一个新的家庭的整合酶与原噬菌体基因组岛内部集成的tRNA核苷合成酶基因（DUSA）

Genomic islands play a key role in prokaryotic genome plasticity. Genomic islands integrate into chromosomal loci such as transfer RNA genes and protein coding genes, whilst retaining various cargo genes that potentially bestow novel functions on the host organism. A gene encoding a putative integrase was identified at a single site within the 5' end of the dusA gene in the genomes of over 200 bacteria. This integrase was discovered to be a component of numerous genomic islands, which appear to share a target site within the dusA gene. dusA encodes the tRNA-dihydrouridine synthase A enzyme, which catalyses the post-transcriptional reduction of uridine to dihydrouridine in tRNA. Genomic islands encoding homologous dusA-associated integrases were found at a much lower frequency within the related dusB and dusC genes, and non-dus genes. Excision of these dusA-associated islands from the chromosome as circularized intermediates was confirmed by polymerase chain reaction. Analysis of the dusA-associated islands indicated that they were highly diverse, with the integrase gene representing the only universal common feature.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4547?rss=1

[详细]

在酵母内转录景观演化

Variations in gene expression have been widely explored in order to obtain an accurate overview of the changes in regulatory networks that underlie phenotypic diversity. Numerous studies have characterized differences in genomic expression between large numbers of individuals of model organisms such as Saccharomyces cerevisiae. To more broadly survey the evolution of the transcriptomic landscape across species, we measured whole-genome expression in a large collection of another yeast species: Lachancea kluyveri (formerly Saccharomyces kluyveri), using RNAseq. Interestingly, this species diverged from the S. cerevisiae lineage prior to its ancestral whole genome duplication. Moreover, L. kluyveri harbors a chromosome-scale compositional heterogeneity due to a 1-Mb ancestral introgressed region as well as a large set of unique unannotated genes. In this context, our comparative transcriptomic analysis clearly showed a link between gene evolutionary history and expression behavior. Indeed, genes that have been recently acquired or under function relaxation tend to be less transcribed show a higher intraspecific variation (plasticity) and are less involved in network (connectivity). Moreover, utilizing this approach in L. kluyveri also highlighted specific regulatory network signatures in aerobic respiration, amino-acid biosynthesis and glycosylation, presumably due to its different lifestyle. Our data set sheds an important light on the evolution of intraspecific transcriptomic variation across distant species.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4558?rss=1

[详细]

2’-氟改性寡核苷酸可引起与PSF p54nrb蛋白的快速降解

Synthetic oligonucleotides are used to regulate gene expression through different mechanisms. Chemical modifications of the backbone of the nucleic acid and/or of the 2' moiety of the ribose can increase nuclease stability and/or binding affinity of oligonucleotides to target molecules. Here we report that transfection of 2'-F-modified phosphorothioate oligonucleotides into cells can reduce the levels of P54nrb and PSF proteins through proteasome-mediated degradation. Such deleterious effects of 2'-F-modified oligonucleotides were observed in different cell types from different species, and were independent of oligonucleotide sequence, positions of the 2'-F-modified nucleotides in the oligonucleotides, method of delivery or mechanism of action of the oligonucleotides. Four 2'-F-modified nucleotides were sufficient to cause the protein reduction. P54nrb and PSF belong to Drosophila behavior/human splicing (DBHS) family. The third member of the family, PSPC1, was also reduced by the 2'-F-modified oligonucleotides. Preferential association of 2'-F-modified oligonucleotides with P54nrb was observed, which is partially responsible for the protein reduction. Consistent with the role of DBHS proteins in double-strand DNA break (DSB) repair, elevated DSBs were observed in cells treated with 2'-F-modified oligonucleotides, which contributed to severe impairment in cell proliferation. These results suggest that oligonucleotides with 2'-F modifications can cause non-specific loss of cellular protein(s).

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4569?rss=1

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对tRNA模仿真核细胞释放因子eRF1 l123遗传分析，氨基酸残基为终止密码子的识别关键

In eukaryotes, the tRNA-mimicking polypeptide-chain release factor, eRF1, decodes stop codons on the ribosome in a complex with eRF3; this complex exhibits striking structural similarity to the tRNA–eEF1A–GTP complex. Although amino acid residues or motifs of eRF1 that are critical for stop codon discrimination have been identified, the details of the molecular mechanisms involved in the function of the ribosomal decoding site remain obscure. Here, we report analyses of the position-123 amino acid of eRF1 (L123 in Saccharomyces cerevisiae eRF1), a residue that is phylogenetically conserved among species with canonical and variant genetic codes. In vivo readthrough efficiency analysis and genetic growth complementation analysis of the residue-123 systematic mutants suggested that this amino acid functions in stop codon discrimination in a manner coupled with eRF3 binding, and distinctive from previously reported adjacent residues. Furthermore, aminoglycoside antibiotic sensitivity analysis and ribosomal docking modeling of eRF1 in a quasi-A/T state suggested a functional interaction between the side chain of L123 and ribosomal residues critical for codon recognition in the decoding site, as a molecular explanation for coupling with eRF3. Our results provide insights into the molecular mechanisms underlying stop codon discrimination by a tRNA-mimicking protein on the ribosome.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4591?rss=1

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影响因素的cmob羧甲基转移酶用于选择性摆动修改

Enzyme-mediated modifications at the wobble position of tRNAs are essential for the translation of the genetic code. We report the genetic, biochemical and structural characterization of CmoB, the enzyme that recognizes the unique metabolite carboxy-S-adenosine-L-methionine (Cx-SAM) and catalyzes a carboxymethyl transfer reaction resulting in formation of 5-oxyacetyluridine at the wobble position of tRNAs. CmoB is distinctive in that it is the only known member of the SAM-dependent methyltransferase (SDMT) superfamily that utilizes a naturally occurring SAM analog as the alkyl donor to fulfill a biologically meaningful function. Biochemical and genetic studies define the in vitro and in vivo selectivity for Cx-SAM as alkyl donor over the vastly more abundant SAM. Complementary high-resolution structures of the apo- and Cx-SAM bound CmoB reveal the determinants responsible for this remarkable discrimination. Together, these studies provide mechanistic insight into the enzymatic and non-enzymatic feature of this alkyl transfer reaction which affords the broadened specificity required for tRNAs to recognize multiple synonymous codons.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4602?rss=1

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BLM展开四链体不同结构环境中通过不同的机制

Mutations in the RecQ DNA helicase gene BLM give rise to Bloom's syndrome, which is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition. BLM helicase is highly active in binding and unwinding G-quadruplexes (G4s), which are physiological targets for BLM, as revealed by genome-wide characterizations of gene expression of cells from BS patients. With smFRET assays, we studied the molecular mechanism of BLM-catalyzed G4 unfolding and showed that ATP is required for G4 unfolding. Surprisingly, depending on the molecular environments of G4, BLM unfolds G4 through different mechanisms: unfolding G4 harboring a 3'-ssDNA tail in three discrete steps with unidirectional translocation, and unfolding G4 connected to dsDNA by ssDNA in a repetitive manner in which BLM remains anchored at the ss/dsDNA junction, and G4 was unfolded by reeling in ssDNA. This indicates that one BLM molecule may unfold G4s in different molecular environments through different mechanisms.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4614?rss=1

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在大的核糖体RNA的运动的主要中心

Major centers of motion in the rRNAs of Thermus thermophilus are identified by alignment of crystal structures of EF-G bound and EF-G unbound ribosomal subunits. Small rigid helices upstream of these ‘pivots’ are aligned, thereby decoupling their motion from global rearrangements. Of the 21 pivots found, six are observed in the large subunit rRNA and 15 in the small subunit rRNA. Although the magnitudes of motion differ, with only minor exceptions equivalent pivots are seen in comparisons of Escherichia coli structures and one Saccharomyces cerevisiae structure pair. The pivoting positions are typically associated with structurally weak motifs such as non-canonical, primarily U-G pairs, bulge loops and three-way junctions. Each pivot is typically in direct physical contact with at least one other in the set and often several others. Moving helixes include rRNA segments in contact with the tRNA, intersubunit bridges and helices 28, 32 and 34 of the small subunit. These helices are envisioned to form a network. EF-G rearrangement would then provide directional control of this network propagating motion from the tRNA to the intersubunit bridges to the head swivel or along the same path backward.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4640?rss=1

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双链siRNA的细胞内活细胞成像

Intracellular distribution of siRNA after in vitro transfection typically depends on lipopolyplexes, which must release the siRNA into the cytosol. Here, the fate of siRNAs was monitored by FRET-based live cell imaging. Subsequent to in situ observation of uptake and release processes, this approach allowed the observation of a number of hitherto uncharacterized intracellular distribution and degradation processes, commencing with a burst of endosomal releases, followed, in some cases, by fast siRNA influx into the nucleus. The continued observation of intact siRNA against a background of free fluorophores resulting from advanced degradation was possible by a specifically developed imaging algorithm, which identified populations of intact siRNA in pixels based on FRET. This proved to be essential in the end point definition of siRNA distribution, which typically featured partially degraded siRNA pools in perinuclear structures. Our results depict the initial 4 h as a critical time window, characterized by fast initial burst release into the cytosol, which lay the foundations for subsequent intracellular distribution of siRNA. Combination with a subsequent slower, but sustained release from endosomal reservoirs may contribute to the efficiency and duration of RNAi, and explain the success of lipopolyplexes in RNAi experiments in cell culture.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4650?rss=1

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丝氨酸富含精氨酸（SR）剪接因子调节的可变剪接在弓形虫基因一千

Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii, and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine–rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii. Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4661?rss=1

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HIV-1 Rev应答元件（RRE）采用另一种构象，促进病毒复制率不同

The HIV Rev protein forms a complex with a 351 nucleotide sequence present in unspliced and incompletely spliced human immunodeficiency virus (HIV) mRNAs, the Rev response element (RRE), to recruit the cellular nuclear export receptor Crm1 and Ran-GTP. This complex facilitates nucleo-cytoplasmic export of these mRNAs. The precise secondary structure of the HIV-1 RRE has been controversial, since studies have reported alternative structures comprising either four or five stem-loops. The published structures differ only in regions that lie outside of the primary Rev binding site. Using in-gel SHAPE, we have now determined that the wt NL4-3 RRE exists as a mixture of both structures. To assess functional differences between these RRE ‘conformers’, we created conformationally locked mutants by site-directed mutagenesis. Using subgenomic reporters, as well as HIV replication assays, we demonstrate that the five stem-loop form of the RRE promotes greater functional Rev/RRE activity compared to the four stem-loop counterpart.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4676?rss=1

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cwc22依赖剪接和eif4a3结合使外显子连接配合全球沉积

In metazoan cells, spliced mRNAs are marked by the exon junction complex (EJC), a multi-protein complex that serves as a key regulator of post-transcriptional mRNA metabolism. Deposition of EJCs on mRNA is intimately linked to the splicing process. The spliceosomal protein CWC22 directly binds the core EJC-protein eIF4A3, guides it to the spliceosome and initiates EJC assembly. In addition, CWC22 is involved in the splicing process itself, but the molecular details of its dual function remain elusive. Here we analyze the mechanisms, by which CWC22 co-regulates pre-mRNA splicing and EJC assembly. We show that the core of CWC22 is sufficient to mediate both pre-mRNA splicing and EJC assembly. Nonetheless, both processes can be functionally uncoupled with an eIF4A3-binding deficient mutant of CWC22, which impedes EJC assembly. A C-terminal domain of CWC22 strongly enhances its spliceosomal interaction and likely regulates its function. High-throughput RNA-sequencing identifies global defects of pre-mRNA splicing and downregulation of diverse gene expression pathways in CWC22-depleted cells. We propose a model, in which CWC22 represents an integral component of the spliceosome and orchestrates pre-mRNA splicing and eIF4A3 binding to achieve global assembly of exon junction complexes.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4687?rss=1

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蛋白质的降解和动态化微调tRNA翻译在升高的温度下

Maintenance of protein quality control has implications in various processes such as neurodegeneration and ageing. To investigate how environmental insults affect this process, we analysed the proteome of yeast continuously exposed to mild heat stress. In agreement with previous transcriptomics studies, amongst the most marked changes, we found up-regulation of cytoprotective factors; a shift from oxidative phosphorylation to fermentation; and down-regulation of translation. Importantly, we also identified a novel, post-translationally controlled, component of the heat shock response. The abundance of Ncs2p and Ncs6p, two members of the URM1 pathway responsible for the thiolation of wobble uridines in cytoplasmic tRNAs tK^UUU, tQ^UUG and tE^UUC, is down-regulated in a proteasomal dependent fashion. Using random forests we show that this results in differential translation of transcripts with a biased content for the corresponding codons. We propose that the role of this pathway in promoting catabolic and inhibiting anabolic processes, affords cells with additional time and resources needed to attain proper protein folding under periods of stress.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4701?rss=1

[详细]

RNA的基础病变对mRNA翻译的影响

The biological effect of oxidatively damaged RNA, unlike oxidatively damaged DNA, has rarely been investigated, although it poses a threat to any living cell. Here we report on the effect of the commonly known RNA base-lesions 8-oxo-rG, 8-oxo-rA, -rC, -rA, 5-HO-rC, 5-HO-rU and the RNA abasic site (rAS) on ribosomal translation. To this end we have developed an in vitro translation assay based on the mRNA display methodology. A short synthetic mRNA construct containing the base lesion in a predefined position of the open reading frame was ³²P-labeled at the 5'-end and equipped with a puromycin unit at the 3'-end. Upon in vitro translation in rabbit reticulocyte lysates, the encoded peptide chain is transferred to the puromycin unit and the products analyzed by gel electrophoresis. Alternatively, the unlabeled mRNA construct was used and incubated with ³⁵S-methionine to prove peptide elongation of the message. We find that all base-lesions interfere substantially with ribosomal translation. We identified two classes, the first containing modifications at the base coding edge (-rC, -rA and rAS) which completely abolish peptide synthesis at the site of modification, and the second consisting of 8-oxo-rG, 8-oxo-rA, 5-HO-rC and 5-HO-rU that significantly retard full-length peptide synthesis, leading to some abortive peptides at the site of modification.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4713?rss=1

[详细]

许多人类基因的内含子剪接涉及嵌入式网站

The conventional model for splicing involves excision of each intron in one piece; we demonstrate this inaccurately describes splicing in many human genes. First, after switching on transcription of SAMD4A, a gene with a 134 kb-long first intron, splicing joins the 3' end of exon 1 to successive points within intron 1 well before the acceptor site at exon 2 is made. Second, genome-wide analysis shows that >60% of active genes yield products generated by such intermediate intron splicing. These products are present at ~15% the levels of primary transcripts, are encoded by conserved sequences similar to those found at canonical acceptors, and marked by distinctive structural and epigenetic features. Finally, using targeted genome editing, we demonstrate that inhibiting the formation of these splicing intermediates affects efficient exon–exon splicing. These findings greatly expand the functional and regulatory complexity of the human transcriptome.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4721?rss=1

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独特的秀丽隐杆线虫端粒悬垂结构揭示了端粒DNA的进化上保守的特性

There are two basic mechanisms that are associated with the maintenance of the telomere length, which endows cancer cells with unlimited proliferative potential. One mechanism, referred to as alternative lengthening of telomeres (ALT), accounts for approximately 10–15% of all human cancers. Tumours engaged in the ALT pathway are characterised by the presence of the single stranded 5'-C-rich telomeric overhang (C-overhang). This recently identified hallmark of ALT cancers distinguishes them from healthy tissues and renders the C-overhang as a clear target for anticancer therapy. We analysed structures of the 5'-C-rich and 3'-G-rich telomeric overhangs from human and Caenorhabditis elegans, the recently established multicellular in vivo model of ALT tumours. We show that the telomeric DNA from C. elegans and humans forms fundamentally different secondary structures. The unique structural characteristics of C. elegans telomeric DNA that are distinct not only from those of humans but also from those of other multicellular eukaryotes allowed us to identify evolutionarily conserved properties of telomeric DNA. Differences in structural organisation of the telomeric DNA between the C. elegans and human impose limitations on the use of the C. elegans as an ALT tumour model.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4733?rss=1

[详细]

在核糖体合成的rpf2-rrs1复杂的结构和功能分析

Proteins Rpf2 and Rrs1 are required for 60S ribosomal subunit maturation. These proteins are necessary for the recruitment of three ribosomal components (5S ribosomal RNA [rRNA], RpL5 and RpL11) to the 90S ribosome precursor and subsequent 27SB pre-rRNA processing. Here we present the crystal structure of the Aspergillus nidulans (An) Rpf2-Rrs1 core complex. The core complex contains the tightly interlocked N-terminal domains of Rpf2 and Rrs1. The Rpf2 N-terminal domain includes a Brix domain characterized by similar N- and C-terminal architecture. The long α-helix of Rrs1 joins the C-terminal half of the Brix domain as if it were part of a single molecule. The conserved proline-rich linker connecting the N- and C-terminal domains of Rrs1 wrap around the side of Rpf2 and anchor the C-terminal domain of Rrs1 to a specific site on Rpf2. In addition, gel shift analysis revealed that the Rpf2-Rrs1 complex binds directly to 5S rRNA. Further analysis of Rpf2-Rrs1 mutants demonstrated that Saccharomyces cerevisiae Rpf2 R236 (corresponds to R238 of AnRpf2) plays a significant role in this binding. Based on these studies and previous reports, we have proposed a model for ribosomal component recruitment to the 90S ribosome precursor.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4746?rss=1

[详细]

一个可变的DNA识别位点组织建立骗子介导的细胞信封应激反应的肠球菌对达托霉素

LiaR is a ‘master regulator’ of the cell envelope stress response in enterococci and many other Gram-positive organisms. Mutations to liaR can lead to antibiotic resistance to a variety of antibiotics including the cyclic lipopeptide daptomycin. LiaR is phosphorylated in response to membrane stress to regulate downstream target operons. Using DNA footprinting of the regions upstream of the liaXYZ and liaFSR operons we show that LiaR binds an extended stretch of DNA that extends beyond the proposed canonical consensus sequence suggesting a more complex level of regulatory control of target operons. We go on to determine the biochemical and structural basis for increased resistance to daptomycin by the adaptive mutation to LiaR (D191N) first identified from the pathogen Enterococcus faecalis S613. LiaR^D191N increases oligomerization of LiaR to form a constitutively activated tetramer that has high affinity for DNA even in the absence of phosphorylation leading to increased resistance. Crystal structures of the LiaR DNA binding domain complexed to the putative consensus sequence as well as an adjoining secondary sequence show that upon binding, LiaR induces DNA bending that is consistent with increased recruitment of RNA polymerase to the transcription start site and upregulation of target operons.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4758?rss=1

[详细]

大分子拥挤和条件在体外核糖体施工减少影响

In vitro construction of Escherichia coli ribosomes could elucidate a deeper understanding of these complex molecular machines and make possible the production of synthetic variants with new functions. Toward this goal, we recently developed an integrated synthesis, assembly and translation (iSAT) system that allows for co-activation of ribosomal RNA (rRNA) transcription and ribosome assembly, mRNA transcription and protein translation without intact cells. Here, we discovered that macromolecular crowding and reducing agents increase overall iSAT protein synthesis; the combination of 6% w/v Ficoll 400 and 2 mM DTBA yielded approximately a five-fold increase in overall iSAT protein synthesis activity. By utilizing a fluorescent RNA aptamer, fluorescent reporter proteins and ribosome sedimentation analysis, we showed that crowding agents increase iSAT yields by enhancing translation while reducing agents increase rRNA transcription and ribosome assembly. Finally, we showed that iSAT ribosomes possess ~70% of the protein synthesis activity of in vivo-assembled E. coli ribosomes. This work improves iSAT protein synthesis through the addition of crowding and reducing agents, provides a thorough understanding of the effect of these additives within the iSAT system and demonstrates how iSAT allows for manipulation and analysis of ribosome biogenesis in the context of an in vitro transcription-translation system.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4774?rss=1

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Dicer的诸多方面：调节机制的Dicer酶的基因表达和活动的复杂性

There is increasing evidence indicating that the production of small regulatory RNAs is not the only process in which ribonuclease Dicer can participate. For example, it has been demonstrated that this enzyme is also involved in chromatin structure remodelling, inflammation and apoptotic DNA degradation. Moreover, it has become increasingly clear that cellular transcript and protein levels of Dicer must be strictly controlled because even small changes in their accumulation can initiate various pathological processes, including carcinogenesis. Accordingly, in recent years, a number of studies have been performed to identify the factors regulating Dicer gene expression and protein activity. As a result, a large amount of complex and often contradictory data has been generated. None of these data have been subjected to an exhaustive review or critical discussion. This review attempts to fill this gap by summarizing the current knowledge of factors that regulate Dicer gene transcription, primary transcript processing, mRNA translation and enzyme activity. Because of the high complexity of this topic, this review mainly concentrates on human Dicer. This review also focuses on an additional regulatory layer of Dicer activity involving the interactions of protein and RNA factors with Dicer substrates.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4365?rss=1

[详细]

RK的主题和连接段使然Oct4 DNA识别功能之间的相互作用

The POU family transcription factor Oct4 plays pivotal roles in regulating pluripotency and somatic cell reprogramming. Previous studies have indicated an important role for major groove contacts in Oct4–DNA recognition; however, the contributions of the RK motif in the POUh domain and the linker segment joining the two DNA-binding domains remain poorly understood. Here, by combining molecular modelling and functional assays, we find that the RK motif is essential for Oct4–DNA association by recognizing the narrowed DNA minor groove. Intriguingly, computational simulations reveal that the function of the RK motif may be finely tuned by H-bond interactions with the partially disordered linker segment and that breaking these interactions significantly enhances the DNA binding and reprogramming activities of Oct4. These findings uncover a self-regulatory mechanism for specific Oct4–DNA recognition and provide insights into the functional crosstalk at the molecular level that may illuminate mechanistic studies of the Oct protein family and possibly transcription factors in the POU family. Our gain-of-function Oct4 mutants might also be useful tools for use in reprogramming and regenerative medicine.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4381?rss=1

[详细]

揭示了转录子网布线CHINET在先进生物燃料转化酵母耐

Analysis of rewired upstream subnetworks impacting downstream differential gene expression aids the delineation of evolving molecular mechanisms. Cumulative statistics based on conventional differential correlation are limited for subnetwork rewiring analysis since rewiring is not necessarily equivalent to change in correlation coefficients. Here we present a computational method ChiNet to quantify subnetwork rewiring by statistical heterogeneity that enables detection of potential genotype changes causing altered transcription regulation in evolving organisms. Given a differentially expressed downstream gene set, ChiNet backtracks a rewired upstream subnetwork from a super-network including gene interactions known to occur under various molecular contexts. We benchmarked ChiNet for its high accuracy in distinguishing rewired artificial subnetworks, in silico yeast transcription-metabolic subnetworks, and rewired transcription subnetworks for Candida albicans versus Saccharomyces cerevisiae, against two differential-correlation based subnetwork rewiring approaches. Then, using transcriptome data from tolerant S. cerevisiae strain NRRL Y-50049 and a wild-type intolerant strain, ChiNet identified 44 metabolic pathways affected by rewired transcription subnetworks anchored to major adaptively activated transcription factor genes YAP1, RPN4, SFP1 and ROX1, in response to toxic chemical challenges involved in lignocellulose-to-biofuels conversion. These findings support the use of ChiNet in rewiring analysis of subnetworks where differential interaction patterns resulting from divergent nonlinear dynamics abound.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4393?rss=1

[详细]

评估肌分化核糖体分析翻译景观

The formation of skeletal muscles is associated with drastic changes in protein requirements known to be safeguarded by tight control of gene transcription and mRNA processing. The contribution of regulation of mRNA translation during myogenesis has not been studied so far. We monitored translation during myogenic differentiation of C2C12 myoblasts, using a simplified protocol for ribosome footprint profiling. Comparison of ribosome footprints to total RNA showed that gene expression is mostly regulated at the transcriptional level. However, a subset of transcripts, enriched for mRNAs encoding for ribosomal proteins, was regulated at the level of translation. Enrichment was also found for specific pathways known to regulate muscle biology. We developed a dedicated pipeline to identify translation initiation sites (TISs) and discovered 5333 unannotated TISs, providing a catalog of upstream and alternative open reading frames used during myogenesis. We identified 298 transcripts with a significant switch in TIS usage during myogenesis, which was not explained by alternative promoter usage, as profiled by DeepCAGE. Also these transcripts were enriched for ribosomal protein genes. This study demonstrates that differential mRNA translation controls protein expression of specific subsets of genes during myogenesis. Experimental protocols, analytical workflows, tools and data are available through public repositories (http://lumc.github.io/ribosome-profiling-analysis-framework/).

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4408?rss=1

[详细]

在基因组规模的米曲霉DNA与蛋白质相互作用的调查

The genome-scale delineation of in vivo protein–DNA interactions is key to understanding genome function. Only ~5% of transcription factors (TFs) in the Aspergillus genus have been identified using traditional methods. Although the Aspergillus oryzae genome contains >600 TFs, knowledge of the in vivo genome-wide TF-binding sites (TFBSs) in aspergilli remains limited because of the lack of high-quality antibodies. We investigated the landscape of in vivo protein–DNA interactions across the A. oryzae genome through coupling the DNase I digestion of intact nuclei with massively parallel sequencing and the analysis of cleavage patterns in protein–DNA interactions at single-nucleotide resolution. The resulting map identified overrepresented de novo TF-binding motifs from genomic footprints, and provided the detailed chromatin remodeling patterns and the distribution of digital footprints near transcription start sites. The TFBSs of 19 known Aspergillus TFs were also identified based on DNase I digestion data surrounding potential binding sites in conjunction with TF binding specificity information. We observed that the cleavage patterns of TFBSs were dependent on the orientation of TF motifs and independent of strand orientation, consistent with the DNA shape features of binding motifs with flanking sequences.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4429?rss=1

[详细]

综合基因组分析揭示了广泛的增强子调控p53的DNA损伤反应

The tumor suppressor p53 has been studied extensively as a direct transcriptional activator of protein-coding genes. Recent studies, however, have shed light on novel regulatory functions of p53 within noncoding regions of the genome. Here, we use a systematic approach that integrates transcriptome-wide expression analysis, genome-wide p53 binding profiles and chromatin state maps to characterize the global regulatory roles of p53 in response to DNA damage. Notably, our approach identified conserved features of the p53 network in both human and mouse primary fibroblast models. In addition to known p53 targets, we identify many previously unappreciated mRNAs and long noncoding RNAs that are regulated by p53. Moreover, we find that p53 binding occurs predominantly within enhancers in both human and mouse model systems. The ability to modulate enhancer activity offers an additional layer of complexity to the p53 network and greatly expands the diversity of genomic elements directly regulated by p53.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4447?rss=1

[详细]