Journal: Nucleic Acids Research

Article Title: Cloning and expression of new microRNAs from zebrafish

doi: 10.1093/nar/gkl278

Figure Lengend Snippet: Cloning frequency and conservation for all miRNAs cloned from zebrafish small-RNA cDNA libraries. The upper panel depicts the cloning frequency for all small RNAs that were found in the two libraries and that passed our computational pipeline. All 139 known miRNAs (blue data points) were cloned more than once, while 37 out of the 66 new miRNAs (pink dots) were represented by a single sequenced clone. The lower panel shows a scatter plot of the conservation of known (blue dots) and new (pink data dots) miRNAs in 12 vertebrate species (zebrafish, fugu, tetraodon, mouse, rat, human, dog, macaca, opossum, chicken, chimpanzee, cow). Forty-four of the new miRNAs were only found in zebrafish, while most of the known miRNAs were found in several species according to our conservation criteria.

Article Snippet: Two small-RNA cDNA libraries were prepared by Vertis Biotechnologie AG (Freising-Weihenstephan, Germany).

Techniques: Cloning, Clone Assay

Journal: The Plant journal : for cell and molecular biology

Article Title: The PPR-SMR protein PPR53 enhances the stability and translation of specific chloroplast RNAs in maize.

doi: 10.1111/tpj.13093

Figure Lengend Snippet: Figure 5. Analysis of chloroplast rRNAs in ppr53 mutants. (a) RNA gel blot analysis of transcripts from the rrn transcription unit. Replicate blots of seedling leaf RNA were hybridized to the probes indicated on the map. Three heteroallelic ppr53 individuals with distinct pigment phenotypes (iv vir, vpyg vir and vir; see Figure 1) were analyzed. RNAs from the non-photosynthetic mutants atp4 and wtf2 were analyzed for comparison. The wtf2 mutant is shown to illustrate pleiotropic effects resulting from the loss of plastid ribosomes. An image of one of the blots stained with methylene blue is shown below to illustrate equal loading of cytosolic 28S and 18S rRNAs. (b) Primer extension assay to quantify processed transcripts with a 50-end mapping 70 nucleotides upstream of rrn23. A 50-end labeled 24-nucleotide primer starting four nucleotides upstream of mature 23S rRNA was used to prime reverse transcription on 5 lg of seedling leaf RNA. The ribosome-deficient mutant wtf2 (see panel a) as well as two other non-photosynthetic mutants (atp4 and crp1) accumulate increased levels of the 70 23S rRNA precursor (see arrow), whereas it is undetectable in all three ppr53 mutant individuals. The abundance of longer rrn23 processing intermediates is similar in all mutant samples ana- lyzed, as shown also on the RNA gel blot in panel (a) (see probe 2 data). (c) PPR53-dependent sRNA mapping to the 50-end of the PPR53-dependent rrn23 precursor. Screen captures from the Integrated Genome Viewer show reads as pink lines and a histogram of read counts in gray (above). The 50-end of the PPR53-dependent sRNAs corresponds to that of the PPR53-dependent pre-23S rRNA. The sRNA reads from flanking regions serve as internal standards. Data from a ppr4 mutant are shown to control for effects resulting from the loss of plastid ribosomes. The loss of this PPR53-dependent sRNA is accompanied by an increase in the abundance of an sRNA with a 50-end two nucleotides upstream. The basis for this effect is unknown.

Article Snippet: Sequencing libraries were prepared with the NEBNext Multiplex Small RNA Library Prep Set.

Techniques: Western Blot, Comparison, Mutagenesis, Staining, Primer Extension Assay, Labeling, Reverse Transcription, Control

Journal: Genome Research

Article Title: Transcriptional activity and epigenetic regulation of transposable elements in the symbiotic fungus Rhizophagus irregularis

doi: 10.1101/gr.275752.121

Figure Lengend Snippet: Isolation of 2′-O-methylated and Argonaute-loaded small RNA. (A) Schematic representation of the sRNA-seq analysis pipeline used in this study. (B,C) Plot of mean normalized counts against log2 fold change (log2FC) in sRNAs sequenced following enrichment using NaIO4 oxidation (B) or TraPR column purification (C), compared to sRNAs sequenced following no treatment. Points represent individual sRNA loci. Significantly differentially expressed loci are shown in red. Mitochondria-derived sRNA loci are shown in blue. (D–F) Length distribution and first nucleotide bias of the sRNAs in untreated (D), NaIO4-treated (E), and TraPR column-extracted (F) sRNA libraries. (G) Significantly differentially expressed sRNA loci. sRNAs enriched by NaIO4 or column-purification were induced compared to expression in untreated samples, and sRNAs depleted by NaIO4 or column-purification were down-regulated.

Article Snippet: For Lexogen's TraPR Small RNA Isolation, 20 mg of ground spore material was suspended in TRaPR lysis buffer and the standard TRaPR experimental procedure and RNA extraction was carried out (Lexogen).

Techniques: Isolation, Methylation, Purification, Derivative Assay, Expressing

Journal: Genome Research

Article Title: Transcriptional activity and epigenetic regulation of transposable elements in the symbiotic fungus Rhizophagus irregularis

doi: 10.1101/gr.275752.121

Figure Lengend Snippet: Genomic origin of small RNA–producing loci. (A) Genomic location of 3067 R. irregularis sRNA loci. The number of sRNA loci derived from nonexpressed and expressed TEs, unannotated regions, and genes is represented in a pie chart. Bar chart shows the number of genes of each class that produce sRNA. (B) mCG scores of expressed sRNA loci derived from nonexpressed TEs, expressed TEs, unannotated features, and expressed genes. Red dots represent the median mCG values of sRNAs from each feature. (C) Number of sRNA loci associated with different TE superfamilies. Percentage of TE copies from each superfamily that produce sRNA is shown on the right. Green color code indicates the relative age of sRNA-producing TEs, represented in Kimura distance bins between 0 and 40. Expressed sRNAs have been grouped into bins based on Kimura distance and hence relative age, represented using color coding. (D) Distance from sRNA loci derived from unannotated regions and expressed genes to closest known TE. Significance was assessed by a nonparametric Kruskal–Wallis test comparing the distance distribution of unannotated or genic sRNA loci to shuffled loci, respectively. A Kruskal–Wallis H test comparing unannotated and genic sRNA loci to shuffled loci was used to assess significance. (*) P = 2.8 × 10−6, (**) P < 2.2 × 10−16. (E) Schematic diagram of the genomic distribution of sRNA loci hypothesized due to data from this study of the R. irregularis genome, sRNAome, methylome, and transcriptome. sRNA loci, methylation levels, and RNA expression are displayed. The proportion of sRNA loci corresponding to each genomic context is indicated in blue boxes. Curvy lines highlight the proximity between TEs and genic and unannotated sRNA loci. Expression of unannotated regions was not analyzed and is represented by a dotted line.

Article Snippet: For Lexogen's TraPR Small RNA Isolation, 20 mg of ground spore material was suspended in TRaPR lysis buffer and the standard TRaPR experimental procedure and RNA extraction was carried out (Lexogen).

Techniques: Derivative Assay, Methylation, RNA Expression, Expressing