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Novel Intronic RNA Structures Contribute to Maintenance of Phenotype in Saccharomyces cerevisiae

Katarzyna B. Hooks, Samina Naseeb, Steven Parker, Sam Griffiths-Jones and Daniela Delneri
Genetics July 1, 2016 vol. 203 no. 3 1469-1481; https://doi.org/10.1534/genetics.115.185363
Katarzyna B. Hooks
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
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Samina Naseeb
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
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Steven Parker
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
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Sam Griffiths-Jones
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
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  • For correspondence: d.delneri@manchester.ac.uksam.griffiths-jones@manchester.ac.uk
Daniela Delneri
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
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  • For correspondence: d.delneri@manchester.ac.uksam.griffiths-jones@manchester.ac.uk
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    Figure 1

    Predicted RNA structures. (A) For each intron of S. cerevisiae, the number of orthologous introns was counted among the 36 species included in the study. Plotted is the histogram of the number of introns (y-axis) yielding a specific number of orthologs (x-axis). Introns containing known snoRNAs are highlighted by name (snR). (B) Venn diagram showing the number of common RNA structures predicted by RNAz, Cmfinder, and EvoFold. For example, there are 14 introns with structures predicted by all programs. (C) Predicted consensus RNA structures of selected introns. For each chosen intron, an iterative procedure of searching for orthologous sequences and extending the predicted RNA structure resulted in a multiple sequence alignment, which was collapsed to a consensus sequence with a secondary structure. For each structure, the gene name and the length of the predicted structured region are shown. In the case of duplicated ribosomal gene introns where both paralogous introns share a similar structure, both gene names are given. Structure images were prepared using VARNA (Darty et al. 2009).

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    Figure 2

    RT-PCR confirmation of intron fates conducted on total RNA and low-weight-enriched RNA using random priming. PCR of genomic DNA was used as positive control. (A) Agarose gel confirming ncRNAs expressed from introns and maintained in the cell. (B) Agarose gel confirming ncRNA expression accompanied by complete mRNA splicing. (C) Agarose gel confirming ncRNA expression accompanied by alternative splicing. Arrows indicate the expected size of the PCR products according to the key. Lane designations for DNA templates: G, Genomic DNA (positive control); T, total cDNA; L, low-molecular-weight-enriched cDNA; and −, no template negative control. With the exception of GLC7 ncRNA, which was run on a separate gel, the images of mRNAs, introns, and ncRNAs for each gene were cropped from the same agarose gel picture with brightness and contrast applied equally across the entire image (full images available in Figure S1, Figure S2, Figure S3, Figure S4, Figure S5, and Figure S6). For small-size PCR products, cropping included primer dimers.

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    Figure 3

    Properties of loci containing intronic novel RNA predictions or snoRNAs. Quantifications were based on RNA-seq data of the same total RNA as used in Figure 2 (GEO accession no. GSE58884) and on CRAC data (GEO accession no. GSE40046). (A) Box plots showing ribosomal protein intron transcript levels in RPKM without normalization and with normalization to host gene transcript levels. Values are shown for all RP gene introns and 12 RP gene introns with predictions. (B) Association of introns with the exosome. We reanalyzed 16 independent sequencing experiments by Schneider et al. (2012) of RNA fragments cross-linked to exosome components. Reads mapping to each intron were normalized by the host gene expression estimated by RNA-seq. For each CRAC experiment an intron was given a percentile value of how frequently it was bound to an exosome component compared to other introns. Values presented are averaged percentile derived from 16 experiments and are shown for all introns, the 19 introns with predictions, and the eight introns containing snoRNAs. The levels of significance for the Mann–Whitney U-tests are represented as follows: *** P < 10−3; ** P < 0.01; * P < 0.05.

  • Figure 4
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    Figure 4

    Characterization of the size and expression of the ncRNA within GLC7 intron. (A) Northern blot of S. cerevisiae total RNA and oligos mimicking GLC7 intron. The blots were probed with strand-specific probes showing expression of ncRNA in antisense (left panel) and sense orientation (right panel) to the GLC7 gene. The estimation of the sizes is based on the 60-nt oligos visible on the film and a comparison with the markers visible on the membrane. (B) RT-PCR on total RNA and low-weight-enriched RNA using random priming showing the expression of ncRNA within the GLC7 intron. PCR of genomic DNA was used as positive control. Names of the primers are listed on the right and are the same as indicated in C. Gel annotation: G, genomic DNA; T, total cDNA; L, low-molecular-weight-enriched cDNA; and −, no template negative control. (C) Data uploaded into the University of California Santa Cruz genome browser for sequence annotation and data visualization presenting annotated GLC7 intron. Primer names used for RT-PCR are listed next to black, blue, and red boxes indicating their position with respect to the gene annotation below and the size of the corresponding PCR product. Lines joining primers mark the amplified sequences; the regions targeted for deletion by the loxP method are symbolized by the black box; the location of the strand-specific Northern probes are shown in green; and regions with the putative structure predicted by RNAz and CMfinder are shown in gray. All features map directly onto the fragment of the gene structure diagrams. The bottom panel represents the degree of conservation of gene regions among seven yeast species.

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    Figure 5

    Effects of GLC7 intron mutation. (A) Schematic representation of intron mutants used for phenotype studies. (B) The GLC7 ncRNA deletion and GLC7 ncRNA insertion mutants disrupting the structure sequence of the GLC7 intron were compared with the WT strain and the intronic negative control in F1 medium containing 0.9 M NaCl. Values in the box plots present the means of the AUC as determined by the R pracma package. Significance estimated by one-way ANOVA with Dunnett’s multiple comparison test (*** P < 0.0001). (C) Expression levels (average expression with SEM) of GLC7 mRNA in the WT, control mutant, and the GLC7 ncRNA deletion mutant grown in F1 and F1 + 0.9 M NaCl media, assessed by RT-qPCR. Significance estimated by one-way ANOVA with Dunnett’s multiple comparison test (*** P < 0.0001, ** P = 0.005).

Tables

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  • Table 1 Conservation of RNA structure predictions
    No.LocusGeneConservation
    1YBR189WRPS9BSaccharomycetaceae and Candida sp.
    YPL081WRPS9A
    2YDR064WRPS13Saccharomycetaceae
    3YDR381WYRA1Saccharomycetaceae
    4YER133WGLC7Saccharomyces sensu stricto
    5YFL031WHAC1Fungi and Metazoa
    6YFL034C-ARPL22BSaccharomycetaceae
    YLR061WRPL22A
    7YGL076CRPL7ASaccharomycetaceae and Candida sp.
    YPL198WRPL7B
    8YGL103WRPL28Saccharomyces sensu stricto
    9YGL178WMPT5Saccharomyces sensu stricto
    10YLR367WRPS22BSaccharomycetaceae except L. lactis
    5′ UTR
    11YLR367WRPS22BSaccharomycotina and Pezizomycotina
    snR44
    12YML017WPSP2Saccharomyces sensu stricto
    13YML056CIMD4Saccharomycetales and Diptera
    snR54
    14YNL301CRPL18BSaccharomycetaceae and Candida sp.
    YOL120CRPL18A
    15YNR053CNOG2Saccharomycetaceae and Candida sp.
    snR191

Additional Files

  • Figures
  • Tables
  • Supplemental Material for Hooks et al., 2016

    Supporting Information

    • Figure S1 - RT-PCR confirmation of ncRNA expressed from introns. (.pdf, 4 MB)
    • Figure S2 - RT-PCR confirmation of intron expression accompanied by complete mRNA splicing. (.pdf, 3 MB)
    • Figure S3 - RT-PCR confirmation of intron expression accompanied by complete mRNA splicing. (.pdf, 3 MB)
    • Figure S4 - RT-PCR confirmation of alternative splicing. (.pdf, 5 MB)
    • Figure S5 - RT-PCR confirmation of alternative splicing. (.pdf, 5 MB)
    • Figure S6 - Control RT-PCRs confirming mRNA splicing status and intron expression. (.pdf, 1 MB)
    • Figure S7 - Growth of GLC7 mutants in F1 and F1 + NaCl media. (.pdf, 349 KB)
    • Figure S8 - Phenotypic effect of reintroducing ncRNA into GLC7 ncRNA deletion mutant. (.pdf, 187 KB)
    • Table S1 - List of fungal genomes used for BLAST searches. (.pdf, 103 KB)
    • File S1 - List of RNAz, CMfinder and EvoFold predictions for each intron. (.xls, 93 KB)
    • File S2 - Primer sequences and probes used in the study. (.xls, 69 KB)
    • File S3 - Expression of introns and coding sequences of host genes RPKM and the average percentile from exosome targets data (CRAC) for each gene. (.xls, 168 KB)
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Volume 203 Issue 3, July 2016

Genetics: 203 (3)

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Novel Intronic RNA Structures Contribute to Maintenance of Phenotype in Saccharomyces cerevisiae

Katarzyna B. Hooks, Samina Naseeb, Steven Parker, Sam Griffiths-Jones and Daniela Delneri
Genetics July 1, 2016 vol. 203 no. 3 1469-1481; https://doi.org/10.1534/genetics.115.185363
Katarzyna B. Hooks
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
  • Find this author on Google Scholar
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Samina Naseeb
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
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Steven Parker
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
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Sam Griffiths-Jones
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
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  • For correspondence: d.delneri@manchester.ac.uksam.griffiths-jones@manchester.ac.uk
Daniela Delneri
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
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  • For correspondence: d.delneri@manchester.ac.uksam.griffiths-jones@manchester.ac.uk
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Citation

Novel Intronic RNA Structures Contribute to Maintenance of Phenotype in Saccharomyces cerevisiae

Katarzyna B. Hooks, Samina Naseeb, Steven Parker, Sam Griffiths-Jones and Daniela Delneri
Genetics July 1, 2016 vol. 203 no. 3 1469-1481; https://doi.org/10.1534/genetics.115.185363
Katarzyna B. Hooks
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Samina Naseeb
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Steven Parker
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
  • Find this author on Google Scholar
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Sam Griffiths-Jones
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
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  • For correspondence: d.delneri@manchester.ac.uksam.griffiths-jones@manchester.ac.uk
Daniela Delneri
Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: d.delneri@manchester.ac.uksam.griffiths-jones@manchester.ac.uk

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