Hyeshik Chang

Terminal uridylyl transferases (TUTs) are responsible for the post-transcriptional addition of uridyl residues to RNA 3' ends, leading in some cases to altered stability. The Schizosaccharomyces pombe TUT Cid1 is a model enzyme that has been characterized structurally at moderate resolution and provides insights into the larger and more complex mammalian TUTs, ZCCHC6 and ZCCHC11. Here, we report a higher resolution (1.74 Å) crystal structure of Cid1 that provides detailed evidence for uracil selection via the dynamic flipping of a single histidine residue. We also describe a novel closed conformation of the enzyme that may represent an intermediate stage in a proposed product ejection mechanism. The structural insights gained, combined with normal mode analysis and biochemical studies, demonstrate that the plasticity of Cid1, particularly about a hinge region (N164–N165), is essential for catalytic activity, and provide an explanation for its distributive uridylyl transferase activity. We propose a model clarifying observed differences between the in vitro apparently processive activity and in vivo distributive monouridylylation activity of Cid1. We suggest that modulating the flexibility of such enzymes—for example by the binding of protein co-factors—may allow them alternatively to add single or multiple uridyl residues to the 3' termini of RNA molecules.

Ribonucleases (RNases) play a critical role in RNA processing and degradation by hydrolyzing phosphodiester bonds (exo- or endonucleolytically). Many RNases that cut RNA internally exhibit substrate specificity, but their target sites are usually limited to one or a few specific nucleotides in single-stranded RNA and often in a context of a particular three-dimensional structure of the substrate. Thus far, no RNase counterparts of restriction enzymes have been identified which could cleave double-stranded RNA (dsRNA) in a sequence-specific manner. Here, we present evidence for a sequence-dependent cleavage of long dsRNA by RNase Mini-III from Bacillus subtilis (BsMiniIII). Analysis of the sites cleaved by this enzyme in limited digest of bacteriophage 6 dsRNA led to the identification of a consensus target sequence. We defined nucleotide residues within the preferred cleavage site that affected the efficiency of the cleavage and were essential for the discrimination of cleavable versus non-cleavable dsRNA sequences. We have also determined that the loop α5b-α6, a distinctive structural element in Mini-III RNases, is crucial for the specific cleavage, but not for dsRNA binding. Our results suggest that BsMiniIII may serve as a prototype of a sequence-specific dsRNase that could possibly be used for targeted cleavage of dsRNA.

Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni2+-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni2+-NTA–based probes. Unfortunately, previous Ni-NTA–based...

Publication date: 12 March 2015
Source:Cell, Volume 160, Issue 6
Author(s): Vladimir Presnyak , Najwa Alhusaini , Ying-Hsin Chen , Sophie Martin , Nathan Morris , Nicholas Kline , Sara Olson , David Weinberg , Kristian E. Baker , Brenton R. Graveley , Jeff Coller
mRNA degradation represents a critical regulated step in gene expression. Although the major pathways in turnover have been identified, accounting for disparate half-lives has been elusive. We show that codon optimality is one feature that contributes greatly to mRNA stability. Genome-wide RNA decay analysis revealed that stable mRNAs are enriched in codons designated optimal, whereas unstable mRNAs contain predominately non-optimal codons. Substitution of optimal codons with synonymous, non-optimal codons results in dramatic mRNA destabilization, whereas the converse substitution significantly increases stability. Further, we demonstrate that codon optimality impacts ribosome translocation, connecting the processes of translation elongation and decay through codon optimality. Finally, we show that optimal codon content accounts for the similar stabilities observed in mRNAs encoding proteins with coordinated physiological function. This work demonstrates that codon optimization exists as a mechanism to finely tune levels of mRNAs and, ultimately, proteins.

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Teaser

Codon usage impacts gene expression both at the level of translation and mRNA decay, with the balance between optimal and non-optimal codons helping to fine-tune levels of mRNAs and, ultimately, proteins.

Publication date: 12 March 2015
Source:Cell, Volume 160, Issue 6
Author(s): Simon J. Conn , Katherine A. Pillman , John Toubia , Vanessa M. Conn , Marika Salmanidis , Caroline A. Phillips , Suraya Roslan , Andreas W. Schreiber , Philip A. Gregory , Gregory J. Goodall
Circular RNAs (circRNAs), formed by non-sequential back-splicing of pre-mRNA transcripts, are a widespread form of non-coding RNA in animal cells. However, it is unclear whether the majority of circRNAs represent splicing by-products without function or are produced in a regulated manner to carry out specific cellular functions. We show that hundreds of circRNAs are regulated during human epithelial-mesenchymal transition (EMT) and find that the production of over one-third of abundant circRNAs is dynamically regulated by the alternative splicing factor, Quaking (QKI), which itself is regulated during EMT. Furthermore, by modulating QKI levels, we show the effect on circRNA abundance is dependent on intronic QKI binding motifs. Critically, the addition of QKI motifs is sufficient to induce de novo circRNA formation from transcripts that are normally linearly spliced. These findings demonstrate circRNAs are both purposefully synthesized and regulated by cell-type specific mechanisms, suggesting they play specific biological roles in EMT.

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Teaser

The RNA binding protein Quaking (QKI) promotes circRNA biogenesis during epithelial to mesenchymal transition, strongly arguing for their functions.

Publication date: 15 May 2015
Source:Developmental Biology, Volume 401, Issue 2
Author(s): Magdalene Rausch , Matyas Ecsedi , Hrishikesh Bartake , Almuth Müllner , Helge Großhans
The heterochronic pathway controls temporal patterning during Caenorhabditis elegans larval development. The highly conserved let-7 microRNA (miRNA) plays a key role in this pathway, directing the larval-to-adult (L/A) transition. Hence, knowledge of the genetic interactome of let-7 has the potential to provide insight into both control of temporal cell fates and mechanisms of regulation and function of miRNAs. Here, we report the results of a genome-wide, RNAi-based screen for suppressors of let-7 mutant vulval bursting. The 201 genetic interaction partners of let-7 thus identified include genes that promote target silencing activity of let-7, seam cell differentiation, or both. We illustrate the suitability of our approach by uncovering the mitotic cyclin-dependent kinase CDK-1 as a downstream effector of let-7 that affects both seam cell proliferation and differentiation, and by identifying a core set of candidate modulators of let-7 activity, which includes all subunits of the condensin II complex. We propose that the genes identified in our screen thus constitute a valuable resource for studies of the heterochronic pathway and miRNAs.

Background: Histone epigenome data determined by chromatin immunoprecipitation sequencing (ChIP-seq) is used in identifying transcript regions and estimating expression levels. However, this estimation does not always correlate with eventual RNA expression levels measured by RNA sequencing (RNA-seq). Part of the inconsistency may arise from the variance in RNA stability, where the transcripts that are more or less abundant than predicted RNA expression from histone epigenome data are inferred to be more or less stable. However, there is little systematic analysis to validate this assumption. Here, we used stability data of whole transcriptome measured by 5′-bromouridine immunoprecipitation chase sequencing (BRIC-seq), which enabled us to determine the half-lives of whole transcripts including lincRNAs, and we integrated BRIC-seq with ChIP-seq to achieve better estimation of the eventual transcript levels and to understand the importance of post-transcriptional regulation that determine the eventual transcript levels. Results: We identified discrepancies between the RNA abundance estimated by ChIP-seq and measured RNA expression from RNA-seq; for number of genes and estimated that the expression level of 865 genes was controlled at the level of RNA stability in HeLa cells. ENCODE data analysis supported the idea that RNA stability control aids to determine transcript levels in multiple cell types. We identified UPF1, EXOSC5 and STAU1, well-studied RNA degradation factors, as controlling factors for 8% of cases. Computational simulations reasonably explained the changes of eventual mRNA levels attributable to the changes in the rates of mRNA half-lives. In addition, we propose a feedback circuit that includes the regulated degradation of mRNAs encoding transcription factors to maintain the steady state level of RNA abundance. Intriguingly, these regulatory mechanisms were distinct between mRNAs and lincRNAs. Conclusions: Integrative analysis of ChIP-seq, RNA-seq and our BRIC-seq showed that transcriptional regulation and RNA degradation are independently regulated. In addition, RNA stability is an important determinant of eventual transcript levels. RNA binding proteins, such as UPF1, STAU1 and EXOSC5 may play active roles in such controls.

Nature Biotechnology 33, 269 (2015). doi:10.1038/nbt.3154

Authors: Victoria Moignard, Steven Woodhouse, Laleh Haghverdi, Andrew J Lilly, Yosuke Tanaka, Adam C Wilkinson, Florian Buettner, Iain C Macaulay, Wajid Jawaid, Evangelia Diamanti, Shin-Ichi Nishikawa, Nir Piterman, Valerie Kouskoff, Fabian J Theis, Jasmin Fisher & Berthold Göttgens

The proteome of the amoebo-flagellate protozoan Naegleria gruberi is rich in candidate RNA repair enzymes, including 15 putative RNA ligases, one of which, NgrRnl, is a eukaryal homolog of Deinococcus radiodurans RNA ligase, DraRnl. Here we report that purified recombinant NgrRnl seals nicked 3'-OH/5'-PO₄ duplexes in which the 3'-OH strand is RNA. It does so via the "classic" ligase pathway, entailing reaction with ATP to form a covalent NgrRnl–AMP intermediate, transfer of AMP to the nick 5'-PO₄, and attack of the RNA 3'-OH on the adenylylated nick to form a 3'–5' phosphodiester. Unlike members of the four known families of ATP-dependent RNA ligases, NgrRnl lacks a carboxy-terminal appendage to its nucleotidyltransferase domain. Instead, it contains a defining amino-terminal domain that we show is important for 3'-OH/5'-PO₄ nick-sealing and ligase adenylylation, but dispensable for phosphodiester synthesis at a preadenylylated nick. We propose that NgrRnl, DraRnl, and their homologs from diverse bacteria, viruses, and unicellular eukarya comprise a new "Rnl5 family" of nick-sealing ligases with a signature domain organization.

Diversity and selectivity in mRNA translation on the endoplasmic reticulum.

Nat Rev Neurosci. 2015 Mar 4;

Authors: Reid DW, Nicchitta CV

Abstract
Pioneering electron microscopy studies defined two primary populations of ribosomes in eukaryotic cells: one freely dispersed through the cytoplasm and the other bound to the surface of the endoplasmic reticulum (ER). Subsequent investigations revealed a specialized function for each population, with secretory and integral membrane protein-encoding mRNAs translated on ER-bound ribosomes, and cytosolic protein synthesis was widely attributed to free ribosomes. Recent findings have challenged this view, and transcriptome-scale studies of mRNA distribution and translation have now demonstrated that ER-bound ribosomes also function in the translation of a large fraction of mRNAs that encode cytosolic proteins. These studies suggest a far more expansive role for the ER in transcriptome expression, where membrane and secretory protein synthesis represents one element of a multifaceted and dynamic contribution to post-transcriptional gene expression.

PMID: 25735911 [PubMed - as supplied by publisher]

The mammalian cerebral cortex supports cognitive functions such as sensorimotor integration, memory, and social behaviors. Normal brain function relies on a diverse set of differentiated cell types, including neurons, glia, and vasculature. Here, we have used large-scale single-cell RNA sequencing (RNA-seq) to classify cells in the mouse somatosensory cortex and hippocampal CA1 region. We found 47 molecularly distinct subclasses, comprising all known major cell types in the cortex. We identified numerous marker genes, which allowed alignment with known cell types, morphology, and location. We found a layer I interneuron expressing Pax6 and a distinct postmitotic oligodendrocyte subclass marked by Itpr2. Across the diversity of cortical cell types, transcription factors formed a complex, layered regulatory code, suggesting a mechanism for the maintenance of adult cell type identity. Authors: Amit Zeisel, Ana B. Muñoz-Manchado, Simone Codeluppi, Peter Lönnerberg, Gioele La Manno, Anna Juréus, Sueli Marques, Hermany Munguba, Liqun He, Christer Betsholtz, Charlotte Rolny, Gonçalo Castelo-Branco, Jens Hjerling-Leffler, Sten Linnarsson

Nature Structural & Molecular Biology 22, 256 (2015). doi:10.1038/nsmb.2959

Authors: Zhaoyong Li, Chuan Huang, Chun Bao, Liang Chen, Mei Lin, Xiaolin Wang, Guolin Zhong, Bin Yu, Wanchen Hu, Limin Dai, Pengfei Zhu, Zhaoxia Chang, Qingfa Wu, Yi Zhao, Ya Jia, Ping Xu, Huijie Liu & Ge Shan

Nature Structural & Molecular Biology 22, 176 (2015). doi:10.1038/nsmb.2981

Authors: Sue Jinks-Robertson & Hannah L Klein

Mapping of ribonucleotides to single-nucleotide resolution in yeast genomes provides new insight into the enzymology of DNA replication.

Publication date: 10 March 2015
Source:Cell Reports, Volume 10, Issue 9
Author(s): Panagiotis Xenopoulos , Minjung Kang , Alberto Puliafito , Stefano Di Talia , Anna-Katerina Hadjantonakis
The pluripotent epiblast (EPI) is the founder tissue of almost all somatic cells. EPI and primitive endoderm (PrE) progenitors arise from the inner cell mass (ICM) of the blastocyst-stage embryo. The EPI lineage is distinctly identified by its expression of pluripotency-associated factors. Many of these factors have been reported to exhibit dynamic fluctuations of expression in embryonic stem cell cultures. Whether these fluctuations correlating with ICM fate choice occur in vivo remains an open question. Using single-cell resolution quantitative imaging of a Nanog transcriptional reporter, we noted an irreversible commitment to EPI/PrE lineages in vivo. A period of apoptosis occurred concomitantly with ICM cell-fate choice, followed by a burst of EPI-specific cell proliferation. Transitions were occasionally observed from PrE-to-EPI, but not vice versa, suggesting that they might be regulated and not stochastic. We propose that the rapid timescale of early mammalian embryonic development prevents fluctuations in cell fate.

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Xenopoulos et al. use high-resolution live cell imaging to visualize the emergence of the pluripotent lineage in mammalian embryos. Automated quantitative single-cell image analysis reveals that distinct developmental states are not observed to fluctuate in the mouse embryo. Occasionally, cells can change their fate toward a pluripotent identity, which once specified is rapidly expanded.

Nature Methods 12, 203 (2015). doi:10.1038/nmeth.3223

Authors: Jacob O Kitzman, Lea M Starita, Russell S Lo, Stanley Fields & Jay Shendure

Random mutagenesis methods only partially cover the mutational space and are constrained by DNA synthesis length limitations. Here we demonstrate programmed allelic series (PALS), a single-volume, site-directed mutagenesis approach using microarray-programmed oligonucleotides. We created libraries including nearly every missense mutation as singleton events for the yeast transcription factor Gal4 (99.9% coverage) and human tumor suppressor p53 (93.5%). PALS-based comprehensive missense mutational scans may aid structure-function studies, protein engineering, and the interpretation of variants identified by clinical sequencing.

Authors: Tibor Harkany, sa Hagner-McWhirter

Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N6-methyladenosine (m6A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m6A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m6A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner. Authors: Shay Geula, Sharon Moshitch-Moshkovitz, Dan Dominissini, Abed AlFatah Mansour, Nitzan Kol, Mali Salmon-Divon, Vera Hershkovitz, Eyal Peer, Nofar Mor, Yair S. Manor, Moshe Shay Ben-Haim, Eran Eyal, Sharon Yunger, Yishay Pinto, Diego Adhemar Jaitin, Sergey Viukov, Yoach Rais, Vladislav Krupalnik, Elad Chomsky, Mirie Zerbib, Itay Maza, Yoav Rechavi, Rada Massarwa, Suhair Hanna, Ido Amit, Erez Y. Levanon, Ninette Amariglio, Noam Stern-Ginossar, Noa Novershtern, Gideon Rechavi, Jacob H. Hanna

Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.

Here we describe a preparative differential centrifugation protocol for the isolation of ribosomes from a crude cell homogenate. The subcellular fraction obtained is enriched in ribosome monomers and polysomes. The protocol has been optimized for the homogenization and collection of the ribosomal fraction from prokaryotic cells, mammalian and plant tissues, reticulocytes, and chloroplasts. The quality of the ribosomal preparation is enhanced by the removal of the remaining cellular components and adsorbed proteins by pelleting through a sucrose cushion with a high concentration of monovalent salts, NH₄Cl or KCl. The different components of the ribosomal fraction isolated using this protocol can be further purified by sucrose gradient centrifugation.

Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg(egfp), encoding the enhanced green fluorescent protein (GFP) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo.

In this protocol, an RNA sample, fractionated by gel electrophoresis, is transferred from the gel onto a membrane by capillary transfer. Short-wave UV light is used to fix the transferred RNA to the membrane. The membrane is then pretreated to block nonspecific probe-binding sites, and hybridization of the immobilized RNA to a ³²P-labeled DNA or RNA probe specific for the mRNA of interest is performed. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. Because exposure to UV cross-links the RNA to the membrane, the membrane can be stripped and hybridized with other probes. The procedure is suitable for detecting poly(A)⁺-selected mRNA or mRNA in total cellular RNA if the target transcript is relatively abundant. Using DNA or RNA probes labeled to 1 x 10⁸–10 x 10⁸ cpm/µg, it should be possible to detect ~5 pg of a specific RNA.

by Thomas Lengauer, Ruth Nussinov

Steady state cellular microRNA (miRNA) levels represent the balance between miRNA biogenesis and turnover. The kinetics and sequence determinants of mammalian miRNA turnover during and after miRNA maturation are not fully understood. Through a large-scale study on mammalian miRNA turnover, we report the co-existence of multiple cellular miRNA pools with distinct turnover kinetics and biogenesis properties and reveal previously unrecognized sequence features for fast turnover miRNAs. We measured miRNA turnover rates in eight mammalian cell types with a combination of expression profiling and deep sequencing. While most miRNAs are stable, a subset of miRNAs, mostly miRNA*s, turnovers quickly, many of which display a two-step turnover kinetics. Moreover, different sequence isoforms of the same miRNA can possess vastly different turnover rates. Fast turnover miRNA isoforms are enriched for 5' nucleotide bias against Argonaute-(AGO)-loading, but also additional 3' and central sequence features. Modeling based on two fast turnover miRNA*s miR-222-5p and miR-125b-1-3p, we unexpectedly found that while both miRNA*s are associated with AGO, they strongly differ in HSP90 association and sensitivity to HSP90 inhibition. Our data characterize the landscape of genome-wide miRNA turnover in cultured mammalian cells and reveal differential HSP90 requirements for different miRNA*s. Our findings also implicate rules for designing stable small RNAs, such as siRNAs.

During cellular stress, protein synthesis is severely reduced and bulk mRNA is recruited to stress granules (SGs). Previously, we showed that the SG-recruited IGF2 mRNA-binding protein 1 (IGF2BP1) interferes with target mRNA degradation during cellular stress. Whether this requires the formation of SGs remained elusive. Here, we demonstrate that the sustained inhibition of visible SGs requires the concomitant knockdown of TIA1, TIAR and G3BP1. FRAP and photo-conversion studies, however, indicate that these proteins only transiently associate with SGs. This suggests that instead of forming a rigid scaffold for mRNP recruitment, TIA proteins and G3BP1 promote SG-formation by constantly replenishing mRNPs. In contrast, RNA-binding proteins like IGF2BP1 or HUR, which are dispensable for SG-assembly, are stably associated with SGs and the IGF2BP1/HUR-G3BP1 association is increased during stress. The depletion of IGF2BP1 enhances the degradation of target mRNAs irrespective of inhibiting SG-formation, whereas the turnover of bulk mRNA remains unaffected when SG-formation is impaired. Together these findings indicate that the stabilization of mRNAs during cellular stress is facilitated by the formation of stable mRNPs, which are recruited to SGs by TIA proteins and/or G3BP1. Importantly, however, the aggregation of mRNPs to visible SGs is dispensable for preventing mRNA degradation.

SC3-seq: a method for highly parallel and quantitative measurement of single-cell gene expression.

Nucleic Acids Res. 2015 Feb 26;

Authors: Nakamura T, Yabuta Y, Okamoto I, Aramaki S, Yokobayashi S, Kurimoto K, Sekiguchi K, Nakagawa M, Yamamoto T, Saitou M

Abstract
Single-cell mRNA sequencing (RNA-seq) methods have undergone rapid development in recent years, and transcriptome analysis of relevant cell populations at single-cell resolution has become a key research area of biomedical sciences. We here present single-cell mRNA 3-prime end sequencing (SC3-seq), a practical methodology based on PCR amplification followed by 3-prime-end enrichment for highly quantitative, parallel and cost-effective measurement of gene expression in single cells. The SC3-seq allows excellent quantitative measurement of mRNAs ranging from the 10,000-cell to 1-cell level, and accordingly, allows an accurate estimate of the transcript levels by a regression of the read counts of spike-in RNAs with defined copy numbers. The SC3-seq has clear advantages over other typical single-cell RNA-seq methodologies for the quantitative measurement of transcript levels and at a sequence depth required for the saturation of transcript detection. The SC3-seq distinguishes four distinct cell types in the peri-implantation mouse blastocysts. Furthermore, the SC3-seq reveals the heterogeneity in human-induced pluripotent stem cells (hiPSCs) cultured under on-feeder as well as feeder-free conditions, demonstrating a more homogeneous property of the feeder-free hiPSCs. We propose that SC3-seq might be used as a powerful strategy for single-cell transcriptome analysis in a broad range of investigations in biomedical sciences.

PMID: 25722368 [PubMed - as supplied by publisher]

A functional extracellular transcriptome in animals? implications for biology, disease and medicine.

Genome Biol. 2015;16(1):47

Authors: Tewari M

Abstract
Muneesh Tewari shares his views on possible functions and applications of RNA transport by extracellular vesicles in animals.

PMID: 25723626 [PubMed - as supplied by publisher]

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Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation.

Cell Res. 2014 Jul;24(7):842-51

Authors: Han Y, Gao X, Liu B, Wan J, Zhang X, Qian SB

Abstract
The journey of a newly synthesized polypeptide starts in the peptidyltransferase center of the ribosome, from where it traverses the exit tunnel. The interior of the ribosome exit tunnel is neither straight nor smooth. How the ribosome dynamics in vivo is influenced by the exit tunnel is poorly understood. Genome-wide ribosome profiling in mammalian cells reveals elevated ribosome density at the start codon and surprisingly the downstream 5th codon position as well. We found that the highly focused ribosomal pausing shortly after initiation is attributed to the geometry of the exit tunnel, as deletion of the loop region from ribosome protein L4 diminishes translational pausing at the 5th codon position. Unexpectedly, the ribosome variant undergoes translational abandonment shortly after initiation, suggesting that there exists an obligatory step between initiation and elongation commitment. We propose that the post-initiation pausing of ribosomes represents an inherent signature of the translation machinery to ensure productive translation.

PMID: 24903108 [PubMed - indexed for MEDLINE]

Pseudogene-derived lncRNAs: emerging regulators of gene expression.

Front Genet. 2014;5:476

Authors: Milligan MJ, Lipovich L

Abstract
In the more than one decade since the completion of the Human Genome Project, the prevalence of non-protein-coding functional elements in the human genome has emerged as a key revelation in post-genomic biology. Highlighted by the ENCODE (Encyclopedia of DNA Elements) and FANTOM (Functional Annotation of Mammals) consortia, these elements include tens of thousands of pseudogenes, as well as comparably numerous long non-coding RNA (lncRNA) genes. Pseudogene transcription and function remain insufficiently understood. However, the field is of great importance for human disease due to the high sequence similarity between pseudogenes and their parental protein-coding genes, which generates the potential for sequence-specific regulation. Recent case studies have established essential and coordinated roles of both pseudogenes and lncRNAs in development and disease in metazoan systems, including functional impacts of lncRNA transcription at pseudogene loci on the regulation of the pseudogenes' parental genes. This review synthesizes the nascent evidence for regulatory modalities jointly exerted by lncRNAs and pseudogenes in human disease, and for recent evolutionary origins of these systems.

PMID: 25699073 [PubMed]

Nature Reviews Genetics. doi:10.1038/nrg3919

Author: Darren J. Burgess

Nature Genetics 47, 199 (2015). doi:10.1038/ng.3192

Authors: Matthew K Iyer, Yashar S Niknafs, Rohit Malik, Udit Singhal, Anirban Sahu, Yasuyuki Hosono, Terrence R Barrette, John R Prensner, Joseph R Evans, Shuang Zhao, Anton Poliakov, Xuhong Cao, Saravana M Dhanasekaran, Yi-Mi Wu, Dan R Robinson, David G Beer, Felix Y Feng, Hariharan K Iyer & Arul M Chinnaiyan