Hyeshik Chang

A transgenic mouse with fluorescently labeled endogenous β-actin mRNA permits single-molecule analysis in live cells. [Also see Perspective by Akbalik and Schuman] Authors: Hye Yoon Park, Hyungsik Lim, Young J. Yoon, Antonia Follenzi, Chiso Nwokafor, Melissa Lopez-Jones, Xiuhua Meng, Robert H. Singer

Imaging of β-actin messenger RNA (mRNA) in neurons reveals transient alteration of mRNA availability during synaptic plasticity. [Also see Perspective by Akbalik and Schuman] Authors: Adina R. Buxbaum, Bin Wu, Robert H. Singer

Visualization of mRNA dynamics in live neurons reveals its release from granules at synapses during neuronal plasticity. [Also see Reports by Buxbaum et al. and Park et al.] Authors: Güney Akbalik, Erin M. Schuman

Nature Reviews Cancer 14, 73 (2014). doi:10.1038/nrc3676

Author: Nicola McCarthy

Julian Downward and colleagues have found that the Hmga2 RNA contributes to lung cancer development and progression in mice through its function as a competing endogenous RNA (ceRNA).

Nature Reviews Molecular Cell Biology 15, 76 (2014). doi:10.1038/nrm3745

Author: Kirsty Minton

mRNA methylation destabilizes developmental regulators to maintain the properties of mammalian embryonic stem cells.

Nature Protocols 9, 375 (2014). doi:10.1038/nprot.2014.017

Authors: Swathi Arur & Tim Schedl

Post-translational modifications alter protein structure, affecting activity, stability, localization and/or binding partners. Antibodies that specifically recognize post-translationally modified proteins have a number of uses including immunocytochemistry and immunoprecipitation of the modified protein to purify protein-protein and protein-nucleic acid complexes. However, antibodies directed at modified sites

Nature Protocols 9, 410 (2014). doi:10.1038/nprot.2014.023

Authors: Leslie Gay, Kate V Karfilis, Michael R Miller, Chris Q Doe & Kryn Stankunas

Transcriptional profiling is a powerful approach for studying mouse development, physiology and disease models. Here we describe a protocol for mouse thiouracil tagging (TU tagging), a transcriptome analysis technology that includes in vivo covalent labeling, purification and analysis of cell type–specific

Thousands of large intergenic noncoding RNAs (lincRNAs) have been identified in the mammalian genome, many of which have important roles in regulating a variety of biological processes. Here, we used a custom microarray to identify lincRNAs associated with activation of the innate immune response. A panel of 159 lincRNAs was...

RNA-binding proteins (RBPs) bind to their target RNA molecules by recognizing specific RNA sequences and structural contexts. The development of CLIP-seq and related protocols has made it possible to exhaustively identify RNA fragments that bind to RBPs. However, no efficient bioinformatics method exists to reveal the structural specificities of RBP-RNA interactions using these data. We present CapR, an efficient algorithm that calculates the probability that each RNA base position is located within each secondary structural context. Using CapR, we demonstrate that several RBPs bind to their target RNA molecules under specific structural contexts. CapR is available at https://sites.google.com/site/fukunagatsu/software/capr.

We present GraphProt, a computational framework for learning sequence- and structure-binding preferences of RNA-binding proteins (RBPs) from high-throughput experimental data. We benchmark GraphProt, demonstrating that the modeled binding preferences conform to the literature, and showcase the biological relevance and two applications of GraphProt models. First, estimated binding affinities correlate with experimental measurements. Second, predicted Ago2 targets display higher levels of expression upon Ago2 knockdown, whereas control targets do not. Computational binding models, such as those provided by GraphProt, are essential to predict RBP-binding sites and affinities in all tissues. GraphProt is freely available at http://www.bioinf.uni-freiburg.de/Software/GraphProt.

microRNAs (miRNAs) are small endogenous non-coding RNAs that function as the universal specificity factors in post-transcriptional gene silencing. Discovering miRNAs, identifying their targets and further inferring miRNA functions have been a critical strategy for understanding normal biological processes of miRNAs and their roles in the development of disease. In this review, we focus on computational methods of inferring miRNA functions, including miRNA functional annotation and inferring miRNA regulatory modules, by integrating heterogeneous data sources. We also briefly introduce the research in miRNA discovery and miRNA-target identification with an emphasis on the challenges to computational biology.

Processing of microRNA primary transcripts requires heme in mammalian cells.

Proc Natl Acad Sci U S A. 2014 Jan 21;

Authors: Weitz SH, Gong M, Barr I, Weiss S, Guo F

Abstract
DiGeorge syndrome critical region gene 8 (DGCR8) is the RNA-binding partner protein of the nuclease Drosha. DGCR8 and Drosha recognize and cleave primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical microRNAs (miRNAs) in animals. We previously reported that human, frog, and starfish DGCR8 bind heme when expressed in Escherichia coli and that Fe(III) heme activates apoDGCR8 in reconstituted pri-miRNA processing assays. However, the physiological relevance of heme in miRNA maturation has not been clear. Here, we present a live-cell pri-miRNA processing assay that produces robust signals and faithfully indicates DGCR8 and Drosha activities. We demonstrate that all known heme-binding-deficient DGCR8 mutants are defective in pri-miRNA processing in HeLa cells. DGCR8 contains a previously uncharacterized heme-binding motif, "IPCL," that is also required for its activity. Heme availability and biosynthesis in HeLa cells positively affect pri-miRNA processing and production of mature miRNA. These results establish an essential function for heme in pri-miRNA processing in mammalian cells. Our study suggests that abnormal heme biosynthesis and degradation may contribute to diseases via miRNA-mediated gene regulation networks.

PMID: 24449907 [PubMed - as supplied by publisher]

Tagging RNAs with biotin has numerous useful applications including affinity selections. This paper describes three simple and straightforward techniques for biotinylating the 3' ends of RNA molecules.

We are surprised by the concerns raised by Schubert et al. (1) regarding the quantitative nature and methodological approaches presented in our recent PNAS report (2). I will attempt to clarify some of confusion and misstatements, as several errors and misrepresentation of our data are stated in the critique. The...

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The mitochondrial genome encodes abundant small noncoding RNAs.

Cell Res. 2013 Jun;23(6):759-74

Authors: Ro S, Ma HY, Park C, Ortogero N, Song R, Hennig GW, Zheng H, Lin YM, Moro L, Hsieh JT, Yan W

Abstract
Small noncoding RNAs identified thus far are all encoded by the nuclear genome. Here, we report that the murine and human mitochondrial genomes encode thousands of small noncoding RNAs, which are predominantly derived from the sense transcripts of the mitochondrial genes (host genes), and we termed these small RNAs mitochondrial genome-encoded small RNAs (mitosRNAs). DICER inactivation affected, but did not completely abolish mitosRNA production. MitosRNAs appear to be products of currently unidentified mitochondrial ribonucleases. Overexpression of mitosRNAs enhanced expression levels of their host genes in vitro, and dysregulated mitosRNA expression was generally associated with aberrant mitochondrial gene expression in vivo. Our data demonstrate that in addition to 37 known mitochondrial genes, the mammalian mitochondrial genome also encodes abundant mitosRNAs, which may play an important regulatory role in the control of mitochondrial gene expression in the cell.

PMID: 23478297 [PubMed - indexed for MEDLINE]

Nature advance online publication 19 January 2014. doi:10.1038/nature12943

Authors: Anamaria Necsulea, Magali Soumillon, Maria Warnefors, Angélica Liechti, Tasman Daish, Ulrich Zeller, Julie C. Baker, Frank Grützner & Henrik Kaessmann

After his first turn on the world stage ended in scandal, Woo Suk Hwang has quietly rebuilt his scientific career

The multilayered complexity of ceRNA crosstalk and competition

Nature 505, 7483 (2014). doi:10.1038/nature12986

Authors: Yvonne Tay, John Rinn & Pier Paolo Pandolfi

Recent reports have described an intricate interplay among diverse RNA species, including protein-coding messenger RNAs and non-coding RNAs such as long non-coding RNAs, pseudogenes and circular RNAs. These RNA transcripts act as competing endogenous RNAs (ceRNAs) or natural microRNA sponges — they communicate with and

Molecular biology: RNA retrieved from intact tissue

Nature 505, 7483 (2014). doi:10.1038/505264d

A technique can snatch RNA out of a single cell in live, intact tissue, revealing the genes being expressed by that cell.Other methods of single-cell RNA retrieval tend to disrupt the surrounding tissue before the RNA is captured. James Eberwine of the University of

Nature Reviews Genetics. doi:10.1038/nrg3668

Author: Darren J. Burgess

Nature Reviews Genetics. doi:10.1038/nrg3667

Author: Hannah Stower

Nature Reviews Genetics. doi:10.1038/nrg3669

Author: Darren J. Burgess

Andrey L. Karamyshev, Anna E. Patrick, Zemfira N. Karamysheva, Dustin S. Griesemer, Henry Hudson, Sandra Tjon-Kon-Sang, IngMarie Nilsson, Hendrik Otto, Qinghua Liu, Sabine Rospert, Gunnar von Heijne, Arthur E. Johnson, Philip J. Thomas. Misfolded proteins are often cytotoxic, unless cellular systems prevent their accumulation. Data presented here uncover a mechanism by which defects in secretory proteins lead to a dramatic reduct....

Zhuo Du, Anthony Santella, Fei He, Michael Tiongson, Zhirong Bao. Elucidation of complex phenotypes for mechanistic insights presents a significant challenge in systems biology. We report a strategy to automatically infer mechanistic models of cell fate differen....

Background: The majority of non-coding RNAs (ncRNAs) involved in mRNA metabolism in mammals have been believed to downregulate the corresponding mRNA expression level in a pre- or post-transcriptional manner by forming short or long ncRNA-mRNA duplex structures. Information on non-duplex-forming long ncRNAs is now also rapidly accumulating. To examine the directional properties of transcription at the whole-genome level, we performed directional RNA-seq analysis of mouse and chimpanzee tissue samples. Results: We found that there is only about 1% of the genome where both the top and bottom strands are utilized for transcription, suggesting that RNA-RNA duplexes are not abundantly formed. Focusing on transcription start sites (TSSs) of protein-coding genes revealed that a significant fraction of them contain switching-points that separate antisense- and sense-biased transcription, suggesting that head-to-head transcription is more prevalent than previously thought. More than 90% of head-to-head type promoters contain CpG islands. Moreover, CCG and CGG repeats are significantly enriched in the upstream regions and downstream regions, respectively, of TSSs located in head-to-head type promoters. Genes with tissue-specific promoter-associated ncRNAs (pancRNAs) show a positive correlation between the expression of their pancRNA and mRNA, which is in accord with the proposed role of pancRNA in facultative gene activation, whereas genes with constitutive expression generally lack pancRNAs. Conclusions: We propose that single-stranded ncRNA resulting from head-to-head transcription at GC-rich sequences regulates tissue-specific gene expression.

Nature Chemical Biology 10, 87 (2014). doi:10.1038/nchembio.1445

Author: Terry L. Sheppard

by Zohar Bloom-Ackermann, Sivan Navon, Hila Gingold, Ruth Towers, Yitzhak Pilpel, Orna Dahan

Deciphering the architecture of the tRNA pool is a prime challenge in translation research, as tRNAs govern the efficiency and accuracy of the process. Towards this challenge, we created a systematic tRNA deletion library in Saccharomyces cerevisiae, aimed at dissecting the specific contribution of each tRNA gene to the tRNA pool and to the cell's fitness. By harnessing this resource, we observed that the majority of tRNA deletions show no appreciable phenotype in rich medium, yet under more challenging conditions, additional phenotypes were observed. Robustness to tRNA gene deletion was often facilitated through extensive backup compensation within and between tRNA families. Interestingly, we found that within tRNA families, genes carrying identical anti-codons can contribute differently to the cellular fitness, suggesting the importance of the genomic surrounding to tRNA expression. Characterization of the transcriptome response to deletions of tRNA genes exposed two disparate patterns: in single-copy families, deletions elicited a stress response; in deletions of genes from multi-copy families, expression of the translation machinery increased. Our results uncover the complex architecture of the tRNA pool and pave the way towards complete understanding of their role in cell physiology.

The RNA polymerase I regulatory complex promotes dynamic regulation of ribosomal RNA synthesis within the Drosophila germline. Authors: Qiao Zhang, Nevine A. Shalaby, Michael Buszczak

Twice awarded the Nobel Prize, a biochemist's work on protein and DNA structure opened the door to modern biomedical science. Author: Sydney Brenner

The removal of RNA inadvertently incorporated into our DNA is critical for maintaining genome integrity. Author: Keith W. Caldecott