identification of complementary rRNA sites for newly assigned snoRNAs (Table S3): Shown are the sequences of newly assigned C/D box and H/ACA snoRNAs and the putatively modified rRNA regions. In yellow is putative duplex structures, in blue and green is C/D box elements, and in magenta, ACA are highlighted. These sites were not experimentally confirmed.