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. 2014 Sep;42(15):10073-85.
doi: 10.1093/nar/gku664. Epub 2014 Jul 29.

Identification of Discrete Classes of Small Nucleolar RNA Featuring Different Ends and RNA Binding Protein Dependency

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Free PMC article

Identification of Discrete Classes of Small Nucleolar RNA Featuring Different Ends and RNA Binding Protein Dependency

Gabrielle Deschamps-Francoeur et al. Nucleic Acids Res. .
Free PMC article

Abstract

Small nucleolar RNAs (snoRNAs) are among the first discovered and most extensively studied group of small non-coding RNA. However, most studies focused on a small subset of snoRNAs that guide the modification of ribosomal RNA. In this study, we annotated the expression pattern of all box C/D snoRNAs in normal and cancer cell lines independent of their functions. The results indicate that C/D snoRNAs are expressed as two distinct forms differing in their ends with respect to boxes C and D and in their terminal stem length. Both forms are overexpressed in cancer cell lines but display a conserved end distribution. Surprisingly, the long forms are more dependent than the short forms on the expression of the core snoRNP protein NOP58, thought to be essential for C/D snoRNA production. In contrast, a subset of short forms are dependent on the splicing factor RBFOX2. Analysis of the potential secondary structure of both forms indicates that the k-turn motif required for binding of NOP58 is less stable in short forms which are thus less likely to mature into a canonical snoRNP. Taken together the data suggest that C/D snoRNAs are divided into at least two groups with distinct maturation and functional preferences.

Figures

Figure 1.
Figure 1.
Comparison of box C/D snoRNA expression patterns in normal and cancer cell lines. (A) Schematic representation of box C/D snoRNA structure. Box C/D snoRNAs are small non-coding RNAs featuring two short sequence motifs (C: RUGAUGA and D: CUGA) that are aligned together through base pairing to form a characteristic structural k-turn motif. This motif typically involves a bulge upstream from the box C and non-canonical A–G and G–A base pairing between box C and D residues, preceded on the 5′ side by a stem involving canonical base pairing (12). X and R represent any nucleotide and purines, respectively. The 3′ end terminus of the short and long snoRNA forms detected in this study are indicated by arrows. (B) The processing pattern of box C/D snoRNA is conserved in normal and cancer cells. Sequence reads mapping to at least 77% of full-length box C/D snoRNAs in normal (BJ-Tielf, INOF), breast (MCF-7) and ovarian cancer cell lines (SKOV3ip) were counted and plotted with respect to their corresponding boxes C and D for every residue of all box C/D snoRNAs. CPM indicates count per million. All experiments were performed in duplicate. (C) Identification of two distinct forms of C/D snoRNA. Two general forms (long and short) of box C/D snoRNAs were identified according to the distance between their ends and their characteristic C/D motifs. The short forms (snoRNASH) start 4 or 5 nt upstream of their box C and end 2 or 3 nt downstream of their box D, while the long forms (snoRNAL) start 5 or 6 nt upstream of their box C and end 4 or 5 nt downstream of their box D. The number of snoRNAs displaying only short or only long forms, a mix of the two forms or neither long nor short forms (other) was counted in the different cell lines and presented in the form of a histogram. The standard deviation between the duplicate experiments is shown as error bars.
Figure 2.
Figure 2.
The long form of box C/D snoRNA (snoRNAL) features extended base pairing downstream of the k-turn structural motif. (A) The long and short snoRNA forms share the basic structural features of box C/D snoRNA. The characteristic box C, box D and k-turn were identified and their level of sequence conservation in each class of snoRNA was determined using the sequence obtained from the SKOV3ip1 cell line. The number and percent of snoRNAs displaying each feature are shown. Only predominant snoRNA forms expressed above 1 CPM in both replicates were counted. (B) The snoRNAL form features more stable k-turn structure. The length of the external stem was measured using SKOV3ip1 RNA for snoRNAs only expressed as short or only expressed as long forms. The proportion of snoRNAs from these two groups is expressed as a function of the number of paired nucleotides in their external stem. (C) Schematic representation of the external stem observed in snoRNAs only expressed as long or short snoRNA forms. The percentage of snoRNAs containing the different number of base pairs downstream of box D is shown on the right of each form. The orange and blue boxes indicate the position of the C and D motifs, respectively.
Figure 3.
Figure 3.
Depletion of the core box C/D snoRNA binding protein NOP58 preferentially downregulates the expression of the long snoRNA forms. (A) KD of NOP58 inhibits the expression of some box C/D and not H/ACA snoRNAs. NOP58 was knocked down in SKOV3ip1 using two independent siRNAs and the impact on the abundance of RNA shorter than 200 nt (snoRNA, miRNA, snRNA, YRNA and tRNA) was calculated relative to the level in mock-transfected cells (LF). The log2 fold change in RNA abundance is shown in the form of scatter plots. The different small RNA members are indicated by blue dots, while the box C/D (left panel) and H/ACA snoRNA (right panel) are highlighted in red. The dashed lines represent a decrease of 2-fold in the NOP58 depletion. (B) Effect of the NOP58 KD on the different forms of box C/D snoRNA. The number of long (snoRNAL), short (snoRNASH) and other (that differ from the snoRNAL and snoRNASH) predominant forms of box C/D snoRNAs was determined and plotted relative to binned values of the log2 fold change in expression after the NOP58 KD. The enrichment in snoRNAL downregulated by the KD is shown at bottom.
Figure 4.
Figure 4.
The KD of the RBFOX2 splicing factor preferentially downregulates the expression of short snoRNA forms. (A) KD of RBFOX2 inhibits the expression of select box C/D and H/ACA snoRNAs. RBFOX2 was knocked down in SKOV3ip1 using two independent siRNAs and the impact on the abundance of RNA shorter than 200 nt (snoRNA, miRNA, snRNA, YRNA and tRNA) was calculated relative to the level in mock-transfected cells (LF). The log2 fold change in RNA abundance is shown in the form of scatter plots. The different small RNA members are indicated by blue dots, while the box C/D (left panel) and H/ACA snoRNA (right panel) are highlighted in red. The dashed lines represent a decrease of 2-fold in the RBFOX2 depletion. (B) Effect of the RBFOX2 KD on the different forms of box C/D snoRNA. The number of long (snoRNAL), short (snoRNASH) and other (that differ from the snoRNAL and snoRNASH) forms of box C/D snoRNAs was determined and plotted relative to binned values of the log2 fold change in expression after the RBFOX2 KD. The enrichment in snoRNASH downregulated by the KD is shown at bottom.
Figure 5.
Figure 5.
NOP58 and RBFOX2 snoRNA targets are form-dependent. The abundance of the different forms generated from each snoRNA was determined before and after the KD of either NOP58 or RBFOX2 and plotted relative to the number of nucleotides upstream of box C and downstream of box D. Shown are three examples representing snoRNAs producing only long (A), only short (B) or both long and short (C) forms. The snoRNA names are shown on top, while the enrichment of the short and long forms are illustrated at bottom. CPM and LF respectively indicate counts per million reads mapped and mock transfection (Lipofectamine). The data obtained after the transfection of two independent siRNAs targeting either NOP58 (blue bars) or RBFOX2 (red bars) and three mock transfections (black bars) are shown.
Figure 6.
Figure 6.
Intronic snoRNA processing model. (A) Intronic snoRNAs displaying canonical features including a strong k-turn and/or proximity to the downstream exon are more likely to follow the canonical processing pathway including dependency on core box C/D snoRNP proteins such as NOP58. (B) In contrast, snoRNAs displaying non-canonical features are more likely to depend on non-canonical snoRNA binding proteins such as RBFOX2.

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