Hyeshik Chang

A-repeats are the simplest form of tandem repeats and are found ubiquitously throughout genomes. These mononucleotide repeats have been widely believed to be non-functional ‘junk’ DNA. However, studies in yeasts suggest that A-repeats play crucial biological functions, and their role in humans remains largely unknown. Here, we showed a non-random pattern of distribution of sense A- and T-repeats within 20 kb around transcription start sites (TSSs) in the human genome. Different distributions of these repeats are observed upstream and downstream of TSSs. Sense A-repeats are enriched upstream, whereas sense T-repeats are enriched downstream of TSSs. This enrichment directly correlates with repeat size. Genes with different functions contain different lengths of repeats. In humans, tissue-specific genes are enriched for short repeats of <10 bp, whereas housekeeping genes are enriched for long repeats of ≥10 bp. We demonstrated that DICER1 and Argonaute proteins are required for the cis-regulatory role of A-repeats. Moreover, in the presence of a synthetic polymer that mimics an A-repeat, protein binding to A-repeats was blocked, resulting in a dramatic change in the expression of genes containing upstream A-repeats. Our findings suggest a length-dependent cis-regulatory function of A-repeats and that Argonaute proteins serve as trans-acting factors, binding to A-repeats.

Cancerous and aging cells have long been thought to be impacted by transcription errors that cause genetic and epigenetic changes. Until now, a lack of methodology for directly assessing such errors hindered evaluation of their impact to the cells. We report a high-resolution Illumina RNA-seq method that can assess noncoded base substitutions in mRNA at 10^–4–10^–5 per base frequencies in vitro and in vivo. Statistically reliable detection of changes in transcription fidelity through ~10³ nt DNA sites assures that the RNA-seq can analyze the fidelity in a large number of the sites where errors occur. A combination of the RNA-seq and biochemical analyses of the positions for the errors revealed two sequence-specific mechanisms that increase transcription fidelity by Escherichia coli RNA polymerase: (i) enhanced suppression of nucleotide misincorporation that improves selectivity for the cognate substrate, and (ii) increased backtracking of the RNA polymerase that decreases a chance of error propagation to the full-length transcript after misincorporation and provides an opportunity to proofread the error. This method is adoptable to a genome-wide assessment of transcription fidelity.

The role of the mRNA-binding protein human antigen R (HuR) in stabilization and translation of AU-rich elements (ARE) containing mRNAs is well established. However, the trafficking of HuR and bound mRNA cargo, which comprises a fundamental requirement for the aforementioned HuR functions is only poorly understood. By administering different cytoskeletal inhibitors, we found that the protein kinase C (PKC)-triggered accumulation of cytoplasmic HuR by Angiotensin II (AngII) is an actin-myosin driven process functionally relevant for stabilization of ARE-bearing mRNAs. Furthermore, we show that the AngII-induced recruitment of HuR and its bound mRNA from ribonucleoprotein particles to free and cytoskeleton bound polysomes strongly depended on an intact actomyosin cytoskeleton. In addition, HuR allocation to free and cytoskeletal bound polysomes is highly sensitive toward RNase and PPtase and structurally depends on serine 318 (S318) located within the C-terminal RNA recognition motif (RRM3). Conversely, the trafficking of the phosphomimetic HuRS318D, mimicking HuR phosphorylation at S318 by the PKC remained PPtase resistant. Co-immunoprecipitation experiments with truncated HuR proteins revealed that the stimulus-induced association of HuR with myosin IIA is strictly RNA dependent and mediated via the RRM3. Our data implicate a microfilament dependent transport of HuR, which is relevant for stimulus-induced targeting of ARE-bearing mRNAs from translational inactive ribonucleoprotein particles to polysomes.

The EMBO Journal. doi:10.1038/emboj.2013.222

Authors: Kathrin Sophie Fröhlich, Kai Papenfort, Agnes Fekete & Jörg Vogel

The RNA-binding proteins LIN28A and LIN28B have diverse functions in embryonic stem cells, cellular reprogramming, growth, and oncogenesis. Many of these effects occur via direct inhibition of Let-7 microRNAs (miRNAs), although Let-7-independent effects have been surmised. We report that intestine targeted expression of LIN28B causes intestinal hypertrophy, crypt expansion, and Paneth cell loss. Furthermore, LIN28B fosters intestinal polyp and adenocarcinoma formation. To examine potential Let-7-independent functions of LIN28B, we pursued ribonucleoprotein cross-linking, immunoprecipitation, and high-throughput sequencing (CLIP-seq) to identify direct RNA targets. This revealed that LIN28B bound a substantial number of mRNAs and modestly augmented protein levels of these target mRNAs in vivo. Conversely, Let-7 had a profound effect; modulation of Let-7 levels via deletion of the mirLet7c2/mirLet7b genes recapitulated effects of Lin28b overexpression. Furthermore, intestine-specific Let-7 expression could reverse hypertrophy and Paneth cell depletion caused by Lin28b. This was independent of effects on insulin–PI3K–mTOR signaling. Our study reveals that Let-7 miRNAs are critical for repressing intestinal tissue growth and promoting Paneth cell differentiation. Let-7-dependent effects of LIN28B may supersede Let-7-independent effects on intestinal tissue growth. In summary, LIN28B can definitively act as an oncogene in the absence of canonical genetic alterations.

Charlotte Pollard, Stefaan De Koker, Xavier Saelens, Guido Vanham, Johan Grooten.

by Stefan M. Bresson, Nicholas K. Conrad

Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A)-binding protein (PABPN1), the poly(A) polymerases (PAPs), PAPα and PAPγ, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAPα, redundantly with PAPγ, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A) tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A) tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A) tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAPα/γ, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression.

Nature Protocols 8, 2240 (2013). doi:10.1038/nprot.2013.141

Authors: Samuel Peña-Llopis & James Brugarolas

Genomic technologies have revolutionized our understanding of complex Mendelian diseases and cancer. Solid tumors present several challenges for genomic analyses, such as tumor heterogeneity and tumor contamination with surrounding stroma and infiltrating lymphocytes. We developed a protocol to (i) select tissues of high cellular purity

Nature Protocols 8, 2180 (2013). doi:10.1038/nprot.2013.132

Authors: Matthew H Larson, Luke A Gilbert, Xiaowo Wang, Wendell A Lim, Jonathan S Weissman & Lei S Qi

Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory systems. We have recently described an RNA-based method, CRISPR interference (CRISPRi), for targeted silencing of transcription in bacteria and human cells. The CRISPRi

Nature Protocols 8, 2212 (2013). doi:10.1038/nprot.2013.133

Authors: Annemarie H Becker, Eugene Oh, Jonathan S Weissman, Günter Kramer & Bernd Bukau

A plethora of factors is involved in the maturation of newly synthesized proteins, including chaperones, membrane targeting factors and enzymes. Many factors act co-translationally through association with ribosome-nascent chain complexes (RNCs), but their target specificities and modes of action remain poorly understood. We developed selective

Backers say that South Korea's fledgling Institute for Basic Science will lead the country to a new economic model built on homegrown innovations. Critics blast the institute as a "black hole" that will suck resources from existing grant programs. Author: Richard Stone

The debate continues concerning translation in the nucleus. In this work from the Brogna lab, the authors use fluorescent complementation between the large and small subunits of the ribosome to show that ribosome joining can occur in the nucleus, and accumulate in the nucleolus in particular, in fly tissues and cultured cells. Puromycin incorporation occurred at these sites indicating the possibility that the association is functional.

Ribonuclease III mechanisms of double-stranded RNA cleavage.

Wiley Interdiscip Rev RNA. 2013 Sep 30;

Authors: Nicholson AW

Abstract
Double-stranded(ds) RNA has diverse roles in gene expression and regulation, host defense, and genome surveillance in bacterial and eukaryotic cells. A central aspect of dsRNA function is its selective recognition and cleavage by members of the ribonuclease III (RNase III) family of divalent-metal-ion-dependent phosphodiesterases. The processing of dsRNA by RNase III family members is an essential step in the maturation and decay of coding and noncoding RNAs, including miRNAs and siRNAs. RNase III, as first purified from Escherichia coli, has served as a biochemically well-characterized prototype, and other bacterial orthologs provided the first structural information. RNase III family members share a unique fold (RNase III domain) that can dimerize to form a structure that binds dsRNA and cleaves phosphodiesters on each strand, providing the characteristic 2 nt, 3'-overhang product ends. Ongoing studies are uncovering the functions of additional domains, including, inter alia, the dsRNA-binding and PAZ domains that cooperate with the RNase III domain to select target sites, regulate activity, confer processivity, and support the recognition of structurally diverse substrates. RNase III enzymes function in multicomponent assemblies that are regulated by diverse inputs, and at least one RNase III-related polypeptide can function as a noncatalytic, dsRNA-binding protein. This review summarizes the current knowledge of the mechanisms of catalysis and target site selection of RNase III family members, and also addresses less well understood aspects of these enzymes and their interactions with dsRNA. For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article.

PMID: 24124076 [PubMed - as supplied by publisher]

RNA molecules have long been known to adopt unusual conformations. The first crystal structure of an RNA was that of yeast tRNAphe (1, 2), which provided a portent of what was to come. With more structures available now from X-ray crystallography and NMR, that promise has been met beyond expectations....

Ribonucleotides are frequently incorporated into DNA during replication, they are normally removed, and failure to remove them results in replication stress. This stress correlates with DNA polymerase (Pol) stalling during bypass of ribonucleotides in DNA templates. Here we demonstrate that stalling by yeast replicative Pols δ and ε increases as...

The EMBO Journal. doi:10.1038/emboj.2013.227

Authors: Melissa A Mefford, Qundeel Rafiq & David C Zappulla

Michael Y. Sherman, Shu-Bing Qian.

Yun Zhang, Pengyuan Yang, Xiao-Fan Wang.

Nature advance online publication 13 October 2013. doi:10.1038/nature12581

Authors: Audrone Lapinaite, Bernd Simon, Lars Skjaerven, Magdalena Rakwalska-Bange, Frank Gabel & Teresa Carlomagno

Liuqing Yang, Chunru Lin, Wen Liu, Jie Zhang, Kenneth A. Ohgi, Jonathan D. Grinstein, Pieter C. Dorrestein, Michael G. Rosenfeld.

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A CRISPR CASe for high-throughput silencing.

Front Genet. 2013;4:193

Authors: Heintze J, Luft C, Ketteler R

Abstract
Manipulation of gene expression on a genome-wide level is one of the most important systematic tools in the post-genome era. Such manipulations have largely been enabled by expression cloning approaches using sequence-verified cDNA libraries, large-scale RNA interference libraries (shRNA or siRNA) and zinc finger nuclease technologies. More recently, the CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas)9-mediated gene editing technology has been described that holds great promise for future use of this technology in genomic manipulation. It was suggested that the CRISPR system has the potential to be used in high-throughput, large-scale loss of function screening. Here we discuss some of the challenges in engineering of CRISPR/Cas genomic libraries and some of the aspects that need to be addressed in order to use this technology on a high-throughput scale.

PMID: 24109485 [PubMed - as supplied by publisher]

Mai Sun, Björn Schwalb, Nicole Pirkl, Kerstin C. Maier, Arne Schenk, Henrik Failmezger, Achim Tresch, Patrick Cramer. The rates of mRNA synthesis and degradation determine cellular mRNA levels and can be monitored by comparative dynamic transcriptome analysis (cDTA) that uses nonperturbing metabolic RNA labeling.....

Carol J. Wilusz, Jeffrey Wilusz.

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Michael Spilman, Alexis Cocozaki, Caryn Hale, Yaming Shao, Nancy Ramia, Rebeca Terns, Michael Terns, Hong Li, Scott Stagg. Bacterial and archaeal clustered regularly interspaced short palindromic repeat (CRISPR) loci capture virus and plasmid sequences and use them to recognize and eliminate these invaders. CRISPR RNA....

Bijoyita Roy, Allan Jacobson.

Certain mammalian cells can use RNA interference in the innate defense against invading viruses. [Also see Perspective by Sagan and Sarnow] Authors: Yang Li, Jinfeng Lu, Yanhong Han, Xiaoxu Fan, Shou-Wei Ding

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control mechanism that detects aberrant mRNAs containing nonsense codons and induces their rapid degradation. This degradation is mediated by SMG6, an NMD-specific endonuclease, as well as the SMG5 and SMG7 proteins, which recruit general mRNA decay enzymes. However, it remains unknown which specific decay factors are recruited and whether this recruitment is direct. Here, we show that SMG7 binds directly to POP2, a catalytic subunit of the CCR4–NOT deadenylase complex, and elicits deadenylation-dependent decapping and 5'-to-3' decay of NMD targets. Accordingly, a catalytically inactive POP2 mutant partially suppresses NMD in human cells. The SMG7–POP2 interaction is critical for NMD in cells depleted of SMG6, indicating that SMG7 and SMG6 act redundantly to promote the degradation of NMD targets. We further show that UPF1 provides multiple binding sites for decapping factors. These data unveil a missing direct physical link between NMD and the general mRNA decay machinery and indicate that NMD employs diverse and partially redundant mechanisms to ensure robust degradation of aberrant mRNAs.