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Plant U13 orthologues and orphan snoRNAs identified by RNomics of RNA from Arabidopsis nucleoli.

Kim SH, Spensley M, Choi SK, Calixto CP, Pendle AF, Koroleva O, Shaw PJ, Brown JW - Nucleic Acids Res. (2010)

Bottom Line: The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization.The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters.These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants.

View Article: PubMed Central - PubMed

Affiliation: Genetics Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK.

ABSTRACT
Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs. Three related capped snoRNAs with a distinct gene organization and structure were identified as orthologues of animal U13snoRNAs. In addition, eight of the novel genes had no complementarity to rRNAs or snRNAs and are therefore putative orphan snoRNAs potentially reflecting wider functions for these RNAs. The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization. The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters. These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants.

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RT–PCR expression analysis of novel and extended polycistronic snoRNA gene clusters. (A–C) RT–PCR of polycistronic snoRNA clusters. Primers were positioned in the genes indicated above the lanes (see also Supplementary Figures S1 and S2) and the expected RT–PCR product sizes are indicated below the lanes and are consistent with size markers. Some primers pairs were able to amplify more than one related precursor RNA (B). (A and D) Transcripts from single genes were amplified using primers to the 5′ and 3′ ends of the coding sequences.
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Figure 5: RT–PCR expression analysis of novel and extended polycistronic snoRNA gene clusters. (A–C) RT–PCR of polycistronic snoRNA clusters. Primers were positioned in the genes indicated above the lanes (see also Supplementary Figures S1 and S2) and the expected RT–PCR product sizes are indicated below the lanes and are consistent with size markers. Some primers pairs were able to amplify more than one related precursor RNA (B). (A and D) Transcripts from single genes were amplified using primers to the 5′ and 3′ ends of the coding sequences.

Mentions: Many of the novel uncapped snoRNAs were part of polycistronic gene clusters (for example—Figure 2A and B). To confirm that the new and updated clusters are indeed transcribed as polycistronic pre-snoRNAs, we performed RT–PCR with primers to genes in various clusters using total RNA from Arabidopsis cell culture cells. Polycistronic precursor snoRNA transcripts of the expected sizes were detected for all clusters tested (Figure 5A–C and Supplementary Figure S7). In addition, four of the novel genes and some variants appeared to be single genes and expression of snoR148 and U19-1 (intronic) was also confirmed by RT–PCR (Figure 5A and D).Figure 5.


Plant U13 orthologues and orphan snoRNAs identified by RNomics of RNA from Arabidopsis nucleoli.

Kim SH, Spensley M, Choi SK, Calixto CP, Pendle AF, Koroleva O, Shaw PJ, Brown JW - Nucleic Acids Res. (2010)

RT–PCR expression analysis of novel and extended polycistronic snoRNA gene clusters. (A–C) RT–PCR of polycistronic snoRNA clusters. Primers were positioned in the genes indicated above the lanes (see also Supplementary Figures S1 and S2) and the expected RT–PCR product sizes are indicated below the lanes and are consistent with size markers. Some primers pairs were able to amplify more than one related precursor RNA (B). (A and D) Transcripts from single genes were amplified using primers to the 5′ and 3′ ends of the coding sequences.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875012&req=5

Figure 5: RT–PCR expression analysis of novel and extended polycistronic snoRNA gene clusters. (A–C) RT–PCR of polycistronic snoRNA clusters. Primers were positioned in the genes indicated above the lanes (see also Supplementary Figures S1 and S2) and the expected RT–PCR product sizes are indicated below the lanes and are consistent with size markers. Some primers pairs were able to amplify more than one related precursor RNA (B). (A and D) Transcripts from single genes were amplified using primers to the 5′ and 3′ ends of the coding sequences.
Mentions: Many of the novel uncapped snoRNAs were part of polycistronic gene clusters (for example—Figure 2A and B). To confirm that the new and updated clusters are indeed transcribed as polycistronic pre-snoRNAs, we performed RT–PCR with primers to genes in various clusters using total RNA from Arabidopsis cell culture cells. Polycistronic precursor snoRNA transcripts of the expected sizes were detected for all clusters tested (Figure 5A–C and Supplementary Figure S7). In addition, four of the novel genes and some variants appeared to be single genes and expression of snoR148 and U19-1 (intronic) was also confirmed by RT–PCR (Figure 5A and D).Figure 5.

Bottom Line: The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization.The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters.These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants.

View Article: PubMed Central - PubMed

Affiliation: Genetics Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK.

ABSTRACT
Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs. Three related capped snoRNAs with a distinct gene organization and structure were identified as orthologues of animal U13snoRNAs. In addition, eight of the novel genes had no complementarity to rRNAs or snRNAs and are therefore putative orphan snoRNAs potentially reflecting wider functions for these RNAs. The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization. The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters. These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants.

Show MeSH