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NAR Top Articles - Surveys and Summaries

Surveys and Summaries

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February 2015


Single-cell RNA-seq: advances and future challenges
Saliba, AE; Westermann, AJ; Gorski, SA; Vogel, J
Nucleic Acids Res. 2014, 42, 8845-8860
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Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Single-cell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNA-seq protocols have been introduced and are reviewed here--each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field.

H1 histones: current perspectives and challenges
Harshman, SW; Young, NL; Parthun, MR; Freitas, MA
Nucleic Acids Res. 2013, 41, 9593-9609
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H1 and related linker histones are important both for maintenance of higher-order chromatin structure and for the regulation of gene expression. The biology of the linker histones is complex, as they are evolutionarily variable, exist in multiple isoforms and undergo a large variety of posttranslational modifications in their long, unstructured, NH2- and COOH-terminal tails. We review recent progress in understanding the structure, genetics and posttranslational modifications of linker histones, with an emphasis on the dynamic interactions of these proteins with DNA and transcriptional regulators. We also discuss various experimental challenges to the study of H1 and related proteins, including limitations of immunological reagents and practical difficulties in the analysis of posttranslational modifications by mass spectrometry.

Highlights of the DNA cutters: a short history of the restriction enzymes
Loenen, WAM; Dryden, DTF; Raleigh, EA; Wilson, GG; Murray, NE
Nucleic Acids Res. 2014, 42, 3-19
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In the early 1950's, 'host-controlled variation in bacterial viruses' was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e. g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine.

Classification and evolution of type II CRISPR-Cas systems
Chylinski, K; Makarova, KS; Charpentier, E; Koonin, EV
Nucleic Acids Res. 2014, 42, 6091-6105
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The CRISPR-Cas systems of archaeal and bacterial adaptive immunity are classified into three types that differ by the repertoires of CRISPR-associated (cas) genes, the organization of cas operons and the structure of repeats in the CRISPR arrays. The simplest among the CRISPR-Cas systems is type II in which the endonuclease activities required for the interference with foreign deoxyribonucleic acid (DNA) are concentrated in a single multidomain protein, Cas9, and are guided by a co-processed dual-tracrRNA: crRNA molecule. This compact enzymatic machinery and readily programmable site-specific DNA targeting make type II systems top candidates for a new generation of powerful tools for genomic engineering. Here we report an updated census of CRISPR-Cas systems in bacterial and archaeal genomes. Type II systems are the rarest, missing in archaea, and represented in similar to 5% of bacterial genomes, with an over-representation among pathogens and commensals. Phylogenomic analysis suggests that at least three cas genes, cas1, cas2 and cas4, and the CRISPR repeats of the type II-B system were acquired via recombination with a type I CRISPR-Cas locus...

Regulation of alternative splicing by local histone modifications: potential roles for RNA-guided mechanisms
Zhou, HL; Luo, GB; Wise, JA; Lou, H
Nucleic Acids Res. 2014, 42, 701-713
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The molecular mechanisms through which alternative splicing and histone modifications regulate gene expression are now understood in considerable detail. Here, we discuss recent studies that connect these two previously separate avenues of investigation, beginning with the unexpected discoveries that nucleosomes are preferentially positioned over exons and DNA methylation and certain histone modifications also show exonic enrichment. These findings have profound implications linking chromatin structure, histone modification and splicing regulation. Complementary single gene studies provided insight into the mechanisms through which DNA methylation and histones modifications modulate alternative splicing patterns. Here, we review an emerging theme resulting from these studies: RNA-guided mechanisms integrating chromatin modification and splicing. Several groundbreaking papers reported that small noncoding RNAs affect alternative exon usage by targeting histone methyltransferase complexes to form localized facultative heterochromatin. More recent studies provided evidence that pre-messenger RNA itself can serve as a guide to enable precise alternative splicing regulation...

Histone H4 Lysine 20 methylation: key player in epigenetic regulation of genomic integrity
Jorgensen, S; Schotta, G; Sorensen, CS
Nucleic Acids Res. 2013, 41, 2797-2806
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Maintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer. Nuclear DNA is packaged into chromatin, and thus genome maintenance can be influenced by distinct chromatin environments. In particular, post-translational modifications of histones have emerged as key regulators of genomic integrity. Intense research during the past few years has revealed histone H4 lysine 20 methylation (H4K20me) as critically important for the biological processes that ensure genome integrity, such as DNA damage repair, DNA replication and chromatin compaction. The distinct H4K20 methylation states are mediated by SET8/PR-Set7 that catalyses monomethylation of H4K20, whereas SUV4-20H1 and SUV4-20H2 enzymes mediate further H4K20 methylation to H4K20me2 and H4K20me3. Disruption of these H4K20-specific histone methyltransferases leads to genomic instability, demonstrating the important functions of H4K20 methylation in genome maintenance. In this review, we explain molecular mechanisms underlying these defects and discuss novel ideas for furthering our understanding of genome maintenance in higher eukaryotes.

Type II restriction endonucleases--a historical perspective and more
Pingoud, A; Wilson, GG; Wende, W
Nucleic Acids Res. 2014, 42, 7489-7527
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This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss 'Type II' REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since impacted every corner of the life sciences. They became the enabling tools of molecular biology, genetics and biotechnology, and made analysis at the most fundamental levels routine. Hundreds of different REases have been discovered and are available commercially. Their genes have been cloned, sequenced and overexpressed. Most have been characterized to some extent, but few have been studied in depth. Here, we describe the original discoveries in this field, and the properties of the first Type II REases investigated. We discuss the mechanisms of sequence recognition and catalysis, and the varied oligomeric modes in which Type II REases act.

Biases in small RNA deep sequencing data
Raabe, CA; Tang, TH; Brosius, J; Rozhdestvensky, TS
Nucleic Acids Res. 2014, 42, 1414-1426
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High-throughput RNA sequencing (RNA-seq) is considered a powerful tool for novel gene discovery and fine-tuned transcriptional profiling. The digital nature of RNA-seq is also believed to simplify meta-analysis and to reduce background noise associated with hybridization-based approaches. The development of multiplex sequencing enables efficient and economic parallel analysis of gene expression. In addition, RNA-seq is of particular value when low RNA expression or modest changes between samples are monitored. However, recent data uncovered severe bias in the sequencing of small non-protein coding RNA (small RNA-seq or sRNA-seq), such that the expression levels of some RNAs appeared to be artificially enhanced and others diminished or even undetectable. The use of different adapters and barcodes during ligation as well as complex RNA structures and modifications drastically influence cDNA synthesis efficacies and exemplify sources of bias in deep sequencing. In addition, variable specific RNA G/C-content is associated with unequal polymerase chain reaction amplification efficiencies...

Protein-DNA binding: complexities and multi-protein codes
Siggers, T; Gordan, R
Nucleic Acids Res. 2014, 42, 2099-2111
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Binding of proteins to particular DNA sites across the genome is a primary determinant of specificity in genome maintenance and gene regulation. DNA-binding specificity is encoded at multiple levels, from the detailed biophysical interactions between proteins and DNA, to the assembly of multi-protein complexes. At each level, variation in the mechanisms used to achieve specificity has led to difficulties in constructing and applying simple models of DNA binding. We review the complexities in protein-DNA binding found at multiple levels and discuss how they confound the idea of simple recognition codes. We discuss the impact of new high-throughput technologies for the characterization of protein-DNA binding, and how these technologies are uncovering new complexities in protein-DNA recognition. Finally, we review the concept of multi-protein recognition codes in which new DNA-binding specificities are achieved by the assembly of multi-protein complexes.

Type I restriction enzymes and their relatives
Loenen, WAM; Dryden, DTF; Raleigh, EA; Wilson, GG
Nucleic Acids Res. 2014, 42, 20-44
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Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the enzymes and of their S subunits. Finally, we describe examples of Type II restriction-modification systems that have features in common with Type I enzymes...

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