Hyeshik Chang

MicroRNAs shape circadian hepatic gene expression on a transcriptome-wide scale.

Elife. 2014;3:e02510

Authors: Du NH, Arpat AB, De Matos M, Gatfield D

Abstract
A considerable proportion of mammalian gene expression undergoes circadian oscillations. Post-transcriptional mechanisms likely make important contributions to mRNA abundance rhythms. We have investigated how microRNAs (miRNAs) contribute to core clock and clock-controlled gene expression using mice in which miRNA biogenesis can be inactivated in the liver. While the hepatic core clock was surprisingly resilient to miRNA loss, whole transcriptome sequencing uncovered widespread effects on clock output gene expression. Cyclic transcription paired with miRNA-mediated regulation was thus identified as a frequent phenomenon that affected up to 30% of the rhythmic transcriptome and served to post-transcriptionally adjust the phases and amplitudes of rhythmic mRNA accumulation. However, only few mRNA rhythms were actually generated by miRNAs. Overall, our study suggests that miRNAs function to adapt clock-driven gene expression to tissue-specific requirements. Finally, we pinpoint several miRNAs predicted to act as modulators of rhythmic transcripts, and identify rhythmic pathways particularly prone to miRNA regulation.DOI: http://dx.doi.org/10.7554/eLife.02510.001.

PMID: 24867642 [PubMed]

Nature Methods 11, 683 (2014). doi:10.1038/nmeth.2970

Authors: Jacob M Tome, Abdullah Ozer, John M Pagano, Dan Gheba, Gary P Schroth & John T Lis

Nature Methods 11, 597 (2014). doi:10.1038/nmeth.2974

Authors: Martin Krzywinski & Naomi Altman

Good experimental designs limit the impact of variability and reduce sample-size requirements.

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Divergent LIN28-mRNA associations result in translational suppression upon the initiation of differentiation.

Nucleic Acids Res. 2014 May 23;

Authors: Tan SM, Altschuler G, Zhao TY, Ang HS, Yang H, Lim B, Vardy L, Hide W, Thomson AM, Lareu RR

Abstract
LIN28 function is fundamental to the activity and behavior of human embryonic stem cells (hESCs) and induced pluripotent stem cells. Its main roles in these cell types are the regulation of translational efficiency and let-7 miRNA maturation. However, LIN28-associated mRNA cargo shifting and resultant regulation of translational efficiency upon the initiation of differentiation remain unknown. An RNA-immunoprecipitation and microarray analysis protocol, eRIP, that has high specificity and sensitivity was developed to test endogenous LIN28-associated mRNA cargo shifting. A combined eRIP and polysome analysis of early stage differentiation of hESCs with two distinct differentiation cues revealed close similarities between the dynamics of LIN28 association and translational modulation of genes involved in the Wnt signaling, cell cycle, RNA metabolism and proteasomal pathways. Our data demonstrate that change in translational efficiency is a major contributor to early stages of differentiation of hESCs, in which LIN28 plays a central role. This implies that eRIP analysis of LIN28-associated RNA cargoes may be used for rapid functional quality control of pluripotent stem cells under manufacture for therapeutic applications.

PMID: 24860167 [PubMed - as supplied by publisher]

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The pathway of miRNA maturation.

Methods Mol Biol. 2014;1095:3-10

Authors: Sand M

Abstract
MicroRNAs (miRNAs) are a novel class of 17-23 nucleotide short, nonprotein-coding RNA molecules which have emerged to be key players in posttranscriptional gene regulation. In this chapter we give an in-depth review of the classic, canonical mammalian miRNA maturation pathway and discuss new, noncanonical alternatives such as the mirtron pathway which were recently described.

PMID: 24166299 [PubMed - indexed for MEDLINE]

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Assaying Dicer-mediated miRNA maturation by means of fluorescent substrates.

Methods Mol Biol. 2014;1095:95-102

Authors: Hesse M, Davies BP, Arenz C

Abstract
Assaying Dicer-mediated miRNA maturation is a valuable tool not only for validating miRNA maturation itself but also for testing Dicer activity in cell lysate and for screening small molecules inhibiting miRNA maturation in a high-throughput format. The classical assay for miRNA maturation relies on radioactive labeling of a pre-miRNA and subsequent gel electrophoresis and autoradiography. Here we present a fluorescently labeled and quenched pre-miRNA beacon that can be ligated easily out of two single labeled RNA strands. Upon Dicer cleavage of the beacon, fluorophore and quencher are separated, which results in an increase of fluorescence over time. Unlike (32)P-labeled probes, our fluorescently labeled pre-miRNA beacon is stable for at least 5 years under storage conditions. Dicer or miRNA maturation assays can be easily performed in a 384-well plate format, consuming less than 1 pmol of RNA beacon per reaction.

PMID: 24166305 [PubMed - indexed for MEDLINE]

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Detection of microRNA maturation using unmodified pre-microRNA and branched rolling circle amplification.

Methods Mol Biol. 2014;1095:109-19

Authors: Neubacher S, Arenz C

Abstract
The ever-increasing number of different miRNAs and their association with a vast number of cellular dysfunctions and diseases have initiated several groups to investigate miRNA maturation, which ultimately leads to down regulation of a target messenger RNA (mRNA) and its downstream product. A rapid, convenient, and reliable assay to detect the Dicer-mediated miRNA-maturation step may facilitate research in this field. Here we describe the in vitro detection of the Dicer-mediated miRNA maturation step using unmodified pre-miRNA and branched rolling circle amplification.

PMID: 24166307 [PubMed - indexed for MEDLINE]

Nature Genetics 46, 530 (2014). doi:10.1038/ng.2993

Authors: Alexis Sarazin & Olivier Voinnet

Although silent transposons in plants can be reactivated by stress or during development, their potential deleterious effects are prevented by transposon-derived epigenetically activated small interfering RNAs (easiRNAs). A new study shows how serendipitous interactions between reactivated transposons and endogenous microRNAs might initiate easiRNA biogenesis, establishing an unexpected link between these two classes of silencing small RNAs.

Nature Genetics 46, 528 (2014). doi:10.1038/ng.2994

Author: Adele Murrell

Prader-Willi syndrome (PWS) is caused by loss of paternally expressed genes at an imprinted locus on chromosome 15, including the long noncoding RNA IPW. A new study identifies a critical role for IPW in modulating the expression of maternally expressed genes in trans, which has important implications for the understanding of imprinted gene networks.

This report demonstrates that G-quadruplexes with a long central loop are actually found in the 5'-UTRs of human mRNAs. Consideration of these new candidates might aid in elucidating the potentially important biological implications of the G-quadruplex structure.

A draft map of the human proteome

Nature 509, 7502 (2014). doi:10.1038/nature13302

Authors: Min-Sik Kim, Sneha M. Pinto, Derese Getnet, Raja Sekhar Nirujogi, Srikanth S. Manda, Raghothama Chaerkady, Anil K. Madugundu, Dhanashree S. Kelkar, Ruth Isserlin, Shobhit Jain, Joji K. Thomas, Babylakshmi Muthusamy, Pamela Leal-Rojas, Praveen Kumar, Nandini A. Sahasrabuddhe, Lavanya Balakrishnan, Jayshree Advani, Bijesh George, Santosh Renuse, Lakshmi Dhevi N. Selvan, Arun H. Patil, Vishalakshi Nanjappa, Aneesha Radhakrishnan, Samarjeet Prasad, Tejaswini Subbannayya, Rajesh Raju, Manish Kumar, Sreelakshmi K. Sreenivasamurthy, Arivusudar Marimuthu, Gajanan J. Sathe, Sandip Chavan, Keshava K. Datta, Yashwanth Subbannayya, Apeksha Sahu, Soujanya D. Yelamanchi, Savita Jayaram, Pavithra Rajagopalan, Jyoti Sharma, Krishna R. Murthy, Nazia Syed, Renu Goel, Aafaque A. Khan, Sartaj Ahmad, Gourav Dey, Keshav Mudgal, Aditi Chatterjee, Tai-Chung Huang, Jun Zhong, Xinyan Wu, Patrick G. Shaw, Donald Freed, Muhammad S. Zahari, Kanchan K. Mukherjee, Subramanian Shankar, Anita Mahadevan, Henry Lam, Christopher J. Mitchell, Susarla Krishna Shankar, Parthasarathy Satishchandra, John T. Schroeder, Ravi Sirdeshmukh, Anirban Maitra, Steven D. Leach, Charles G. Drake, Marc K. Halushka, T. S. Keshava Prasad, Ralph H. Hruban, Candace L. Kerr, Gary D. Bader, Christine A. Iacobuzio-Donahue, Harsha Gowda & Akhilesh Pandey

The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution

Nature advance online publication 28 May 2014. doi:10.1038/nature13420

Authors: Pietro Genovese, Giulia Schiroli, Giulia Escobar, Tiziano Di Tomaso, Claudia Firrito, Andrea Calabria, Davide Moi, Roberta Mazzieri, Chiara Bonini, Michael C. Holmes, Philip D. Gregory, Mirjam van der Burg, Bernhard Gentner, Eugenio Montini, Angelo Lombardo & Luigi Naldini

Background: A major hurdle to transcriptome profiling by deep-sequencing technologies is that abundant transcripts, such as rRNAs, can overwhelm the libraries, severely reducing transcriptome-wide coverage. Methods for depletion of such unwanted sequences typically require treatment of RNA samples prior to library preparation, are costly and not suited to unusual species and applications. Here we describe Probe-Directed Degradation (PDD), an approach that employs hybridisation to DNA oligonucleotides at the single-stranded cDNA library stage and digestion with Duplex-Specific Nuclease (DSN). Results: Targeting Saccharomyces cerevisiae rRNA sequences in Illumina HiSeq libraries generated by the split adapter method we show that PDD results in efficient removal of rRNA. The probes generate extended zones of depletion as a function of library insert size and the requirements for DSN cleavage. Using intact total RNA as starting material, probes can be spaced at the minimum anticipated library size minus 20 nucleotides to achieve continuous depletion. No off-target bias is detectable when comparing PDD-treated with untreated libraries. We further provide a bioinformatics tool to design suitable PDD probe sets. Conclusion: We find that PDD is a rapid procedure that results in effective and specific depletion of unwanted sequences from deep-sequencing libraries. Because PDD acts at the cDNA stage, handling of fragile RNA samples can be minimised and it should further be feasible to remediate existing libraries. Importantly, PDD preserves the original RNA fragment boundaries as is required for nucleotide-resolution footprinting or base-cleavage studies. Finally, as PDD utilises unmodified DNA oligonucleotides it can provide a low-cost option for large-scale projects, or be flexibly customised to suit different depletion targets, sample types and organisms.

Filed under: Media, MIT, Talks

This paper discusses the provocative hypothesis that functional regions of long noncoding RNAs might be derived from sequences present in transposable elements.

Nature Reviews Molecular Cell Biology 15, 366 (2014). doi:10.1038/nrm3813

Author: Andrea Du Toit

Mammalian Dicer is recruited to loci of endogenously overlapping transcripts to promote the formation of heterochromatin.

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Structure-Specific Ribonucleases for MS-Based Elucidation of Higher-Order RNA Structure.

J Am Soc Mass Spectrom. 2014 May 21;

Authors: Scalabrin M, Siu Y, Asare-Okai PN, Fabris D

Abstract
Supported by high-throughput sequencing technologies, structure-specific nucleases are experiencing a renaissance as biochemical probes for genome-wide mapping of nucleic acid structure. This report explores the benefits and pitfalls of the application of Mung bean (Mb) and V1 nuclease, which attack specifically single- and double-stranded regions of nucleic acids, as possible structural probes to be employed in combination with MS detection. Both enzymes were found capable of operating in ammonium-based solutions that are preferred for high-resolution analysis by direct infusion electrospray ionization (ESI). Sequence analysis by tandem mass spectrometry (MS/MS) was performed to confirm mapping assignments and to resolve possible ambiguities arising from the concomitant formation of isobaric products with identical base composition and different sequences. The observed products grouped together into ladder-type series that facilitated their assignment to unique regions of the substrate, but revealed also a certain level of uncertainty in identifying the boundaries between paired and unpaired regions. Various experimental factors that are known to stabilize nucleic acid structure, such as higher ionic strength, presence of Mg(II), etc., increased the accuracy of cleavage information, but did not completely eliminate deviations from expected results. These observations suggest extreme caution in interpreting the results afforded by these types of reagents. Regardless of the analytical platform of choice, the results highlighted the need to repeat probing experiments under the most diverse possible conditions to recognize potential artifacts and to increase the level of confidence in the observed structural information.

PMID: 24845355 [PubMed - as supplied by publisher]

Article

Non-coding RNAs are widely expressed, yet their functions remain poorly understood. Here, Leong et al . identify a set of antisense RNAs elevated during the yeast stress response that directly correlate with reduced protein levels, indicating a general regulatory effect of antisense expression.

Nature Communications doi: 10.1038/ncomms4947

Authors: Hui Sun Leong, Keren Dawson, Chris Wirth, Yaoyong Li, Yvonne Connolly, Duncan L. Smith, Caroline R. M. Wilkinson, Crispin J. Miller

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A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA.

RNA. 2014 May 22;

Authors: Burke JM, Kelenis DP, Kincaid RP, Sullivan CS

Abstract
Processing of primary microRNA (pri-miRNA) stem-loops by the Drosha-DGCR8 complex is the initial step in miRNA maturation and crucial for miRNA function. Nonetheless, the underlying mechanism that determines the Drosha cleavage site of pri-miRNAs has remained unclear. Two prevalent but seemingly conflicting models propose that Drosha-DGCR8 anchors to and directs cleavage a fixed distance from either the basal single-stranded (ssRNA) or the terminal loop. However, recent studies suggest that the basal ssRNA and/or the terminal loop may influence the Drosha cleavage site dependent upon the sequence/structure of individual pri-miRNAs. Here, using a panel of closely related pri-miRNA variants, we further examine the role of pri-miRNA structures on Drosha cleavage site selection in cells. Our data reveal that both the basal ssRNA and terminal loop influence the Drosha cleavage site within three pri-miRNAs, the Simian Virus 40 (SV40) pri-miRNA, pri-miR-30a, and pri-miR-16. In addition to the flanking ssRNA regions, we show that an internal loop within the SV40 pri-miRNA stem strongly influences Drosha cleavage position and efficiency. We further demonstrate that the positions of the internal loop, basal ssRNA, and the terminal loop of the SV40 pri-miRNA cooperatively coordinate Drosha cleavage position and efficiency. Based on these observations, we propose that the pri-miRNA stem, defined by internal and flanking structural elements, guides the binding position of Drosha-DGCR8, which consequently determines the cleavage site. This study provides mechanistic insight into pri-miRNA processing in cells that has numerous biological implications and will assist in refining Drosha-dependent shRNA design.

PMID: 24854622 [PubMed - as supplied by publisher]

This paper describes an informatic analysis of RNA-seq data relevant to levels of mRNAs encoding ribosomal proteins. Major findings are that the vast majority of molar ratios of these mRNAs are within threefold of one another. In addition, some of these mRNAs, apparently transcribed from "pseudogenes," are expressed in a tissue-specific manner.

The RNA-binding protein with a multiple splicing (RBPMS) family of proteins has been implicated in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. This study defines the transcriptome-wide RNA targets of RBPMS using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing binding sites in exonic mRNA, intronic pre-mRNA, and repeat-annotated sequences. The RBPMS family of proteins binds a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to some other mRNA-binding proteins, RBPMS proteins relocalize to cytoplasmic stress granules under oxidative stress, suggestive of a support function for mRNA localization in large and/or multinucleate cells, where it is preferentially expressed.

This paper describes the finding that inactivating the exosome in yeast leads to up-regulation of the iron uptake regulon. The up-regulation is caused by increased levels of reactive oxygen species. These observations demonstrate that the exosome in some way affects the physiology of an organism.

Publication date: September 2014
Source:Trends in Cell Biology, Volume 24, Issue 9
Author(s): David Jee , Eric C. Lai
miRNAs are enclosed within Argonaute (Ago) proteins, the downstream effectors of small RNA-mediated gene silencing. Because miRNAs mediate extensive networks of post-transcriptional control, cells have evolved multiple strategies to control their activity with precision. A growing theme of recent years is how post-translational modifications of Ago proteins, such as prolyl hydroxylation, phosphorylation, ubiquitination, and poly-ADP-ribosylation, alter miRNA activity at global or specific levels. In this review, we discuss recent advances in Ago modifications in mammalian cells and emphasize how such alterations modulate small RNA function to coordinate appropriate downstream cellular responses. These findings provide a framework to understand how Ago protein modifications are linked to reorganization of post-transcriptional regulatory networks, enabling dynamic responses to diverse external stimuli and changing environmental conditions.

Nature Protocols 9, 1428 (2014). doi:10.1038/nprot.2014.083

Authors: Wenxiu Ma, William S Noble & Timothy L Bailey

MEME-ChIP is a web-based tool for analyzing motifs in large DNA or RNA data sets. It can analyze peak regions identified by ChIP-seq, cross-linking sites identified by CLIP-seq and related assays, as well as sets of genomic regions selected using other criteria. MEME-ChIP performs de

Publication date: 22 May 2014
Source:Molecular Cell, Volume 54, Issue 4
Author(s): Sarah F. Mitchell , Roy Parker
The proper processing, export, localization, translation, and degradation of mRNAs are necessary for regulation of gene expression. These processes are controlled by mRNA-specific regulatory proteins, noncoding RNAs, and core machineries common to most mRNAs. These factors bind the mRNA in large complexes known as messenger ribonucleoprotein particles (mRNPs). Herein, we review the components of mRNPs, how they assemble and rearrange, and how mRNP composition differentially affects mRNA biogenesis, function, and degradation. We also describe how properties of the mRNP “interactome” lead to emergent principles affecting the control of gene expression.

Long intergenic noncoding RNA (lincRNA) is transcriptionally complex and pervasive across mammalian cells, but aside from a handful of specific examples, its general functional significance remains uncertain. Complementary to individual lincRNA functional analyses, the authors here test lincRNA developmental expression conservation in the human and macaque prefrontal cortex as a criterion for lincRNA function. Remarkably, lincRNA expression patterns were as conserved between humans and macaques as those of protein-coding genes, and ectopic expression of a development-associated lincRNA affected gene expression in a sequence-specific manner. These results support the potential functionality of lincRNA in primate development.

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RNA: riboSNitches reveal heredity in RNA secondary structure.

Nat Rev Genet. 2014 Apr;15(4):219

Authors: Lokody I

PMID: 24535248 [PubMed - indexed for MEDLINE]

Publication date: June 2014
Source:Trends in Biotechnology, Volume 32, Issue 6
Author(s): Cor van der Weele , Johannes Tramper
Rising global demand for meat will result in increased environmental pollution, energy consumption, and animal suffering. Cultured meat, produced in an animal-cell cultivation process, is a technically feasible alternative lacking these disadvantages, provided that an animal-component-free growth medium can be developed. Small-scale production looks particularly promising, not only technologically but also for societal acceptance. Economic feasibility, however, emerges as the real obstacle.

Structural and functional implications of the QUA2 domain on RNA recognition by GLD-1.

Nucleic Acids Res. 2014 May 16;

Authors: Daubner GM, Brümmer A, Tocchini C, Gerhardy S, Ciosk R, Zavolan M, Allain FH

Abstract
The STAR family comprises ribonucleic acid (RNA)-binding proteins that play key roles in RNA-regulatory processes. RNA recognition is achieved by a KH domain with an additional α-helix (QUA2) that seems to extend the RNA-binding surface to six nucleotides for SF1 (Homo sapiens) and seven nucleotides for GLD-1 (Caenorhabditis elegans). To understand the structural basis of this probable difference in specificity, we determined the solution structure of GLD-1 KH-QUA2 with the complete consensus sequence identified in the tra-2 gene. Compared to SF1, the GLD-1 KH-QUA2 interface adopts a different conformation resulting indeed in an additional sequence-specific binding pocket for a uracil at the 5'end. The functional relevance of this binding pocket is emphasized by our bioinformatics analysis showing that GLD-1 binding sites with this 5'end uracil are more predictive for the functional response of the messenger RNAs to gld-1 knockout. We further reveal the importance of the KH-QUA2 interface in vitro and that its alteration in vivo affects the level of translational repression dependent on the sequence of the GLD-1 binding motif. In conclusion, we demonstrate that the QUA2 domain distinguishes GLD-1 from other members of the STAR family and contributes more generally to the modulation of RNA-binding affinity and specificity of KH domain containing proteins.

PMID: 24838563 [PubMed - as supplied by publisher]

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Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues.

BMC Biotechnol. 2013;13:99

Authors: Zaghlool A, Ameur A, Nyberg L, Halvardson J, Grabherr M, Cavelier L, Feuk L

Abstract
BACKGROUND: The starting material for RNA sequencing (RNA-seq) studies is usually total RNA or polyA+ RNA. Both forms of RNA represent heterogeneous pools of RNA molecules at different levels of maturation and processing. Such heterogeneity, in addition to the biases associated with polyA+ purification steps, may influence the analysis, sensitivity and the interpretation of RNA-seq data. We hypothesize that subcellular fractions of RNA may provide a more accurate picture of gene expression.
RESULTS: We present results for sequencing of cytoplasmic and nuclear RNA after cellular fractionation of tissue samples. In comparison with conventional polyA+ RNA, the cytoplasmic RNA contains a significantly higher fraction of exonic sequence, providing increased sensitivity in expression analysis and splice junction detection, and in improved de novo assembly of RNA-seq data. Conversely, the nuclear fraction shows an enrichment of unprocessed RNA compared with total RNA-seq, making it suitable for analysis of nascent transcripts and RNA processing dynamics.
CONCLUSION: Our results show that cellular fractionation is a more rapid and cost effective approach than conventional polyA+ enrichment when studying mature RNAs. Thus, RNA-seq of separated cytosolic and nuclear RNA can significantly improve the analysis of complex transcriptomes from mammalian tissues.

PMID: 24225116 [PubMed - indexed for MEDLINE]