Hyeshik Chang

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A truncated form of dicer tilts the balance of RNA interference pathways.

Cell Rep. 2013 Aug 15;4(3):454-63

Authors: Sawh AN, Duchaine TF

Abstract
The RNase III enzyme Dicer is responsible for key steps in the biogenesis of small RNA species in multiple RNA interference pathways. Here, we show that, in the adult C. elegans soma, half of the total DCR-1 protein is expressed as a truncated, stable C-terminal fragment named small DCR-1 (sDCR-1). sDCR-1 operates independently of full-length DCR-1 in two distinct RNAi pathways; it enhances exogenous RNAi (exoRNAi) and concurrently acts as a negative regulator of microRNA (miRNA) biogenesis. Enhancement of exoRNAi relies on sDCR-1 catalytic activity, whereas impinging on miRNA processing does not. Instead, sDCR-1 competes with pre-miRNA processing by interacting with the miRNA-dedicated Argonautes ALG-1 and ALG-2. Finally, triggering a strong exoRNAi response in the presence of elevated levels of sDCR-1 exacerbates the miRNA processing defect. Our results unveil a surprising role for a truncated form of DCR-1 in the modulation of multiple RNAi activities and in the regulation of mechanistic boundaries between pathways.

PMID: 23933256 [PubMed - indexed for MEDLINE]

4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells.

Genome Biol. 2014 May 16;15(5):R69

Authors: Fuchs G, Voichek Y, Benjamin S, Gilad S, Amit I, Oren M

Abstract
Although transcriptional elongation by RNA polymerase II is coupled with many RNA-related processes, genomewide elongation rates remain unknown. We describe a method, called 4sUDRB-seq, based on reversible inhibition of transcription elongation coupled with tagging newly transcribed RNA with 4-thiouridine and high throughput sequencing to measure simultaneously with high confidence genome-wide transcription elongation rates in cells. We find that most genes are transcribed at about 3.5 kb/min, with elongation rates varying between 2 kb/min - 6 kb/min. 4sUDRB-seq can facilitate genomewide exploration of the involvement of specific elongation factors in transcription and the contribution of deregulated transcription elongation to various pathologies.

PMID: 24887486 [PubMed - as supplied by publisher]

Widespread Changes in the Posttranscriptional Landscape at the Drosophila Oocyte-to-Embryo Transition.

Cell Rep. 2014 May 28;

Authors: Kronja I, Yuan B, Eichhorn SW, Dzeyk K, Krijgsveld J, Bartel DP, Orr-Weaver TL

Abstract
The oocyte-to-embryo transition marks the onset of development. The initial phase of this profound change from the differentiated oocyte to the totipotent embryo occurs in the absence of both transcription and mRNA degradation. Here we combine global polysome profiling, ribosome-footprint profiling, and quantitative mass spectrometry in a comprehensive approach to delineate the translational and proteomic changes that occur during this important transition in Drosophila. Our results show that PNG kinase is a critical regulator of the extensive changes in the translatome, acting uniquely at this developmental window. Analysis of the proteome in png mutants provided insights into the contributions of translation to changes in protein levels, revealing a compensatory dynamic between translation and protein turnover during proteome remodeling at the return to totipotency. The proteome changes additionally suggested regulators of meiosis and early embryogenesis, including the conserved H3K4 demethylase LID, which we demonstrated is required during this period despite transcriptional inactivity.

PMID: 24882012 [PubMed - as supplied by publisher]

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MicroRNAs and spermatogenesis.

Fertil Steril. 2014 Jun;101(6):1552-1562

Authors: Kotaja N

Abstract
In mammals, male gametes are produced inside the testis by spermatogenesis, which has three phases: mitotic proliferation of spermatogonia, meiosis of spermatocytes, and haploid differentiation of spermatids. The genome of male germ cells is actively transcribed to produce phase-specific gene expression patterns. Male germ cells have a complex transcriptome. In addition to protein-coding messenger RNAs, many noncoding RNAs, including microRNAs (miRNAs), are produced. The miRNAs are important regulators of gene expression. They function mainly post-transcriptionally to control the stability or translation of their target messenger RNAs. The miRNAs are expressed in a cell-specific manner during spermatogenesis to participate in the control of each step of male germ cell differentiation. Genetically modified mouse models have demonstrated the importance of miRNA pathways for normal spermatogenesis, and functional studies have been designed to dissect the roles of specific miRNAs in distinct cell types. Clinical studies have exploited the well-defined expression profiles of miRNAs, and human spermatozoal or seminal plasma miRNAs have been explored as potential biomarkers for male factor infertility. This review article discusses the current findings that support the central role of miRNAs in the regulation of spermatogenesis and male fertility.

PMID: 24882619 [PubMed - as supplied by publisher]

We have prepared L- and D-deoxypolypeptides (DOPPs) by selective reduction of appropriately protected polyhistidines with borane, reducing the carbonyl groups to methylenes. The result is a chiral polyamine, not amide, with a mainly protonated backbone and chirally mounted imidazolylmethylene side chains that are mostly unprotonated at neutrality because of the...

Nature Structural & Molecular Biology 21, 552 (2014). doi:10.1038/nsmb.2827

Authors: Eleanor White, Margarita Schlackow, Kinga Kamieniarz-Gdula, Nick J Proudfoot & Monika Gullerova

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Comparison of RNA-Seq by poly (A) capture, ribosomal RNA depletion, and DNA microarray for expression profiling.

BMC Genomics. 2014 Jun 2;15(1):419

Authors: Zhao W, He X, Hoadley KA, Parker JS, Hayes DN, Perou CM

Abstract
BACKGROUND: RNA sequencing (RNA-Seq) is often used for transcriptome profiling as well as the identification of novel transcripts and alternative splicing events. Typically, RNA-Seq libraries are prepared from total RNA using poly(A) enrichment of the mRNA (mRNA-Seq) to remove ribosomal RNA (rRNA), however, this method fails to capture non-poly(A) transcripts or partially degraded mRNAs. Hence, a mRNA-Seq protocol will not be compatible for use with RNAs coming from Formalin-Fixed and Paraffin-Embedded (FFPE) samples.
RESULTS: To address the desire to perform RNA-Seq on FFPE materials, we evaluated two different library preparation protocols that could be compatible for use with small RNA fragments. We obtained paired Fresh Frozen (FF) and FFPE RNAs from multiple tumors and subjected these to different gene expression profiling methods. We tested 11 human breast tumor samples using: (a) FF RNAs by microarray, mRNA-Seq, Ribo-Zero-Seq and DSN-Seq (Duplex-Specific Nuclease) and (b) FFPE RNAs by Ribo-Zero-Seq and DSN-Seq. We also performed these different RNA-Seq protocols using 10 TCGA tumors as a validation set.The data from paired RNA samples showed high concordance in transcript quantification across all protocols and between FF and FFPE RNAs. In both FF and FFPE, Ribo-Zero-Seq removed rRNA with comparable efficiency as mRNA-Seq, and it provided an equivalent or less biased coverage on gene 3[prime] ends. Compared to mRNA-Seq where 69% of bases were mapped to the transcriptome, DSN-Seq and Ribo-Zero-Seq contained significantly fewer reads mapping to the transcriptome (20-30%); in these RNA-Seq protocols, many if not most reads mapped to intronic regions. Approximately 14 million reads in mRNA-Seq and 45-65 million reads in Ribo-Zero-Seq or DSN-Seq were required to achieve the same gene detection levels as a standard Agilent DNA microarray.
CONCLUSIONS: Our results demonstrate that compared to mRNA-Seq and microarrays, Ribo-Zero-Seq provides equivalent rRNA removal efficiency, coverage uniformity, genome-based mapped reads, and consistently high quality quantification of transcripts. Moreover, Ribo-Zero-Seq and DSN-Seq have consistent transcript quantification using FFPE RNAs, suggesting that RNA-Seq can be used with FFPE-derived RNAs for gene expression profiling.

PMID: 24888378 [PubMed - as supplied by publisher]

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One hundred million adenosine-to-inosine RNA editing sites: Hearing through the noise.

Bioessays. 2014 May 30;

Authors: Ulbricht RJ, Emeson RB

Abstract
The most recent work toward compiling a comprehensive database of adenosine-to-inosine RNA editing events suggests that the potential for RNA editing is much more pervasive than previously thought; indeed, it is manifest in more than 100 million potential editing events located primarily within Alu repeat elements of the human transcriptome. Pairs of inverted Alu repeats are found in a substantial number of human genes, and when transcribed, they form long double-stranded RNA structures that serve as optimal substrates for RNA editing enzymes. A small subset of edited Alu elements has been shown to exhibit diverse functional roles in the regulation of alternative splicing, miRNA repression, and cis-regulation of distant RNA editing sites. The low level of editing for the remaining majority may be non-functional, yet their persistence in the primate genome provides enhanced genomic flexibility that may be required for adaptive evolution.

PMID: 24889193 [PubMed - as supplied by publisher]

Study challenges conventional wisdom around morning meal

Laboratory training: Experimentation needs theory, too

Nature 510, 7503 (2014). doi:10.1038/510035e

Author: Min-Liang Wong

John Skoyles emphasizes the importance of practical experimental work for the developing scientist (Nature508, 319; 201410.1038/508319e). But theory is crucial too — for interpreting the results and for advancing research.In 1928, British physicist Paul Dirac came up with

miR-34/449 miRNAs are required for motile ciliogenesis by repressing cp110

Nature 510, 7503 (2014). doi:10.1038/nature13413

Authors: Rui Song, Peter Walentek, Nicole Sponer, Alexander Klimke, Joon Sub Lee, Gary Dixon, Richard Harland, Ying Wan, Polina Lishko, Muriel Lize, Michael Kessel & Lin He

The mir-34/449 family consists of six homologous miRNAs at three genomic loci. Redundancy of miR-34/449 miRNAs and their dominant expression in multiciliated epithelia suggest a functional significance in ciliogenesis. Here we report that mice deficient for all miR-34/449 miRNAs exhibited postnatal mortality, infertility and

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CAP-miRSeq: a comprehensive analysis pipeline for microRNA sequencing data.

BMC Genomics. 2014 Jun 3;15(1):423

Authors: Sun Z, Evans J, Bhagwate A, Middha S, Bockol M, Yan H, Kocher JP

Abstract
BACKGROUND: miRNAs play a key role in normal physiology and various diseases. miRNA profiling through next generation sequencing (miRNA-seq) has become the main platform for biological research and biomarker discovery. However, analyzing miRNA sequencing data is challenging as it needs significant amount of computational resources and bioinformatics expertise. Several web based analytical tools have been developed but they are limited to processing one or a pair of samples at time and are not suitable for a large scale study. Lack of flexibility and reliability of these web applications are also common issues.
RESULTS: We developed a Comprehensive Analysis Pipeline for microRNA Sequencing data (CAP-miRSeq) that integrates read pre-processing, alignment, mature/precursor/novel miRNA detection and quantification, data visualization, variant detection in miRNA coding region, and more flexible differential expression analysis between experimental conditions. According to computational infrastructure, users can install the package locally or deploy it in Amazon Cloud to run samples sequentially or in parallel for a large number of samples for speedy analyses. In either case, summary and expression reports for all samples are generated for easier quality assessment and downstream analyses. Using well characterized data, we demonstrated the pipeline's superior performances, flexibility, and practical use in research and biomarker discovery.
CONCLUSIONS: CAP-miRSeq is a powerful and flexible tool for users to process and analyze miRNA-seq data scalable from a few to hundreds of samples. The results are presented in the convenient way for investigators or analysts to conduct further investigation and discovery.

PMID: 24894665 [PubMed - as supplied by publisher]

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Computational Analysis of Conserved RNA Secondary Structure in Transcriptomes and Genomes.

Annu Rev Biophys. 2014 May 6;43:433-456

Authors: Eddy SR

Abstract
Transcriptomics experiments and computational predictions both enable systematic discovery of new functional RNAs. However, many putative noncoding transcripts arise instead from artifacts and biological noise, and current computational prediction methods have high false positive rates. I discuss prospects for improving computational methods for analyzing and identifying functional RNAs, with a focus on detecting signatures of conserved RNA secondary structure. An interesting new front is the application of chemical and enzymatic experiments that probe RNA structure on a transcriptome-wide scale. I review several proposed approaches for incorporating structure probing data into the computational prediction of RNA secondary structure. Using probabilistic inference formalisms, I show how all these approaches can be unified in a well-principled framework, which in turn allows RNA probing data to be easily integrated into a wide range of analyses that depend on RNA secondary structure inference. Such analyses include homology search and genome-wide detection of new structural RNAs.

PMID: 24895857 [PubMed - as supplied by publisher]

A few of these molecules are clearly important, but just how many? Author: Elizabeth Pennisi

by Fabienne F. V. Chevance, Soazig Le Guyon, Kelly T. Hughes

We developed a bacterial genetic system based on translation of the his operon leader peptide gene to determine the relative speed at which the ribosome reads single or multiple codons in vivo. Low frequency effects of so-called “silent” codon changes and codon neighbor (context) effects could be measured using this assay. An advantage of this system is that translation speed is unaffected by the primary sequence of the His leader peptide. We show that the apparent speed at which ribosomes translate synonymous codons can vary substantially even for synonymous codons read by the same tRNA species. Assaying translation through codon pairs for the 5′- and 3′- side positioning of the 64 codons relative to a specific codon revealed that the codon-pair orientation significantly affected in vivo translation speed. Codon pairs with rare arginine codons and successive proline codons were among the slowest codon pairs translated in vivo. This system allowed us to determine the effects of different factors on in vivo translation speed including Shine-Dalgarno sequence, rate of dipeptide bond formation, codon context, and charged tRNA levels.

Nature Medicine 20, 577 (2014). doi:10.1038/nm.3578

Authors: Benjamin Cogné, Richard Snyder, Pierre Lindenbaum, Jean-Baptiste Dupont, Richard Redon, Philippe Moullier & Adrien Leger

Nature Medicine 20, 595 (2014). doi:10.1038/nm.3604

Author: Michael Basson

Systematics balances uneasily between realism and nominalism, uncommitted as to whether biological taxa are discoveries or inventions. If the former, they might be taken as natural kinds. I briefly review some philosophers’ concepts of natural kinds and then argue that several of these apply well enough to "eukaryote." Although there are some sticky issues around genomic chimerism and when eukaryotes first appeared, if we allow for degrees in the naturalness of kinds, existing eukaryotes rank highly, higher than prokaryotes. Most biologists feel this intuitively: All I attempt to do here is provide some conceptual justification.

The antimetabolite 5-fluorouracil is a widely used chemotherapeutic for the treatment of several solid cancers. However, resistance to 5-fluorouracil remains a major drawback in its clinical use. In this study we report that treatment of HeLa cells with 5-fluorouracil resulted in de novo assembly of stress granules. Moreover, we revealed that stress granule assembly under stress conditions as well as disassembly is altered in cells treated with 5-fluorouracil. Notably, we discovered that RACK1, a protein mediating cell survival and apoptosis, is a component of 5-fluorouracil-induced stress granules. To explore the mode of action of 5-fluorouracil accountable for de novo stress granule assembly, we analyzed 5-fluorouracil metabolites and noticed that stress granule assembly is caused by RNA, not DNA incorporating 5-fluorouracil metabolites. Interestingly, we observed that other RNA incorporating drugs also cause assembly of stress granules. Thus, our results suggest that incorporation of chemotherapeutics into RNA may result in stress granule assembly with potential significance in chemoresistance.

Structural information is crucial in ribonucleic acid (RNA) analysis and functional annotation; nevertheless, how to include such structural data is still a debated problem. Dot-bracket notation is the most common and simple representation for RNA secondary structures but its simplicity leads also to ambiguity requiring further processing steps to dissolve. Here we present BEAR (Brand nEw Alphabet for RNA), a new context-aware structural encoding represented by a string of characters. Each character in BEAR encodes for a specific secondary structure element (loop, stem, bulge and internal loop) with specific length. Furthermore, exploiting this informative and yet simple encoding in multiple alignments of related RNAs, we captured how much structural variation is tolerated in RNA families and convert it into transition rates among secondary structure elements. This allowed us to compute a substitution matrix for secondary structure elements called MBR (Matrix of BEAR-encoded RNA secondary structures), of which we tested the ability in aligning RNA secondary structures. We propose BEAR and the MBR as powerful resources for the RNA secondary structure analysis, comparison and classification, motif finding and phylogeny.

This protocol is used to label RNA molecules (in vitro–synthesized or in vivo–purified RNA molecules) that have free 3'-hydroxyl termini. The reaction is performed in 10 min using yeast poly(A) polymerase and 3'-deoxyadenosine 5'-[α-³²P]triphosphate (cordycepin 5'-[α-³²P]triphosphate), a chain-terminating nucleotide. At the end of the procedure, the reaction is desalted by gel filtration to remove any unincorporated nucleotides.

The binding of a protein to an RNA sequence protects that the region of the RNA from ribonuclease (RNase) digestion; this protected region is known as the protein's "footprint." In this protocol, end-labeled RNAs with and without bound protein are digested with RNase, and the products of digestion are analyzed by gel electrophoresis on denaturing polyacrylamide gels. If the experiment is performed properly, a comparison of the banding patterns from the two samples will reveal the binding site of the protein. The binding site—or footprint—will be detected as a region without bands in the protein-bound sample. In the sample without bound protein, the bands should cover the entirety of the RNA molecule. To establish appropriate digestion conditions for the procedure (i.e., ≤1 cleavage event per molecule), it is necessary to titrate the amount of RNase under a range of time and temperature conditions. RNase I cleaves after every nucleotide of RNA and works well under many assay conditions, but other enzymes with different cleavage specificities can also be used. RNase VI is preferable when analyzing structured RNA; RNase A is preferable when using pyrimidine-rich RNAs; and RNase T1 is useful for G-rich RNAs. Choosing enzymes with preference for double-stranded (such as RNase VI) versus single-stranded (such as RNase I) RNA may be helpful. Often, a combination of nucleases is advantageous.

Nuclease protection of a site-specifically labeled RNA by an RNA-binding protein is an extremely powerful method for determining the site of an RNA–protein interaction. If a protein binds to the region that contains the site-specific label, it will protect the label and adjoining sequences from nuclease digestion. The protected region, or "footprint," can then be characterized by extensive fragmentation and gel electrophoresis. As the name implies, reverse footprinting produces a mirror image of a conventional footprint; the footprint is revealed by the presence, not the absence, of bands. This method has a distinct advantage over conventional footprinting in that only a small fraction of the labeled RNA must be bound.

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Poly(A) polymerase (PAP) diversity in gene expression--star-PAP vs canonical PAP.

FEBS Lett. 2014 Jun 27;588(14):2185-97

Authors: Laishram RS

Abstract
Almost all eukaryotic mRNAs acquire a poly(A) tail at the 3'-end by a concerted RNA processing event: cleavage and polyadenylation. The canonical PAP, PAPα, was considered the only nuclear PAP involved in general polyadenylation of mRNAs. A phosphoinositide-modulated nuclear PAP, Star-PAP, was then reported to regulate a select set of mRNAs in the cell. In addition, several non-canonical PAPs have been identified with diverse cellular functions. Further, canonical PAP itself exists in multiple isoforms thus illustrating the diversity of PAPs. In this review, we compare two nuclear PAPs, Star-PAP and PAPα with a general overview of PAP diversity in the cell. Emerging evidence suggests distinct niches of target pre-mRNAs for the two PAPs and that modulation of these PAPs regulates distinct cellular functions.

PMID: 24873880 [PubMed - in process]

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siPools: highly complex but accurately defined siRNA pools eliminate off-target effects.

Nucleic Acids Res. 2014 May 29;

Authors: Hannus M, Beitzinger M, Engelmann JC, Weickert MT, Spang R, Hannus S, Meister G

Abstract
Short interfering RNAs (siRNAs) are widely used as tool for gene inactivation in basic research and therapeutic applications. One of the major shortcomings of siRNA experiments are sequence-specific off-target effects. Such effects are largely unpredictable because siRNAs can affect partially complementary sequences and function like microRNAs (miRNAs), which inhibit gene expression on mRNA stability or translational levels. Here we demonstrate that novel, enzymatically generated siRNA pools-referred to as siPools-containing up to 60 accurately defined siRNAs eliminate off-target effects. This is achieved by the low concentration of each individual siRNA diluting sequence-specific off-target effects below detection limits. In fact, whole transcriptome analyses reveal that single siRNA transfections can severely affect global gene expression. However, when complex siRNA pools are transfected, almost no transcriptome alterations are observed. Taken together, we present enzymatically produced complex but accurately defined siRNA pools with potent on-target silencing but without detectable off-target effects.

PMID: 24875475 [PubMed - as supplied by publisher]

Cooperative gene regulation by microRNA pairs and their identification using a computational workflow.

Nucleic Acids Res. 2014 May 29;

Authors: Schmitz U, Lai X, Winter F, Wolkenhauer O, Vera J, Gupta SK

Abstract
MicroRNAs (miRNAs) are an integral part of gene regulation at the post-transcriptional level. Recently, it has been shown that pairs of miRNAs can repress the translation of a target mRNA in a cooperative manner, which leads to an enhanced effectiveness and specificity in target repression. However, it remains unclear which miRNA pairs can synergize and which genes are target of cooperative miRNA regulation. In this paper, we present a computational workflow for the prediction and analysis of cooperating miRNAs and their mutual target genes, which we refer to as RNA triplexes. The workflow integrates methods of miRNA target prediction; triplex structure analysis; molecular dynamics simulations and mathematical modeling for a reliable prediction of functional RNA triplexes and target repression efficiency. In a case study we analyzed the human genome and identified several thousand targets of cooperative gene regulation. Our results suggest that miRNA cooperativity is a frequent mechanism for an enhanced target repression by pairs of miRNAs facilitating distinctive and fine-tuned target gene expression patterns. Human RNA triplexes predicted and characterized in this study are organized in a web resource at www.sbi.uni-rostock.de/triplexrna/.

PMID: 24875477 [PubMed - as supplied by publisher]

Gene expression is regulated in a context-dependent, cell-type-specific manner. Condition-specific transcription is dependent on the presence of transcription factors (TFs) that can activate or inhibit its target genes (global context). Additional factors, such as chromatin structure, histone, or DNA modifications, also influence the activity of individual target genes (individual context). The role of the global and individual context for post-transcriptional regulation has not systematically been investigated on a large scale and is poorly understood. Here we show that global and individual context dependency is a pervasive feature of microRNA-mediated regulation. Our comprehensive and highly consistent data set from several high-throughput technologies (PAR-CLIP, RIP-chip, 4sU-tagging, and SILAC) provides strong evidence that context-dependent microRNA target sites (CDTS) are as frequent and functionally relevant as constitutive target sites (CTS). Furthermore, we found the global context to be insufficient to explain the CDTS, and that flanking sequence motifs provide individual context that is an equally important factor. Our results demonstrate that, similar to TF-mediated regulation, global and individual context dependency are prevalent in microRNA-mediated gene regulation, implying a much more complex post-transcriptional regulatory network than is currently known. The necessary tools to unravel post-transcriptional regulations and mechanisms need to be much more involved, and much more data will be needed for particular cell types and cellular conditions in order to understand microRNA-mediated regulation and the context-dependent post-transcriptional regulatory network.

Nature advance online publication 01 June 2014. doi:10.1038/nature13395

Authors: Evgeny Z. Kvon, Tomas Kazmar, Gerald Stampfel, J. Omar Yáñez-Cuna, Michaela Pagani, Katharina Schernhuber, Barry J. Dickson & Alexander Stark

Transcriptional enhancers are crucial regulators of gene expression and animal development and the characterization of their genomic organization, spatiotemporal activities and sequence properties is a key goal in modern biology. Here we characterize the in vivo activity of 7,705 Drosophila melanogaster enhancer candidates covering 13.5% of the non-coding non-repetitive genome throughout embryogenesis. 3,557 (46%) candidates are active, suggesting a high density with 50,000 to 100,000 developmental enhancers genome-wide. The vast majority of enhancers display specific spatial patterns that are highly dynamic during development. Most appear to regulate their neighbouring genes, suggesting that the cis-regulatory genome is organized locally into domains, which are supported by chromosomal domains, insulator binding and genome evolution. However, 12 to 21 per cent of enhancers appear to skip non-expressed neighbours and regulate a more distal gene. Finally, we computationally identify cis-regulatory motifs that are predictive and required for enhancer activity, as we validate experimentally. This work provides global insights into the organization of an animal regulatory genome and the make-up of enhancer sequences and confirms and generalizes principles from previous studies. All enhancer patterns are annotated manually with a controlled vocabulary and all results are available through a web interface (http://enhancers.starklab.org), including the raw images of all microscopy slides for manual inspection at arbitrary zoom levels.

Prediction of Bacterial microRNAs and possible targets in human cell transcriptome.

J Microbiol. 2014 Jun;52(6):482-9

Authors: Shmaryahu A, Carrasco M, Valenzuela PD

Abstract
Recent studies have examined gene transfer from bacteria to humans that would result in vertical inheritance. Bacterial DNA appears to integrate into the human somatic genome through an RNA intermediate, and such integrations are detected more frequently in tumors than normal samples and in RNA than DNA samples. Also, vertebrate viruses encode products that interfere with the RNA silencing machinery, suggesting that RNA silencing may indeed be important for antiviral responses in vertebrates. RNA silencing in response to virus infection could be due to microRNAs encoded by either the virus or the host. We hypothesized that bacterial expression of RNA molecules with secondary structures is potentially able to generate miRNA molecules that can interact with the human host mRNA during bacterial infection. To test this hypothesis, we developed a pipelinebased bioinformatics approach to identify putative micro-RNAs derived from bacterial RNAs that may have the potential to regulate gene expression of the human host cell. Our results suggest that 68 bacterial RNAs predicted from 37 different bacterial genomes have predicted secondary structures potentially able to generate putative microRNAs that may interact with messenger RNAs of genes involved in 47 different human diseases. As an example, we examined the effect of transfecting three putative microRNAs into human embryonic kidney 293 (HEK293) cells. The results show that the bacterially derived microRNA sequence can significantly regulate the expression of the respective target human gene. We suggest that the study of these predicted microRNAs may yield important clues as to how the human host cell processes involved in human diseases like cancer, diabetes, rheumatoid arthritis, and others may respond to a particular bacterial environment.

PMID: 24871974 [PubMed - in process]

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MicroRNA-29c mediates initiation of gastric carcinogenesis by directly targeting ITGB1.

Gut. 2014 May 28;

Authors: Han TS, Hur K, Xu G, Choi B, Okugawa Y, Toiyama Y, Oshima H, Oshima M, Lee HJ, Kim VN, Chang AN, Goel A, Yang HK

Abstract
OBJECTIVE: Gastric cancer (GC) remains difficult to cure due to heterogeneity in a clinical challenge and the molecular mechanisms underlying this disease are complex and not completely understood. Accumulating evidence suggests that microRNAs (miRNAs) play an important role in GC, but the role of specific miRNAs involved in this disease remains elusive. We performed next generation sequencing (NGS)-based whole-transcriptome profiling to discover GC-specific miRNAs, followed by functional validation of results.
DESIGN: NGS-based miRNA profiles were generated in matched pairs of GCs and adjacent normal mucosa (NM). Quantitative RT-PCR validation of miR-29c expression was performed in 274 gastric tissues, which included two cohorts of matched GC and NM specimens. Functional validation of miR-29c and its gene targets was undertaken in cell lines, as well as K19-C2mE and K19-Wnt1/C2mE transgenic mice.
RESULTS: NGS analysis revealed four GC-specific miRNAs. Among these, miR-29c expression was significantly decreased in GC versus NM tissues (p<0.001). Ectopic expression of miR-29c mimics in GC cell lines resulted in reduced proliferation, adhesion, invasion and migration. High miR-29c expression suppressed xenograft tumour growth in nude mice. Direct interaction between miR-29c and its newly discovered target, ITGB1, was identified in cell lines and transgenic mice. MiR-29c expression demonstrated a stepwise decrease in wild type hyperplasia-dysplasia cascade in transgenic mice models of GC.
CONCLUSIONS: MiR-29c acts as a tumour suppressor in GC by directly targeting ITGB1. Loss of miR-29c expression is an early event in the initiation of gastric carcinogenesis and may serve as a diagnostic and therapeutic biomarker for patients with GC.

PMID: 24870620 [PubMed - as supplied by publisher]