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Identification of discrete classes of small nucleolar RNA featuring different ends and RNA binding protein dependency.

Deschamps-Francoeur G, Garneau D, Dupuis-Sandoval F, Roy A, Frappier M, Catala M, Couture S, Barbe-Marcoux M, Abou-Elela S, Scott MS - Nucleic Acids Res. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Département de biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec J1E 4K8, Canada.

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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.
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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.

Mentions: Box C/D snoRNAs are 50–100 nucleotides (nt)-long transcripts featuring conserved box C (RUGAUGA where R is a purine) and box D (CUGA) motifs that align to form a characteristic structural motif called the kink-turn or K-turn motif (4,7,9–11), as illustrated in Figure 1A. K-turn motifs are widespread in many classes of RNA and involve non-canonical G–A base pairing causing a tight kink in the axis of double-stranded RNA (12,13). The k-turn motif is typically flanked by a 5’ canonical stem composed of regular base pairs and by a 3’ non-canonical stem consisting of the G–A base pairs (11,13). The minor grooves of the canonical and non-canonical stems can interact, coordinated by metal ions or binding proteins, stabilizing the structure (11). In box C/D snoRNAs, constrained by the sequences of the boxes C and D, the non-canonical G–A stem is typically followed by one pair of U–U mismatched nucleotides and two canonical base pairs (Figure 1A). Both the canonical stem and the extended non-canonical stem appear to be important for proper processing of the snoRNA (14) and assembly of the snoRNP complex. In vertebrates, most snoRNAs are encoded in introns and co-transcribed from the promoter of their host genes. The assembly of the pre-snoRNP complex typically occurs on spliced and debranched introns and is initiated by the recognition of the k-turn structure by the core binding protein 15.5K (15–20). The binding of 15.5K to the k-turn provides a scaffold for the assembly of the box C/D snoRNP complex which includes additional core binding proteins NOP56, NOP58 and the methyltransferase fibrillarin (15,21–26). The resulting pre-snoRNPs are exonucleolytically trimmed from both the 5′ and 3′ ends, generating mature snoRNPs (19,20,27). The binding of the core proteins likely protects the snoRNAs from further trimming and determines their exact termini (14). Disruption of the k-turn and flanking stems prevents stable accumulation of snoRNAs (14,22), underlining the importance of these motifs for the appropriate processing and the stability of C/D snoRNAs and the proper structure and assembly C/D snoRNPs.


Identification of discrete classes of small nucleolar RNA featuring different ends and RNA binding protein dependency.

Deschamps-Francoeur G, Garneau D, Dupuis-Sandoval F, Roy A, Frappier M, Catala M, Couture S, Barbe-Marcoux M, Abou-Elela S, Scott MS - Nucleic Acids Res. (2014)

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.
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Related In: Results  -  Collection

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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.
Mentions: Box C/D snoRNAs are 50–100 nucleotides (nt)-long transcripts featuring conserved box C (RUGAUGA where R is a purine) and box D (CUGA) motifs that align to form a characteristic structural motif called the kink-turn or K-turn motif (4,7,9–11), as illustrated in Figure 1A. K-turn motifs are widespread in many classes of RNA and involve non-canonical G–A base pairing causing a tight kink in the axis of double-stranded RNA (12,13). The k-turn motif is typically flanked by a 5’ canonical stem composed of regular base pairs and by a 3’ non-canonical stem consisting of the G–A base pairs (11,13). The minor grooves of the canonical and non-canonical stems can interact, coordinated by metal ions or binding proteins, stabilizing the structure (11). In box C/D snoRNAs, constrained by the sequences of the boxes C and D, the non-canonical G–A stem is typically followed by one pair of U–U mismatched nucleotides and two canonical base pairs (Figure 1A). Both the canonical stem and the extended non-canonical stem appear to be important for proper processing of the snoRNA (14) and assembly of the snoRNP complex. In vertebrates, most snoRNAs are encoded in introns and co-transcribed from the promoter of their host genes. The assembly of the pre-snoRNP complex typically occurs on spliced and debranched introns and is initiated by the recognition of the k-turn structure by the core binding protein 15.5K (15–20). The binding of 15.5K to the k-turn provides a scaffold for the assembly of the box C/D snoRNP complex which includes additional core binding proteins NOP56, NOP58 and the methyltransferase fibrillarin (15,21–26). The resulting pre-snoRNPs are exonucleolytically trimmed from both the 5′ and 3′ ends, generating mature snoRNPs (19,20,27). The binding of the core proteins likely protects the snoRNAs from further trimming and determines their exact termini (14). Disruption of the k-turn and flanking stems prevents stable accumulation of snoRNAs (14,22), underlining the importance of these motifs for the appropriate processing and the stability of C/D snoRNAs and the proper structure and assembly C/D snoRNPs.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Département de biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec J1E 4K8, Canada.

Show MeSH
Related in: MedlinePlus