Hyeshik Chang

A-to-I editing in the miRNA seed region regulates target mRNA selection and silencing efficiency.

Nucleic Acids Res. 2014 Jul 23;

Authors: Kume H, Hino K, Galipon J, Ui-Tei K

Abstract
Hydrolytic deamination of adenosine to inosine (A-to-I) by adenosine deaminases acting on RNA (ADARs) is a post-transcriptional modification which results in a discrepancy between genomic DNA and the transcribed RNA sequence, thus contributing to the diversity of the transcriptome. Inosine preferentially base pairs with cytidine, meaning that A-to-I modifications in the mRNA sequences may be observed as A-to-G substitutions by the protein-coding machinery. Genome-wide studies have revealed that the majority of editing events occur in non-coding RNA sequences, but little is known about their functional meaning. MiRNAs are small non-coding RNAs that regulate the expression of target mRNAs with complementarities to their seed region. Here, we confirm that A-to-I editing in the miRNA seed duplex globally reassigns their target mRNAs in vivo, and reveal that miRNA containing inosine in the seed region exhibits a different degree of silencing efficiency compared to the corresponding miRNA with guanosine at the same position. The difference in base-pairing stability, deduced by melting temperature measurements, between seed-target duplexes containing either C:G or I:C pairs may account for the observed silencing efficiency. These findings unequivocally show that C:G and I:C pairs are biologically different in terms of gene expression regulation by miRNAs.

PMID: 25056317 [PubMed - as supplied by publisher]

Background: The recent reports of two circular RNAs (circRNAs) with strong potential to act as microRNA (miRNA) sponges suggest that circRNAs might play important roles in regulating gene expression. However, the global properties of circRNAs are not well understood. Results: We developed a computational pipeline to identify circRNAs and quantify their relative abundance from RNA-seq data. Applying this pipeline to a large set of non-poly(A)-selected RNA-seq data from the ENCODE project, we annotated 7,112 human circRNAs that were estimated to comprise at least 10% of the transcripts accumulating from their loci. Most circRNAs are expressed in only a few cell types and at low abundance, but they are no more cell-type?specific than are mRNAs with similar overall expression levels. Although most circRNAs overlap protein-coding sequences, ribosome profiling provides no evidence for their translation. We also annotated 635 mouse circRNAs, and although 20% of them are orthologous to human circRNAs, the sequence conservation of these circRNA orthologs is no higher than that of their neighboring linear exons. The previously proposed miR-7 sponge, CDR1as, is one of only two circRNAs with more miRNA sites than expected by chance, with the next best miRNA-sponge candidate deriving from a gene encoding a primate-specific zinc-finger protein, ZNF91. Conclusions: Our results provide a new framework for future investigation of this intriguing topological isoform while raising doubts regarding a biological function of most circRNAs.

Expanded identification and characterization of mammalian circular RNAs.

Genome Biol. 2014 Jul 29;15(7):409

Authors: Guo JU, Agarwal V, Guo H, Bartel DP

Abstract
BackgroundThe recent reports of two circular RNAs (circRNAs) with strong potential to act as microRNA (miRNA) sponges suggest that circRNAs might play important roles in regulating gene expression. However, the global properties of circRNAs are not well understood.ResultsWe developed a computational pipeline to identify circRNAs and quantify their relative abundance from RNA-seq data. Applying this pipeline to a large set of non-poly(A)-selected RNA-seq data from the ENCODE project, we annotated 7,112 human circRNAs that were estimated to comprise at least 10% of the transcripts accumulating from their loci. Most circRNAs are expressed in only a few cell types and at low abundance, but they are no more cell-type¿specific than are mRNAs with similar overall expression levels. Although most circRNAs overlap protein-coding sequences, ribosome profiling provides no evidence for their translation. We also annotated 635 mouse circRNAs, and although 20% of them are orthologous to human circRNAs, the sequence conservation of these circRNA orthologs is no higher than that of their neighboring linear exons. The previously proposed miR-7 sponge, CDR1as, is one of only two circRNAs with more miRNA sites than expected by chance, with the next best miRNA-sponge candidate deriving from a gene encoding a primate-specific zinc-finger protein, ZNF91.ConclusionsOur results provide a new framework for future investigation of this intriguing topological isoform while raising doubts regarding a biological function of most circRNAs.

PMID: 25070500 [PubMed - as supplied by publisher]

The sequence of events leading to stress granule assembly in stressed cells remains elusive. We show here, using isotope labeling and ion microprobe, that proportionally more RNA than proteins are present in stress granules than in surrounding cytoplasm. We further demonstrate that the delivery of single strand polynucleotides, mRNA and ssDNA, to the cytoplasm can trigger stress granule assembly. On the other hand, increasing the cytoplasmic level of mRNA-binding proteins like YB-1 can directly prevent the aggregation of mRNA by forming isolated mRNPs, as evidenced by atomic force microscopy. Interestingly, we also discovered that enucleated cells do form stress granules, demonstrating that the translocation to the cytoplasm of nuclear prion-like RNA-binding proteins like TIA-1 is dispensable for stress granule assembly. The results lead to an alternative view on stress granule formation based on the following sequence of events: after the massive dissociation of polysomes during stress, mRNA-stabilizing proteins like YB-1 are outnumbered by the burst of nonpolysomal mRNA. mRNA freed of ribosomes thus becomes accessible to mRNA-binding aggregation-prone proteins or misfolded proteins, which induces stress granule formation. Within the frame of this model, the shuttling of nuclear mRNA-stabilizing proteins to the cytoplasm could dissociate stress granules or prevent their assembly.

Messenger RNA (mRNA) localization is coupled to the translational repression of transcripts during their transport. It is still unknown if this coupling depends on physical interactions between translational control and mRNA localization machineries, and how these interactions are established at the molecular level. In yeast, localization of transcripts like ASH1 to the bud depends on the RNA-binding protein She2. During its transport, ASH1 mRNA translation is repressed by Puf6. Herein, we report that She2 recruits Puf6 on ASH1 co-transcriptionally. The recruitment of Puf6 depends on prior co-transcriptional loading of Loc1, an exclusively nuclear protein. These proteins form a ternary complex, in which Loc1 bridges Puf6 to She2, that binds the ASH1 3'UTR. Using a genome-wide ChIP-chip approach, we identified over 40 novel targets of Puf6, including several bud-localized mRNAs. Interestingly, the co-transcriptional recruitment of Puf6 on genes coding for these bud-localized mRNAs is also She2- and Loc1-dependent. Our results suggest a coordinated assembly of localization and translational control machineries on localized mRNAs during transcription, and underline the importance of co-transcriptional events in establishing the cytoplasmic fate of mRNAs.

Mammalian mRNAs are generated by complex and coordinated biogenesis pathways and acquire 5'-end m⁷G caps that play fundamental roles in processing and translation. Here we show that several selenoprotein mRNAs are not recognized efficiently by translation initiation factor eIF4E because they bear a hypermethylated cap. This cap modification is acquired via a 5'-end maturation pathway similar to that of the small nucle(ol)ar RNAs (sn- and snoRNAs). Our findings also establish that the trimethylguanosine synthase 1 (Tgs1) interacts with selenoprotein mRNAs for cap hypermethylation and that assembly chaperones and core proteins devoted to sn- and snoRNP maturation contribute to recruiting Tgs1 to selenoprotein mRNPs. We further demonstrate that the hypermethylated-capped selenoprotein mRNAs localize to the cytoplasm, are associated with polysomes and thus translated. Moreover, we found that the activity of Tgs1, but not of eIF4E, is required for the synthesis of the GPx1 selenoprotein in vivo.

Imaging of protein–protein and RNA–protein interactions in vivo, especially in live animals, is still challenging. Here we developed far-red mNeptune-based bimolecular fluorescence complementation (BiFC) and trimolecular fluorescence complementation (TriFC) systems with excitation and emission above 600 nm in the ‘tissue optical window’ for imaging of protein–protein and RNA–protein interactions in live cells and mice. The far-red mNeptune BiFC was first built by selecting appropriate split mNeptune fragments, and then the mNeptune-TriFC system was built based on the mNeptune-BiFC system. The newly constructed mNeptune BiFC and TriFC systems were verified as useful tools for imaging protein–protein and mRNA–protein interactions, respectively, in live cells and mice. We then used the new mNeptune-TriFC system to investigate the interactions between human polypyrimidine-tract-binding protein (PTB) and HIV-1 mRNA elements as PTB may participate in HIV mRNA processing in HIV activation from latency. An interaction between PTB and the 3'long terminal repeat region of HIV-1 mRNAs was found and imaged in live cells and mice, implying a role for PTB in regulating HIV-1 mRNA processing. The study provides new tools for in vivo imaging of RNA–protein and protein–protein interactions, and adds new insight into the mechanism of HIV-1 mRNA processing.

Multiplicity of 5' Cap Structures Present on Short RNAs.

PLoS One. 2014;9(7):e102895

Authors: Abdelhamid RF, Plessy C, Yamauchi Y, Taoka M, de Hoon M, Gingeras TR, Isobe T, Carninci P

Abstract
Most RNA molecules are co- or post-transcriptionally modified to alter their chemical and functional properties to assist in their ultimate biological function. Among these modifications, the addition of 5' cap structure has been found to regulate turnover and localization. Here we report a study of the cap structure of human short (<200 nt) RNAs (sRNAs), using sequencing of cDNA libraries prepared by enzymatic pretreatment of the sRNAs with cap sensitive-specificity, thin layer chromatographic (TLC) analyses of isolated cap structures and mass spectrometric analyses for validation of TLC analyses. Processed versions of snoRNAs and tRNAs sequences of less than 50 nt were observed in capped sRNA libraries, indicating additional processing and recapping of these annotated sRNAs biotypes. We report for the first time 2,7 dimethylguanosine in human sRNAs cap structures and surprisingly we find multiple type 0 cap structures (mGpppC, 7mGpppG, GpppG, GpppA, and 7mGpppA) in RNA length fractions shorter than 50 nt. Finally, we find the presence of additional uncharacterized cap structures that wait determination by the creation of needed reference compounds to be used in TLC analyses. These studies suggest the existence of novel biochemical pathways leading to the processing of primary and sRNAs and the modifications of their RNA 5' ends with a spectrum of chemical modifications.

PMID: 25079783 [PubMed - as supplied by publisher]

Related Articles

Effects of genetic variations on microRNA:target interactions.

Nucleic Acids Res. 2014 Jul 31;

Authors: Liu C, Rennie WA, Carmack CS, Kanoria S, Cheng J, Lu J, Ding Y

Abstract
Genetic variations within microRNA (miRNA) binding sites can affect miRNA-mediated gene regulation, which may lead to phenotypes and diseases. We perform a transcriptome-scale analysis of genetic variants and miRNA:target interactions identified by CLASH. This analysis reveals that rare variants tend to reside in CDSs, whereas common variants tend to reside in the 3' UTRs. miRNA binding sites are more likely to reside within those targets in the transcriptome with lower variant densities, especially target regions in which nucleotides have low mutation frequencies. Furthermore, an overwhelming majority of genetic variants within or near miRNA binding sites can alter not only the potential of miRNA:target hybridization but also the structural accessibility of the binding sites and flanking regions. These suggest an interpretation for certain associations between genetic variants and diseases, i.e. modulation of miRNA-mediated gene regulation by common or rare variants within or near miRNA binding sites, likely through target structure alterations. Our data will be valuable for discovering new associations among miRNAs, genetic variations and human diseases.

PMID: 25081214 [PubMed - as supplied by publisher]

Related Articles

The impact of microRNA expression on cellular proliferation.

Hum Genet. 2014 Jul;133(7):931-8

Authors: Lenkala D, LaCroix B, Gamazon ER, Geeleher P, Im HK, Huang RS

Abstract
As an important class of non-coding regulatory RNAs, microRNAs (miRNAs) play a key role in a range of biological processes. These molecules serve as post-transcriptional regulators of gene expression and their regulatory activity has been implicated in disease pathophysiology and pharmacological traits. We sought to investigate the impact of miRNAs on cellular proliferation to gain insight into the molecular basis of complex traits that depend on cellular growth, including, most prominently, cancer. We examined the relationship between miRNA expression and intrinsic cellular growth (iGrowth) in the HapMap lymphoblastoid cell lines derived from individuals of different ethnic backgrounds. We found a substantial enrichment for miRNAs (53 miRNAs, FDR < 0.05) correlated with cellular proliferation in pooled CEU (Caucasian of northern and western European descent) and YRI (individuals from Ibadan, Nigeria) samples. Specifically, 119 miRNAs (59 %) were significantly correlated with iGrowth in YRI; of these miRNAs, 18 were correlated with iGrowth in CEU. To gain further insight into the effect of miRNAs on cellular proliferation in cancer, we showed that over-expression of miR-22, one of the top iGrowth-associated miRNAs, leads to growth inhibition in an ovarian cancer cell line (SKOV3). Furthermore, over-expression of miR-22 down-regulates the expression of its target genes (MXI1 and SLC25A37) in this ovarian cancer cell line, highlighting an miRNA-mediated regulatory network potentially important for cellular proliferation. Importantly, our study identified miRNAs that can be used as molecular targets in cancer therapy.

PMID: 24609542 [PubMed - indexed for MEDLINE]

Motivation: Circular RNAs (circRNAs) are an abundant class of highly stable RNAs that can affect gene regulation by binding and preventing microRNAs (miRNAs) from regulating their messenger RNA (mRNA) targets. Mammals have thousands of circRNAs with predicted miRNA binding sites, but only two circRNAs have been verified as being actual miRNA sponges. As it is unclear whether these thousands of predicted miRNA binding sites are functional, we investigated whether miRNA seed sites within human circRNAs are under selective pressure.

Results: Using SNP data from the 1000 Genomes Project, we found a significant decrease in SNP density at miRNA seed sites compared with flanking sequences and random sites. This decrease was similar to that of miRNA seed sites in 3' untranslated regions, suggesting that many of the predicted miRNA binding sites in circRNAs are functional and under similar selective pressure as miRNA binding sites in mRNAs.

Contact: pal.satrom@ntnu.no

Supplementary information: Supplementary data are available at Bioinformatics online.

miRdentify: high stringency miRNA predictor identifies several novel animal miRNAs.

Nucleic Acids Res. 2014 Jul 22;

Authors: Hansen TB, Venø MT, Kjems J, Damgaard CK

Abstract
During recent years, miRNAs have been shown to play important roles in the regulation of gene expression. Accordingly, much effort has been put into the discovery of novel uncharacterized miRNAs in various organisms. miRNAs are structurally defined by a hairpin-loop structure recognized by the two-step processing apparatus, Drosha and Dicer, necessary for the production of mature ∼22-nucleotide miRNA guide strands. With the emergence of high-throughput sequencing applications, tools have been developed to identify miRNAs and profile their expression based on sequencing reads. However, as the read depth increases, false-positive predictions increase using established algorithms, underscoring the need for more stringent approaches. Here we describe a transparent pipeline for confident miRNA identification in animals, termed miRdentify. We show that miRdentify confidently discloses more than 400 novel miRNAs in humans, including the first male-specific miRNA, which we successfully validate. Moreover, novel miRNAs are predicted in the mouse, the fruit fly and nematodes, suggesting that the pipeline applies to all animals. The entire software package is available at www.ncrnalab.dk/mirdentify.

PMID: 25053842 [PubMed - as supplied by publisher]

Experimental Validation of Predicted Mammalian MicroRNAs of Mirtron Origin.

Methods Mol Biol. 2014;1182:245-63

Authors: Schamberger A, Orbán TI

Abstract
MicroRNAs (miRNAs) are ~22 nucleotide-long noncoding RNAs influencing many cellular processes by their regulatory functions on gene expression. MiRNAs of mirtron origin represent the most prominent group of the alternatively processed miRNAs. They reside in short introns, which are essentially equivalent to the precursor form of the given miRNA. Consequently, their maturation is independent of the Drosha/DGCR8 complex, while depends on the mechanism of mRNA splicing. The number of predicted human mirtron sequences increases as a consequence of the growing deep sequencing data and refined bioinformatics tools. However, experimental validations of particular sequences are also essential. In this chapter, we intend to provide detailed protocols for the investigation of predicted mirtron sequences. First, we use the Sleeping Beauty transposon-based gene-delivery system for the development of cell lines stably overexpressing mirtrons. The processing of functional mature miRNAs is then detected by a luciferase assay using a very strict "triple control" system. In addition, bona fide mirtron features are confirmed by demonstrating splicing dependency through splice site mutations, while Drosha/DGCR8 independency is assessed in DGCR8 deficient cell line. Finally, the presence of mirtron-derived mature miRNAs is detected by quantitative real-time PCR.

PMID: 25055917 [PubMed - in process]

A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription–replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics.

Related Articles

SCOPE++: Sequence Classification Of homoPolymer Emissions.

Genomics. 2014 Jul 31;

Authors: Morton JT, Abrudan P, Figueroa N, Liang C, Karro JE

Abstract
BACKGROUND: mRNA polyadenylation, the addition of a poly(A) tail to the 3'-end of pre-mRNA, is a process critical to gene expression and regulation in eukaryotes. To understand the molecular mechanisms governing polyadenylation and other relevant biological processes, it is important to identify these poly(A) tails accurately in transcriptome sequencing data and differentiate them from artificial adapter sequences added in the sequencing process. But the annotation of these tails is complicated by the presence of sequencing errors and post-transcriptional modifications. While determining that a tail is present in a given transcript fragment is straight-forward, these obfuscations make the problem of boundary identification a challenge; conventional seed-and-extend algorithms struggle to accurately identify these poly(A) tail end-points. Further, all existing tools that we are aware of focus exclusively on the trimming of poly(A) tails, failing to provide the detailed information needed for studying the polyadenylation process.
RESULTS: We have created SCOPE++, a tool for finding the precise border of poly(A) tails and other homopolymers in raw mRNA sequence reads. Based on a Hidden Markov Model (HMM) approach, SCOPE++ accurately identifies specific homopolymer sequences in error-prone EST/cDNA data or RNA-Seq data at a speed appropriate for large sequence sets.
CONCLUSIONS: We demonstrate that our tool can precisely identify poly(A) tails with near perfect accuracy at the speed required for high-throughput applications, providing a valuable resource for polyadenylation research.

PMID: 25087770 [PubMed - as supplied by publisher]

Background: The polyadenylation of RNA is critical for gene functioning, but the conserved sequence motifs (often called signal or signature motifs), motif locations and abundances, and base composition patterns around mRNA polyadenylation [poly(A)] sites are still uncharacterized in most species. The evolutionary tendency for poly(A) site selection is still largely unknown. Results: We analyzed the poly(A) site regions of 31 species or phyla. Different groups of species showed different poly(A) signal motifs: UUACUU at the poly(A) site in the parasite Trypanosoma cruzi; UGUAAC (approximately 13 bases upstream of the site) in the alga Chlamydomonas reinhardtii; UGUUUG (or UGUUUGUU) at mainly the fourth base downstream of the poly(A) site in the parasite Blastocystis hominis; and AAUAAA at approximately 16 bases and approximately 19 bases upstream of the poly(A) site in animals and plants, respectively. Polyadenylation signal motifs are usually several hundred times more abundant around poly(A) sites than in whole genomes. These predominant motifs usually had very specific locations, whether upstream of, at, or downstream of poly(A) sites, depending on the species or phylum. The poly(A) site was usually an adenosine (A) in all analyzed species except for B. hominis, and there was weak A predominance in C. reinhardtii. Fungi, animals, plants, and the protist Phytophthora infestans shared a general base abundance pattern (or base composition pattern) of “U-rich—A-rich—U-rich—Poly(A) site—U-rich regions”, or U-A-U-A-U for short, with some variation for each kingdom or subkingdom. Conclusion: This study identified the poly(A) signal motifs, motif locations, and base composition patterns around mRNA poly(A) sites in protists, fungi, plants, and animals and provided insight into poly(A) site evolution.

lncRNAs: Linking RNA to Chromatin.

Cold Spring Harb Perspect Biol. 2014;6(8)

Authors: Rinn JL

Abstract
Numerous studies over the past decade have identified increasing numbers of long noncoding RNAs (lncRNAs) across many organisms. Research since has shown that lncRNAs constitute an important layer of genome regulation in diverse biological processes and disease. Here, we discuss the common emerging theme of lncRNAs interfacing with epigenetic machinery. This, in turn, modulates the activity and localization of the epigenetic machinery during cell fate specification.

PMID: 25085913 [PubMed - as supplied by publisher]

Nature advance online publication 03 August 2014. doi:10.1038/nature13578

Authors: Brian M. Zid & Erin K. O’Shea

A universal feature of the response to stress and nutrient limitation is transcriptional upregulation of genes that encode proteins important for survival. Under many such conditions, the overall protein synthesis level is reduced, thereby dampening the stress response at the level of protein expression. For example, during glucose starvation in Saccharomyces cerevisiae (yeast), translation is rapidly repressed, yet the transcription of many stress- and glucose-repressed genes is increased. Here we show, using ribosomal profiling and microscopy, that this transcriptionally upregulated gene set consists of two classes: one class produces messenger RNAs that are translated during glucose starvation and are diffusely localized in the cytoplasm, including many heat-shock protein mRNAs; and the other class produces mRNAs that are not efficiently translated during glucose starvation and are concentrated in foci that co-localize with P bodies and stress granules, a class that is enriched for mRNAs involved in glucose metabolism. Surprisingly, the information specifying the differential localization and protein production of these two classes of mRNA is encoded in the promoter sequence: promoter responsiveness to heat-shock factor 1 (Hsf1) specifies diffuse cytoplasmic localization and higher protein production on glucose starvation. Thus, promoter sequences can influence not only the levels of mRNAs but also the subcellular localization of mRNAs and the efficiency with which they are translated, enabling cells to tailor protein production to the environmental conditions.

Nature advance online publication 03 August 2014. doi:10.1038/nature13553

Authors: Christopher R. Faehnle, Jack Walleshauser & Leemor Joshua-Tor

The pluripotency factor Lin28 inhibits the biogenesis of the let-7 family of mammalian microRNAs. Lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development, glucose metabolism and tissue regeneration. Overexpression of Lin28 is correlated with the onset of numerous cancers, whereas let-7, a tumour suppressor, silences several human oncogenes. Lin28 binds to precursor let-7 (pre-let-7) hairpins, triggering the 3′ oligo-uridylation activity of TUT4 and TUT7 (refs 10, 11, 12). The oligoU tail added to pre-let-7 serves as a decay signal, as it is rapidly degraded by Dis3l2 (refs 13, 14), a homologue of the catalytic subunit of the RNA exosome. The molecular basis of Lin28-mediated recruitment of TUT4 and TUT7 to pre-let-7 and its subsequent degradation by Dis3l2 is largely unknown. To examine the mechanism of Dis3l2 substrate recognition we determined the structure of mouse Dis3l2 in complex with an oligoU RNA to mimic the uridylated tail of pre-let-7. Three RNA-binding domains form an open funnel on one face of the catalytic domain that allows RNA to navigate a path to the active site different from that of its exosome counterpart. The resulting path reveals an extensive network of uracil-specific interactions spanning the first 12 nucleotides of an oligoU-tailed RNA. We identify three U-specificity zones that explain how Dis3l2 recognizes, binds and processes uridylated pre-let-7 in the final step of the Lin28–let-7 pathway.

Publication date: 31 July 2014
Source:Cell, Volume 158, Issue 3
Author(s): Thomas Trimarchi , Erhan Bilal , Panagiotis Ntziachristos , Giulia Fabbri , Riccardo Dalla-Favera , Aristotelis Tsirigos , Iannis Aifantis
Notch signaling is a key developmental pathway that is subject to frequent genetic and epigenetic perturbations in many different human tumors. Here we investigate whether long noncoding RNA (lncRNA) genes, in addition to mRNAs, are key downstream targets of oncogenic Notch1 in human T cell acute lymphoblastic leukemia (T-ALL). By integrating transcriptome profiles with chromatin state maps, we have uncovered many previously unreported T-ALL-specific lncRNA genes, a fraction of which are directly controlled by the Notch1/Rpbjκ activator complex. Finally we have shown that one specific Notch-regulated lncRNA, LUNAR1, is required for efficient T-ALL growth in vitro and in vivo due to its ability to enhance IGF1R mRNA expression and sustain IGF1 signaling. These results confirm that lncRNAs are important downstream targets of the Notch signaling pathway, and additionally they are key regulators of the oncogenic state in T-ALL.

Graphical abstract

Teaser

Manual annotation of lncRNA genes revealed many previously unreported T-ALL-specific lncRNAs. A Notch-regulated lncRNA, LUNAR1, controls the expression of the IGF1R gene through chromosomal looping and thereby IGF1 signaling and growth of T-ALL.

Publication date: 31 July 2014
Source:Cell, Volume 158, Issue 3

As part of Cell’s 40^th anniversary celebration, we are spotlighting 40 principal investigators under the age of 40. See the full profiles of all of these young scientists and their responses to this and other questions at http://www.cell.com/40/under40.

Publication date: 31 July 2014
Source:Cell, Volume 158, Issue 3
Author(s): Jessica Nouws , Gerald S. Shadel
Regulation of gene expression in mammalian mitochondria by microRNAs is reported by Zhang et al. During muscle cell differentiation, localization of a miRNA is increased within mitochondria, where it interacts with Ago2 to selectively activate translation of mtDNA-encoded mRNAs. The findings represent a new mitochondrial regulatory pathway and a potentially powerful means to purposefully manipulate mtDNA expression.

Teaser

Regulation of gene expression in mammalian mitochondria by microRNAs is reported by Zhang et al. During muscle cell differentiation, localization of a miRNA is increased within mitochondria, where it interacts with Ago2 to selectively activate translation of mtDNA-encoded mRNAs. The findings represent a new mitochondrial regulatory pathway and a potentially powerful means to purposefully manipulate mtDNA expression.

Publication date: 31 July 2014
Source:Cell, Volume 158, Issue 3
Author(s): Xiaorong Zhang , Xinxin Zuo , Bo Yang , Zongran Li , Yuanchao Xue , Yu Zhou , Jie Huang , Xiaolu Zhao , Jie Zhou , Yun Yan , Huiqiong Zhang , Peipei Guo , Hui Sun , Lin Guo , Yi Zhang , Xiang-Dong Fu
MicroRNAs are well known to mediate translational repression and mRNA degradation in the cytoplasm. Various microRNAs have also been detected in membrane-compartmentalized organelles, but the functional significance has remained elusive. Here, we report that miR-1, a microRNA specifically induced during myogenesis, efficiently enters the mitochondria where it unexpectedly stimulates, rather than represses, the translation of specific mitochondrial genome-encoded transcripts. We show that this positive effect requires specific miR:mRNA base-pairing and Ago2, but not its functional partner GW182, which is excluded from the mitochondria. We provide evidence for the direct action of Ago2 in mitochondrial translation by crosslinking immunoprecipitation coupled with deep sequencing (CLIP-seq), functional rescue with mitochondria-targeted Ago2, and selective inhibition of the microRNA machinery in the cytoplasm. These findings unveil a positive function of microRNA in mitochondrial translation and suggest a highly coordinated myogenic program via miR-1-mediated translational stimulation in the mitochondria and repression in the cytoplasm.

Graphical abstract

Teaser

Ago2 and microRNA are present in mitochondria where they promote translation of target transcripts during muscle differentiation. The same miRNA that enhances target translation in mitochondria has a repressive role in the cytoplasm.

Article

Dendritic protein synthesis is implicated in synaptic plasticity and memory storage. Ainsley et al. , develop a method for collecting ribosome-bound mRNAs from mouse brain dendrites, and use RNA sequencing to characterize dendritic mRNAs that bind to ribosomes after mice experience a novel environment.

Nature Communications doi: 10.1038/ncomms5510

Authors: Joshua A. Ainsley, Laurel Drane, Jonathan Jacobs, Kara A. Kittelberger, Leon G. Reijmers

Nature Methods 11, 809 (2014). doi:10.1038/nmeth.3014

Authors: Pieter Mestdagh, Nicole Hartmann, Lukas Baeriswyl, Ditte Andreasen, Nathalie Bernard, Caifu Chen, David Cheo, Petula D'Andrade, Mike DeMayo, Lucas Dennis, Stefaan Derveaux, Yun Feng, Stephanie Fulmer-Smentek, Bernhard Gerstmayer, Julia Gouffon, Chris Grimley, Eric Lader, Kathy Y Lee, Shujun Luo, Peter Mouritzen, Aishwarya Narayanan, Sunali Patel, Sabine Peiffer, Silvia Rüberg, Gary Schroth, Dave Schuster, Jonathan M Shaffer, Elliot J Shelton, Scott Silveria, Umberto Ulmanella, Vamsi Veeramachaneni, Frank Staedtler, Thomas Peters, Toumy Guettouche, Linda Wong & Jo Vandesompele

Publication date: 7 August 2014
Source:Molecular Cell, Volume 55, Issue 3
Author(s): Agnieszka Tudek , Odil Porrua , Tomasz Kabzinski , Michael Lidschreiber , Karel Kubicek , Andrea Fortova , François Lacroute , Stepanka Vanacova , Patrick Cramer , Richard Stefl , Domenico Libri
The Nrd1-Nab3-Sen1 (NNS) complex is essential for controlling pervasive transcription and generating sn/snoRNAs in S. cerevisiae. The NNS complex terminates transcription of noncoding RNA genes and promotes exosome-dependent processing/degradation of the released transcripts. The Trf4-Air2-Mtr4 (TRAMP) complex polyadenylates NNS target RNAs and favors their degradation. NNS-dependent termination and degradation are coupled, but the mechanism underlying this coupling remains enigmatic. Here we provide structural and functional evidence demonstrating that the same domain of Nrd1p interacts with RNA polymerase II and Trf4p in a mutually exclusive manner, thus defining two alternative forms of the NNS complex, one involved in termination and the other in degradation. We show that the Nrd1-Trf4 interaction is required for optimal exosome activity in vivo and for the stimulation of polyadenylation of NNS targets by TRAMP in vitro. We propose that transcription termination and RNA degradation are coordinated by switching between two alternative partners of the NNS complex.

Graphical abstract

Teaser

The Nrd1-Nab3-Sen1 complex controls pervasive transcription in yeast by terminating transcription of cryptic transcripts and directing these RNAs to degradation by the exosome. Tudek et al. show that the same domain of Nrd1p mediates mutually exclusive interactions with RNAPII or the exosome cofactor TRAMP to coordinate termination and RNA degradation.

The nematode Caenorhabditis elegans is an important model for studies of germ cell biology, including the meiotic cell cycle, gamete specification as sperm or oocyte and gamete development. Fundamental to those studies is a genome-level knowledge of the germline transcriptome. Here we use RNA-Seq to identify genes expressed in isolated XX gonads, which are roughly 95% germline and 5% somatic gonadal tissue. We generate data from mutants making either sperm [fem-3(q96)] or oocytes [fog-2(q71)], both grown at 22°. Our dataset identifies a total of 10,754 mRNAs in the polyadenylated transcriptome of XX gonads, with 2,748 enriched in spermatogenic gonads, 1,732 enriched in oogenic gonads and the remaining 6,274 not enriched in either. These spermatogenic, oogenic and gender-neutral gene datasets compare well with those of earlier studies, but double the number of genes identified. A comparison of the additional genes found in our study with in situ hybridization patterns in the Kohara database suggests that most are expressed in the germline. We also query our RNA-Seq data for differential exon usage and find 351 mRNAs with sex-enriched isoforms. We suggest that this new dataset will prove useful for studies focusing on C. elegans germ cell biology.

Multifunctional Roles for the Protein Translocation Machinery in RNA Anchoring to the Endoplasmic Reticulum.

J Biol Chem. 2014 Jul 25;

Authors: Jagannathan S, Hsu JC, Reid DW, Chen Q, Thompson WJ, Moseley AM, Nicchitta CV

Abstract
mRNAs encoding cytosolic and signal sequence-bearing proteins are translated by free and endoplasmic reticulum (ER)-bound ribosomes, respectively. Recent ribosome footprinting studies have also demonstrated translation of cytosolic protein-encoding mRNAs on ER-bound ribosomes, findings that raise important questions regarding the mechanism of ribosome and mRNA localization and association with the ER. Using a semi- intact HeLa cell model, we performed a polysome solubilization screen and identified conditions that biochemically distinguish polysomes engaged in the translation of distinct cohorts of mRNAs. RNA-protein UV photocrosslinking studies revealed numerous ER integral membrane proteins with RNA binding activity, consistent with direct RNA anchoring functions. Quantitative proteomic analyses of HeLa cytosolic and two classes of ER-bound polysomes identified translocon components as selective polysome-interacting proteins. Notably, the Sec61 complex was highly enriched in polysomes engaged in the translation of endomembrane organelle proteins whereas whereas translocon accessory proteins such as ribophorin I were present in all subpopulations of ER-associated polysomes. Analyses of the protein composition of oligo(dT)-selected UV photocrosslinked protein-RNA adducts identified Sec61α , β and ribophorin I, suggesting roles for the protein translocation and modification machinery in mRNA anchoring to the ER. We propose that multiple mechanisms of mRNA and ribosome association with ER operate to enable a transcriptome-wide function for the ER in cellular protein synthesis.

PMID: 25063809 [PubMed - as supplied by publisher]

Non-coding RNA and the complex regulation of the trypanosome life cycle.

Curr Opin Microbiol. 2014 Jul 23;20C:146-152

Authors: Michaeli S

Abstract
The protozoan parasite Trypanosoma brucei is the causative agent of African sleeping sickness and the wasting disease, nagana, in cattle. The parasite is transmitted to the mammalian host by the bite of a tsetse fly. These parasites lack transcriptional regulation, and their gene expression is mainly regulated post-transcriptionally. Changes in the transcriptome and proteome suggest that mRNA stability and translation regulation operate to control cycling between the hosts. The review discusses the small RNome of T. brucei, and the potential involvement of these molecules in shaping the adaptation of the parasites to their hosts.

PMID: 25063970 [PubMed - as supplied by publisher]

Nature advance online publication 27 July 2014. doi:10.1038/nature13485

Authors: Andrew L. Wolfe, Kamini Singh, Yi Zhong, Philipp Drewe, Vinagolu K. Rajasekhar, Viraj R. Sanghvi, Konstantinos J. Mavrakis, Man Jiang, Justine E. Roderick, Joni Van der Meulen, Jonathan H. Schatz, Christina M. Rodrigo, Chunying Zhao, Pieter Rondou, Elisa de Stanchina, Julie Teruya-Feldstein, Michelle A. Kelliher, Frank Speleman, John A. Porco, Jerry Pelletier, Gunnar Rätsch & Hans-Guido Wendel