Systematic characterization of artificial small RNA-mediated inhibition of Escherichia coli growth.

A new screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli was constructed. In this system, we used a plasmid library to express RNAs of ∼120 nucleotides, each with a random 30-nucleotide sequence that can recognize its target mRNA(s). After approximately 60,000 independent colonies were screened, several plasmids that inhibited bacterial growth were isolated. To understand the inhibitory mechanism, we focused on one sRNA, S-20, that exerted a strong inhibitory effect. A time-course analysis of the proteome of S-20-expressing E. coli and a bioinformatic analysis were used to identify potential S-20 target mRNAs, and suggested that S-20 binds the translation initiation sites of several mRNAs encoding enzymes such as peroxiredoxin (osmC), glycyl-tRNA synthetase α subunit (glyQ), uncharacterized protein ygiM, and tryptophan synthase ß chain (trpB). An in vitro translation analysis of chimeric luciferase-encoding mRNAs, each containing a potential S-20 target sequence, indicated that the translation of these mRNAs was inhibited in the presence of S-20. A gel shift analysis combined with the analysis of a series of S-20 mutants suggested that S-20 targets multiple mRNAs that are responsible for inhibiting E. coli growth. These data also suggest that S-20 acts like an endogenous sRNA and that E. coli can utilize artificial sRNAs.

Precise mapping and dynamics of tRNA-derived fragments (tRFs) in the development of Triops cancriformis (tadpole shrimp).

BACKGROUND: In a deep sequencing analysis of small RNAs prepared from a living fossil, the tadpole shrimp Triops cancriformis, a 32-nt small RNA was specifically detected in the adult stage. A nucleotide sequence comparison between the 32-nt small RNA and predicted tRNA sequences in the draft nuclear genomic DNA showed that the small RNA was derived from tRNA(Gly)(GCC). To determine the overall features of the tRNA-derived fragments (tRFs) of T. cancriformis, the small RNA sequences in each of the six developmental stages (egg, 1st-4th instar larvae, and adult) were compared with the mitochondrial and nuclear tRNA sequences. RESULTS: We found that the tRFs were derived from mitochondrial and nuclear tRNAs corresponding to 16 and 39 anticodons, respectively. The total read number of nuclear tRFs was approximately 400 times larger than the number of mitochondrial tRFs. Interestingly, the main regions in each parental tRNA from which these tRFs were derived differed, depending on the parental anticodon. Mitochondrial tRF(Ser)(GCU)s were abundantly produced from the 5' half regions of the parental tRNA, whereas mitochondrial tRF(Val)(UAC)s were mainly produced from the 3' end regions. Highly abundant nuclear tRFs, tRF(Gly)(GCC)s, tRF(Gly)(CCC)s, tRF(Glu)(CUC)s, and tRF(Lys)(CUU)s were derived from the 5' half regions of the parental tRNAs. Further analysis of the tRF read counts in the individual developmental stages suggested that the expression of mitochondrial and nuclear tRFs differed during the six stages. Based on these data, we precisely summarized the positions of the tRFs in their parental tRNAs and their expression changes during development. CONCLUSIONS: Our results reveal the entire dynamics of the tRFs from both the nuclear and mitochondrial genomes of T. cancriformis and indicate that the majority of tRFs in the cell are derived from nuclear tRNAs. This study provides the first examples of developmentally expressed mitochondrial tRFs.

Identification, expression, and molecular evolution of microRNAs in the "living fossil" Triops cancriformis (tadpole shrimp).

MicroRNAs have been identified and analyzed in various model species, but an investigation of miRNAs in nonmodel species is required for a more complete understanding of miRNA evolution. In this study, we investigated the miRNAs of the nonmodel species Triops cancriformis (tadpole shrimp), a "living fossil," whose morphological form has not changed in almost 200 million years. Dramatic ontogenetic changes occur during its development. To clarify the evolution of miRNAs, we comparatively analyzed its miRNAs and the components of its RNAi machinery. We used deep sequencing to analyze small RNA libraries from the six different developmental stages of T. cancriformis (egg, first-fourth instars, and adult), and also analyzed its genomic DNA with deep sequencing. We identified 180 miRNAs (87 conserved miRNAs and 93 novel candidate miRNAs), and deduced the components of its RNAi machinery: the DICER1, AGO1-3, PIWI, and AUB proteins. A comparative miRNA analysis of T. cancriformis and Drosophila melanogaster showed inconsistencies in the expression patterns of four conserved miRNAs. This suggests that although the miRNA sequences of the two species are very similar, their roles differ across the species. An miRNA conservation analysis revealed that most of the conserved T. cancriformis miRNAs share sequence similarities with those of arthropods, although T. cancriformis is called a "living fossil." However, we found that let-7 and DICER1 of T. cancriformis are more similar to those of the vertebrates than to those of the arthropods. These results suggest that miRNA systems of T. cancriformis have evolved in a unique fashion.

Cul8/Rtt101 forms a variety of protein complexes that regulate DNA damage response and transcriptional silencing.

The budding yeast, Saccharomyces cerevisiae, has three cullin proteins, which act as platforms for Cullin-based E3 ubiquitin ligases. Genetic evidence indicates that Cul8, together with Mms1, Mms22, and Esc4, is involved in the repair of DNA damage that can occur during DNA replication. Cul8 is thought to form a complex with these proteins, but the composition and the function of Cul8-based E3 ubiquitin ligases remain largely uncharacterized. Herein, we report a comprehensive biochemical analysis of Cul8 complexes. Cul8 was found to form a Cul8-Mms1-Mms22-Esc4 complex under physiological conditions, with Mms1 bridging Cul8 and Mms22 and Mms22 bridging Mms1 and Esc4. Domain analysis demonstrated that the N-terminal region of Mms1 and the C-terminal region of Mms22 are required for the Mms1-Mms22 interaction, whereas the N-terminal region of Mms22 is required for the Mms22-Esc4 interaction. We also found other Cul8-Mms1-binding proteins Ctf4, Esc2, and Orc5 using yeast two-hybrid screening. Esc4 and Ctf4 bound to Mms22 directly and bound to Cul8-Mms1 in the presence of Mms22, whereas Esc2 and Orc5 interacted with both Cul8 and Mms1, independently. We found that Cul8, Mms1, and Mms22 participated in the regulation of transcriptional silencing of yeast telomeres. These results suggest that Cul8-Mms1, as part of various protein complexes, is involved in the regulation of chromatin metabolism.