Detection of 5′- and 3′-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli

Evidence is accumulating that small, noncoding RNAs are important regulatory molecules. Computational and experimental searches have led to the identification of ∼60 small RNA genes in Escherichia coli. However, most of these studies focused on the intergenic regions and assumed that small RNAs were >50 nt. Thus, the previous screens missed small RNAs encoded on the antisense strand of protein-coding genes and small RNAs of <50 nt. To identify additional small RNAs, we carried out a cloning-based screen focused on RNAs of 30–65 nt. In this screen, we identified RNA species corresponding to fragments of rRNAs, tRNAs and known small RNAs. Several of the small RNAs also corresponded to 5′- and 3′-untranslated regions (UTRs) and internal fragments of mRNAs. Four of the 3′-UTR-derived RNAs were highly abundant and two showed expression patterns that differed from the corresponding mRNAs, suggesting independent functions for the 3′-UTR-derived small RNAs. We also detected three previously unidentified RNAs encoded in intergenic regions and RNAs from the long direct repeat and hok/sok elements. In addition, we identified a few small RNAs that are expressed opposite protein-coding genes and could base pair with 5′ or 3′ ends of the mRNAs with perfect complementarity

pre-miRNA profiles obtained through application of locked nucleic acids and deep sequencing reveals complex 5′/3′ arm variation including concomitant cleavage and polyuridylation patterns

Recent research hints at an underappreciated complexity in pre-miRNA processing and regulation. Global profiling of pre-miRNA and its potential to increase understanding of the pre-miRNA landscape is impeded by overlap with highly expressed classes of other non coding (nc) RNA. Here, we present a data set excluding these RNA before sequencing through locked nucleic acids (LNA), greatly increasing pre-miRNA sequence counts with no discernable effect on pre-miRNA or mature miRNA sequencing. Analysis of profiles generated in total, nuclear and cytoplasmic cell fractions reveals that pre-miRNAs are subject to a wide range of regulatory processes involving loci-specific 3′- and 5′-end variation entailing complex cleavage patterns with co-occurring polyuridylation. Additionally, examination of nuclear-enriched flanking sequences of pre-miRNA, particularly those derived from polycistronic miRNA transcripts, provides insight into miRNA and miRNA-offset (moRNA) production, specifically identifying novel classes of RNA potentially functioning as moRNA precursors. Our findings point to particularly intricate regulation of the let-7 family in many ways reminiscent of DICER1-independent, pre-mir-451-like processing, introduce novel and unify known forms of pre-miRNA regulation and processing, and shed new light on overlooked products of miRNA processing pathways

Detection of low-level promoter activity within open reading frame sequences of Escherichia coli

The search for promoters has largely been confined to sequences upstream of open reading frames (ORFs) or stable RNA genes. Here we used a cloning approach to discover other potential promoters in Escherichia coli. Chromosomal fragments of ∼160 bp were fused to a promoterless lacZ reporter gene on a multi-copy plasmid. Eight clones were deliberately selected for high activity and 105 clones were selected at random. All eight of the high-activity clones carried promoters that were located upstream of an ORF. Among the randomly-selected clones, 56 had significantly elevated activity. Of these, 7 had inserts which also mapped upstream of an ORF, while 49 mapped within or downstream of ORFs. Surprisingly, the eight promoters selected for high activity matched the canonical σ(70) −35 and −10 sequences no better than sequences from the randomly-selected clones. For six of the nine most active sequences with orientations opposite to that of the ORF, chromosomal expression was detected by RT–PCR, but defined transcripts were not detected by northern analysis. Our results indicate that the E.coli chromosome carries numerous −35 and −10 sequences with weak promoter activity but that most are not productively expressed because other features needed to enhance promoter activity and transcript stability are absent

Comprehensive analysis of liver and blood miRNA in precancerous conditions

Streptozotocin administration to mice (STZ-mice) induces type I diabetes and hepatocellular carcinoma (HCC). We attempted to elucidate the carcinogenic mechanism and the miRNA expression status in the liver and blood during the precancerous state. Serum and liver tissues were collected from STZ-mice and non-treated mice (CTL-mice) at 6, 10, and 12 W. The exosome enriched fraction extracted from serum was used. Hepatic histological examination and hepatic and exosomal miRNA expression analysis were serially performed using next-generation sequencing (NGS). Human miRNA expression analysis of chronic hepatitis liver tissue and exosomes, which were collected before starting the antiviral treatment, were also performed. No inflammation or fibrosis was found in the liver of CTL-mice during the observation period. In STZ-mice, regeneration and inflammation of hepatocytes was found at 6 W and nodules of atypical hepatocytes were found at 10 and 12 W. In the liver tissue, during 6–12 W, the expression levels of let-7f-5p, miR-143-3p, 148a-3p, 191-5p, 192-5p, 21a-5p, 22-3p, 26a-5p, and 92a-3p was significantly increased in STZ-mice, and anti-oncogenes of their target gene candidates were down-regulated. miR-122-5p was also significantly down-regulated in STZ-mice. Fifteen exosomal miRNAs were upregulated in STZ-mice. Six miRNAs (let-7f-5p, miR-10b-5p, 143-3p, 191-5p, 21a-5p, and 26a-5p) were upregulated, similarly to human HCC cases. From the precancerous state, aberrant expression of hepatic miRNAs has already occurred, and then, it can promote carcinogenesis. In exosomes, the expression pattern of common miRNAs between mice and humans before carcinogenesis was observed and can be expected to be developed as a cancer predictive marker

Involvement of an intracellular vesicular transport process in naked-sgRNA-mediated TRUE gene silencing.

tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is an RNA-mediated gene expression control technology with therapeutic potential. Recently, our group demonstrated that a heptamer, mh1 (Bcl‑2), targeting human Bcl-2 mRNA, can be taken up by cells without the use of any transfection reagents and can induce the apoptosis of leukemia cells. However, little is known regarding the mechanism of naked small guide (sg)RNA uptake by cultured cells. Therefore, in the present study the effects of various inhibitors on the induction of apoptosis by naked sgRNA treatment were investigated in order to identify the uptake pathway required for sgRNA function in cultured cells. Addition of the endocytosis inhibitors chlorpromazine, nystatin or methyl‑β‑cyclodextrin together with naked effective sgRNA was unable to diminish the apoptosis‑inducing effects of naked sgRNA or the reduction in target mRNA, suggesting that functional uptake of sgRNA by cells is clathrin‑, caveolae‑ and raft‑independent. Next, chloroquine, an inhibitor of lysosome acidification, and brefeldin A, an inhibitor that blocks protein transport from the Golgi apparatus to the endoplasmic reticulum were administered. In the presence of these compounds, the apoptosis‑inducing effects of naked sgRNA were reduced. These results suggest that a vesicular transport process is involved in sgRNA‑mediated TRUE gene silencing. A greater understanding of how naked sgRNAs enter cells and how they reach their target RNAs may aid in the design of more specifically‑targeted and potent sgRNA drugs

Circulating osteocyte-derived exosomes contain miRNAs which are enriched in exosomes from MLO-Y4 cells

Signaling molecules produced by osteocytes have been proposed to serve as soluble factors that contribute to bone remodeling, as well as to homeostasis of other organs. However, to the best of our knowledge, there are currently no studies investigating the role of osteocyte-secreted exosomes. In the present study, ablation of osteocytes in mice [osteocyte-less (OL)] was used to examine the microRNA (miRNA) levels of plasma-circulating exosomes. In order to investigate the function of osteocyte-secreted exosomes, exosomes derived from MLO-Y4 cells were extracted and their miRNA expression levels were examined using miRNA array analysis and deep sequencing. Comparison of miRNA expression levels between plasma exosomes from OL mouse plasma and MLO-Y4-derived exosomes revealed that decreases in the number of miRNAs from exosomes circulating in the OL mouse plasma may be caused by a decrease in secretion of exosomes from osteocytes. These results suggest that osteocytes secrete exosomes containing characterized miRNAs and then circulate in the blood, and may thus transfer their components, including miRNAs, to recipient cells where they function as signaling molecules in other organs and/ or tissues to regulate biological responses

Novel Small Noncoding RNAs in Mouse Spermatozoa, Zygotes and Early Embryos

The recent discovery of a significant amount of RNA in spermatozoa contradicted the previously held belief that paternal contribution was limited to one copy of the genome. Furthermore, detection of RNA in sperm raised the intriguing question of its possible role in embryonic development. The possibility that RNAs may serve as epigenetic determinants was supported by experiments showing inheritance of epigenetic traits in mice mediated by RNA. We used high-throughput, large-scale sequencing technology to analyze sperm RNA. The RNA sequences generated were diverse in terms of length and included mRNAs, rRNAs, piRNAs, and miRNAs. We studied two small noncoding RNAs enriched in mature sperm, designated sperm RNAs (spR) -12 and -13. They are both encoded in a piRNA locus on chromosome 17, but neither their length (20-21 nt), nor their sequences correspond to known piRNAs or miRNAs. They are resistant to periodate-oxidation-mediated reaction, implying that they undergo terminal post-transcriptional modification. Both were detected in sperm and ovulated unfertilized oocytes, present in one-cell embryos and maintained in preimplantation stages, but not at later differentiation stages. These findings offer a new perspective regarding a possibly important role for gamete-specific small RNAs in early embryogenesis