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Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core proteins and small RNA sequencing

Shivendra Kishore, Andreas R Gruber, Dominik J Jedlinski, Afzal P Syed, Hadi Jorjani and Mihaela Zavolan*

Author Affiliations

Computational and Systems Biology, Biozentrum, University of Basel, Klingelbergstrasse 50-70, 4056 Basel, Switzerland

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Genome Biology 2013, 14:R45  doi:10.1186/gb-2013-14-5-r45

Published: 26 May 2013

Additional files

Additional file 1:

Profiles of PAR-CLIPs reads obtained with various core snoRNP proteins for snoRNAs and scaRNAs. The proteins and normalized read counts are shown on the y-axis. The snoRNA and location of boxes are shown at the bottom. Red bars in the profiles indicate the number of T→C mutations observed at individual nucleotides in the PAR-CLIP reads.

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Additional file 2:

List of novel C/D box, C/D box-like snoRNAs and mini-snoRNAs obtained in this study.

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Additional file 3:

List of novel H/ACA snoRNAs or homologs of known snoRNAs (indicated in the 'BLAST hits' column) that were obtained in this study.

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Additional file 4:

RNA-seq read profiles from selected ENCODE small RNA-seq samples along the novel C/D box and H/ACA box snoRNA loci identified in our study.

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Additional file 5:

Northern blots for selected novel C/D box snoRNAs. Among the 20 most abundantly expressed (in the small RNA-seq data) novel C/D box snoRNAs we could confirm the presence of ZL1, ZL2, ZL8, ZL11, ZL63, ZL107, ZL116, ZL126 and ZL127 by Northern blotting.

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Additional file 6:

Expression of C/D box and C/D box-like snoRNAs in our small RNA-seq run (20 to 200 nucleotides; sequenced 150 cycles). Only reads that cover at least 50% of the snoRNA locus were considered.

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Additional file 7:

SCARNA21 has a C/D box H/ACA box hybrid structure. (A) Screenshot from the UCSC genome browser showing conserved C and D box elements. (B) Northern blot probing for H/ACA box structure only (left) and for the hybrid structure (right).

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Additional file 8:

Primer extension assays for U2 snRNA. Primer extension assay reveals a 2'-O-methyl modification site for nucleotide U47.

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Additional file 9:

Primer extension assays for non-canonical snoRNA targets. Primer extension runs reveal 2'-O-methyl (A-C) and pseudouridine (D-G) modification sites in several non-canonical RNAs. (A) SNORA61: G50. (B) VTRNA1-2: G30, U31, C33, A34. (C) 7SK RNA: C137, G139, C141, G148, C150, G151. (D) SNORD16: U52, U55. (E) SNORD35A: U26, U31, U37, U43, U45, U51. (F) 7SK RNA: U250. (G) 7SL RNA: U226, U233, U236, U266, U273.

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Additional file 10:

Summary of nucleotide modifications detected by primer extension assays and predicted guide snoRNA-target interactions.

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Additional file 11:

Analysis of PAR-CLIP clusters overlapping with mRNA exon annotation. Shown are genome coordinates, host transcript and exon identifier, the number of C and D boxes predicted within the genomic region, snoRNAs to whose guide regions these mRNA fragments are complementary and the number of (normalized) reads obtained from the regions in various PAR-CLIP libraries.

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Additional file 12:

Detailed list of reads mapping to snoRNA loci in Ago2 IP-seq libraries.

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Additional file 13:

Supplementary materials and methods. Detailed information about the experimental methods (PAR-CLIP library preparation, Northern blotting, primer extension assays, mitotic shake-off and Ago2 immunoprecipitation and sequencing). In addition, the annotated C/D and H/ACA snoRNAs used in this study are listed.

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Additional file 14:

Primer extension assays on spliceosomal RNA U6. (A) Primer extension assay on spliceosomal RNA U6 detected documented 2'-O-methylation as well as potentially novel 2'-O-methylation sites. (B) Primer extension assay detected documented pseudouridine sites in U6. CTRL indicates the untreated sample, +CMC the sample treated with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC).

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Additional file 15:

Asynchronous and mitotic GFP-tagged HeLa cells. Green fluorescent protein appears in green and cell boundaries in orange. (A) In an asynchronous cell culture only a few cells are in the mitotic phase, which can be seen from the condensed chromatin and the rounded cell morphology. (B) Cell obtained with mitotic shake-off. The procedure enriches for round cells containing condensed chromatin.

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Additional file 16:

Extended version of Figure 4B showing all snoRNA genes expressed in HEK293 cells.

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