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: 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