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Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans . Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state. What non-coding RNA does Mammalian genomes encode many classes of RNA that do not correspond to messenger (protein-coding) RNAs, transfer RNAs or ribosomal RNAs. Whether these non-coding RNAs have a function, and what that might be, remain outstanding questions. Lander and colleagues have performed a systematic loss-of-function analysis of the long intergenic non-coding RNAs (lincRNAs) in mouse embryonic stem cells. Some of these are found to affect known regulators of the pluripotent state, indicating that they are functional. This work sets the stage for experiments to determine the precise mechanistic roles of lincRNAs.

RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA–protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5′ ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.

Paired-end sequencing of human genomic DNA reveals at least 2.8 Mb of new sequence at 720 distinct loci. Complete sequencing of 1.67 Mb at 192 loci reveals extensive copy-number variation and provides a resource for genotyping these 'missing' sequences. The extent of human genomic structural variation suggests that there must be portions of the genome yet to be discovered, annotated and characterized at the sequence level. We present a resource and analysis of 2,363 new insertion sequences corresponding to 720 genomic loci. We found that a substantial fraction of these sequences are either missing, fragmented or misassigned when compared to recent de novo sequence assemblies from short-read next-generation sequence data. We determined that 18–37% of these new insertions are copy-number polymorphic, including loci that show extensive population stratification among Europeans, Asians and Africans. Complete sequencing of 156 of these insertions identified new exons and conserved noncoding sequences not yet represented in the reference genome. We developed a method to accurately genotype these new insertions by mapping next-generation sequencing datasets to the breakpoint, thereby providing a means to characterize copy-number status for regions previously inaccessible to single-nucleotide polymorphism microarrays.

Array-based comparative genomic hybridization (CGH) measures copy-number variations at multiple loci simultaneously, providing an important tool for studying cancer and developmental disorders and for developing diagnostic and therapeutic targets. Arrays for CGH based on PCR products representing assemblies of BAC or cDNA clones typically require maintenance, propagation, replication, and verification of large clone sets. Furthermore, it is difficult to control the specificity of the hybridization to the complex sequences that are present in each feature of such arrays. To develop a more robust and flexible platform, we created probedesign methods and assay protocols that make oligonucleotide microarrays synthesized in situ by inkjet technology compatible with array-based comparative genomic hybridization applications employing samples of total genomic DNA. Hybridization of a series of cell lines with variable numbers of X chromosomes to arrays designed for CGH measurements gave median ratios for X-chromosome probes within 6% of the theoretical values (0.5 for XY/XX, 1.0 for XX/XX, 1.4 for XXX/XX, 2.1 for XXXX/XX, and 2.6 for XXXXX/XX). Furthermore, these arrays detected and mapped regions of single-copy losses, homozygous deletions, and amplicons of various sizes in different model systems, including diploid cells with a chromosomal breakpoint that has been mapped and sequenced to a precise nucleotide and tumor cell lines with highly variable regions of gains and losses. Our results demonstrate that oligonucleotide arrays designed for CGH provide a robust and precise platform for detecting chromosomal alterations throughout a genome with high sensitivity even when using full-complexity genomic samples.

Copy number variants affect both disease and normal phenotypic variation, but those lying within heavily duplicated, highly identical sequence have been difficult to assay. By analyzing short-read mapping depth for 159 human genomes, we demonstrated accurate estimation of absolute copy number for duplications as small as 1.9 kilobase pairs, ranging from 0 to 48 copies. We identified 4.1 million \"singly unique nucleotide\" positions informative in distinguishing specific copies and used them to genotype the copy and content of specific paralogs within highly duplicated gene families. These data identify human-specific expansions in genes associated with brain development, reveal extensive population genetic diversity, and detect signatures consistent with gene conversion in the human species. Our approach makes ~1000 genes accessible to genetic studies of disease association.

Improved efficiency of CRISPR/Cas editing with chemically modified synthetic sgRNAs. CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34 + hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.

Genetic variation among individual humans occurs on many different scales, ranging from gross alterations in the human karyotype to single nucleotide changes. Here we explore variation on an intermediate scale—particularly insertions, deletions and inversions affecting from a few thousand to a few million base pairs. We employed a clone-based method to interrogate this intermediate structural variation in eight individuals of diverse geographic ancestry. Our analysis provides a comprehensive overview of the normal pattern of structural variation present in these genomes, refining the location of 1,695 structural variants. We find that 50% were seen in more than one individual and that nearly half lay outside regions of the genome previously described as structurally variant. We discover 525 new insertion sequences that are not present in the human reference genome and show that many of these are variable in copy number between individuals. Complete sequencing of 261 structural variants reveals considerable locus complexity and provides insights into the different mutational processes that have shaped the human genome. These data provide the first high-resolution sequence map of human structural variation—a standard for genotyping platforms and a prelude to future individual genome sequencing projects. Pieces of eight genomes Clone-based sequencing of the genomes of eight unrelated individuals — four African and four non-African — has been used to build a picture of human genetic variation. The study concentrated on intermediate-scale variations a few thousand to a few million base pairs long. The results confirm the finding that African genomes are more diverse than other groups, and suggest that previous estimates of the incidence of 'copy-number variant' base pairs have been too high. The data suggest that, despite recent evidence to the contrary, non-allelic homologous recombination is the dominant process in promoting structural variation in the genome. Studies of this type provide benchmarks for the many genome sequences that will be generated by next-generation technologies. This paper examines eight individual genomes using a clone-based sequencing approach, for structural variants of 8,000 nucleotides or more. One of the first high-quality inversion maps for the human genome is generated, and it is demonstrated that previous estimates of variation of this sort have been too high.

THE cytoplasmic proteins β-catenin of vertebrates and armadillo of Drosophila have two functions: they link the cadherin cell-adhesion molecules to the cytoskeleton 1–4 , and they participate in the wnt/wingless signalling pathway 5–7 . Here we show, in a yeast two-hybrid screen, that the architectural transcription factor LEF-1 (for lymphoid enhancer-binding factor) 8–10 interacts with β-catenin. In mammalian cells, coexpressed LEF-1 and β-catenin form a complex that is localized to the nucleus and can be detected by immunoprecipitation. Moreover, LEF-1 and β-catenin form a ternary complex with DNA that displays an altered DNA bend. Microinjection of LEF-1 into Xenopus embryos induces axis duplication, which is augmented by interaction with β-catenin. Thus β-catenin regulates gene expression by direct interaction with transcription factors such as LEF-1, providing a molecular mechanism for the transmission of signals from cell-adhesion components or wnt protein to the nucleus.