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Characteristically, cancer cells metabolize glucose through aerobic glycolysis, known as the Warburg effect. Accumulating evidence suggest that during cancer formation, microRNAs (miRNAs) could regulate such metabolic reprogramming. In the present study, miR-9-1 was identified as significantly hypermethylated in nasopharyngeal carcinoma (NPC) cell lines and clinical tissues. Ectopic expression of miR-9-1 inhibited NPC cell growth and glycolytic metabolism, including reduced glycolysis, by reducing lactate production, glucose uptake, cellular glucose-6-phosphate levels, and ATP generation in vitro and tumor proliferation in vivo. HK2 (encoding hexokinase 2) was identified as a direct target of miR-9-1 using luciferase reporter assays and Western blotting. In NPC cells, hypermethylation regulates miR-9-1 expression and inhibits HK2 translation by directly targeting its 3' untranslated region. MiR-9-1 overexpression markedly reduced HK2 protein levels. Restoration of HK2 expression attenuated the inhibitory effect of miR-9-1 on NPC cell proliferation and glycolysis. Fluorescence in situ hybridization results indicated that miR-9-1 expression was an independent prognostic factor in NPC. Our findings revealed the role of the miR-9-1/HK2 axis in the metabolic reprogramming of NPC, providing a potential therapeutic strategy for NPC., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Cyclobutane pyrimidine dimers (CPDs) are the major products of DNA produced by direct absorption of UV light, and result in C to T mutations linked to human skin cancers. Most recently a new pathway to CPDs in melanocytes has been discovered that has been proposed to arise from a chemisensitized pathway involving a triplet sensitizer that increases mutagenesis by increasing the percentage of C-containing CPDs. To investigate how triplet sensitization may differ from direct UV irradiation, CPD formation was quantified in a 129-mer DNA designed to contain all 64 possible NYYN sequences. CPD formation with UVB light varied about 2-fold between dipyrimidines and 12-fold with flanking sequence and was most frequent at YYYR and least frequent for GYYN sites in accord with a charge transfer quenching mechanism. In contrast, photosensitized CPD formation greatly favored TT over C-containing sites, more so for norfloxacin (NFX) than acetone, in accord with their differing triplet energies. While the sequence dependence for photosensitized TT CPD formation was similar to UVB light, there were significant differences, especially between NFX and acetone that could be largely explained by the ability of NFX to intercalate into DNA., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nibbler (Nbr) is a 3'-to-5' exoribonuclease whose catalytic 3'-end trimming activity impacts microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis. Here, we report on structural and functional studies to decipher the contributions of Nbr's N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3'-end trimming. We have solved the crystal structures of the NTD core and EXO domains of Nbr, both in the apo-state. The NTD-core domain of Aedes aegypti Nbr adopts a HEAT-like repeat scaffold with basic patches constituting an RNA-binding surface exhibiting a preference for binding double-strand RNA (dsRNA) over single-strand RNA (ssRNA). Structure-guided functional assays in Drosophila S2 cells confirmed a principal role of the NTD in exonucleolytic miRNA trimming, which depends on basic surface patches. Gain-of-function experiments revealed a potential role of the NTD in recruiting Nbr to Argonaute-bound small RNA substrates. The EXO domain of A. aegypti and Drosophila melanogaster Nbr adopt a mixed α/β-scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. We demonstrate that Nbr's EXO domain exhibits Mn 2+ -dependent ssRNA-specific 3'-to-5' exoribonuclease activity. Modeling of a 3' terminal Uridine into the catalytic pocket of Nbr EXO indicates that 2'- O -methylation of the 3'-U would result in a steric clash with a tryptophan side chain, suggesting that 2'- O -methylation protects small RNAs from Nbr-mediated trimming. Overall, our data establish that Nbr requires its NTD as a substrate recruitment platform to execute exonucleolytic miRNA maturation, catalyzed by the ribonuclease EXO domain., Competing Interests: The authors declare no competing interest.

We improved miR-143, which inhibits the growth of cancer cells, by the replacement of the passenger strand. As a result, new miR-143 variants were developed with a single mismatch at the 4th position from the 3'-terminal of the guide strand and an RNA passenger strand with a G-rich flanking DNA region. A reporter gene assay showed that the 80% inhibitory concentration of the new miR-143, long miR-143, was 69 pM, which was three times lower than that of natural miR-143. Long miR-143 inhibited the growth of two cancer cell lines, HeLa-S3 and MIAPaCa-2, more effectively than natural miR-143. This method could be applied to other miRNA families and should be useful for the development of miRNA drugs., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Thousands of eukaryotic protein-coding genes generate circular RNAs that have covalently linked ends and are resistant to degradation by exonucleases. To prove their circularity as well as biochemically enrich these transcripts, it has become standard in the field to use the 3'-5' exonuclease RNase R. Here, we demonstrate that standard protocols involving RNase R can fail to digest >20% of all highly expressed linear RNAs, but these shortcomings can largely be overcome. RNAs with highly structured 3' ends, including snRNAs and histone mRNAs, are naturally resistant to RNase R, but can be efficiently degraded once a poly(A) tail has been added to their ends. In addition, RNase R stalls in the body of many polyadenylated mRNAs, especially at G-rich sequences that have been previously annotated as G-quadruplex (G4) structures. Upon replacing K+ (which stabilizes G4s) with Li+ in the reaction buffer, we find that RNase R is now able to proceed through these sequences and fully degrade the mRNAs in their entirety. In total, our results provide important improvements to the current methods used to isolate circular RNAs as well as a way to reveal RNA structures that may naturally inhibit degradation by cellular exonucleases., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The t(8;21) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML). We found that t(8;21) AML were extremely sensitive to THZ1, which triggered apoptosis after only 4 h. We used precision nuclear run-on transcription sequencing (PROseq) to define the global effects of THZ1 and other CDK inhibitors on RNA polymerase II dynamics. Inhibition of CDK7 using THZ1 caused wide-spread loss of promoter-proximal paused RNA polymerase. This loss of 5' pausing was associated with accumulation of polymerases in the body of a large number of genes. However, there were modest effects on genes regulated by 'super-enhancers'. At the 3' ends of genes, treatment with THZ1 suppressed RNA polymerase 'read through' at the end of the last exon, which resembled a phenotype associated with a mutant RNA polymerase with slower elongation rates. Consistent with this hypothesis, polyA site-sequencing (PolyA-seq) did not detect differences in poly A sites after THZ1 treatment. PROseq analysis after short treatments with THZ1 suggested that these 3' effects were due to altered CDK7 activity at the 5' end of long genes, and were likely to be due to slower rates of elongation., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: Alu elements are retroposons that invaded the primate genome and shaped its biology. Some Alus inserted recently and are polymorphic in the human population. It is these Alus that are being sought after in disease association studies and regulatory biology. Discovering polymorphic Alus in the human genome can open areas of new research in these fields., Results: Using the polymerase chain reaction on genomic DNA, we identified a polymorphic Alu in the flanking region of the TFAP2B and TFAP2D genes. The new insert was found in higher frequency in Europeans (0.4) and Asians (0.38) and lower frequency in Africans (0.25). We also show this Alu to be part of a 3 Alu cassette that is human specific. The TFAP2B and TFAP2D genes encode members of the transcription factor AP-2, which plays a role in organ development. The insertion of this Alu cassette flanking the transcription factor genes distinguishes humans from the primates. This cassette can possibly affect the regulation of both genes or alternately provoke genomic deletions, which we have shown in this study. Its presence in such a location is intriguing and unquestionably opens an investigational window in disease association studies and in the field of gene regulation.

The post-transcriptional modification 2'-O-Methyl (2'OMe) could be present on the ribose of all four ribonucleosides, and is highly prevalent in a wide variety of RNA species, including the 5' RNA cap of viruses and higher eukaryotes, as well as internally in transfer RNA and ribosomal RNA. Recent studies have suggested that 2'OMe is also located internally in low-abundance RNA species such as viral RNA and mRNA. To profile 2'OMe on different RNA species, we have developed Nm-seq, which could identify 2'OMe sites at single base resolution. Nm-seq is particularly useful for identifying 2'OMe sites located at the 3' terminal ends of small RNAs. Here, we present an optimized protocol for Nm-seq and a protocol for applying Nm-seq to identify 2'OMe sites on small RNA 3' terminal ends., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The use of RNA-seq as a generalized tool to measure the differential expression of genes has essentially replaced the use of the microarray. Despite the acknowledged technical advantages to this approach, RNA-seq library preparation remains mostly conducted by core facilities rather than in the laboratory due to the infrastructure, expertise and time required per sample. We have recently described two 'click-chemistry' based library construction methods termed ClickSeq and Poly(A)-ClickSeq (PAC-seq) as alternatives to conventional RNA-seq that are both cost effective and rely on straightforward reagents readily available to most labs. ClickSeq is random-primed and can sequence any (unfragmented) RNA template, while PAC-seq is targeted to poly(A) tails of mRNAs. Here, we further develop PAC-seq as a platform that allows for simultaneous mapping of poly(A) sites and the measurement of differential expression of genes. We provide a detailed protocol, descriptions of appropriate computational pipelines, and a proof-of-principle dataset to illustrate the technique. PAC-seq offers a unique advantage over other 3' end mapping protocols in that it does not require additional purification, selection, or fragmentation steps allowing sample preparation directly from crude total cellular RNA. We have shown that PAC-seq is able to accurately and sensitively count transcripts for differential gene expression analysis, as well as identify alternative poly(A) sites and determine the precise nucleotides of the poly(A) tail boundaries., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Post-transcriptional modification of RNA, the so-called 'Epitranscriptome', can regulate RNA structure, stability, localization, and function. Numerous modifications have been identified in virtually all classes of RNAs, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), microRNAs (miRNAs), and other noncoding RNAs (ncRNAs). These modifications may occur internally (by base or sugar modifications) and include RNA methylation at different nucleotide positions, or by the addition of various nucleotides at the 3'-end of certain transcripts by a family of terminal nucleotidylyl transferases. Developing methods to specifically and accurately detect and map these modifications is essential for understanding the molecular function(s) of individual RNA modifications and also for identifying and characterizing the proteins that may read, write, or erase them. Here, we focus on the characterization of RNA species targeted by 3' terminal uridylyl transferases (TUTases) (TUT4/7, also known as Zcchc11/6) and a 3'-5' exoribonuclease, Dis3l2, in the recently identified Dis3l2-mediated decay (DMD) pathway - a dedicated quality control pathway for a subset of ncRNAs. We describe the detailed methods used to precisely identify 3'-end modifications at nucleotide level resolution with a particular focus on the U1 and U2 small nuclear RNA (snRNA) components of the Spliceosome. These tools can be applied to investigate any RNA of interest and should facilitate studies aimed at elucidating the functional relevance of 3'-end modifications., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In addition to phosphodiester bond formation, RNA polymerase II has an RNA endonuclease activity, stimulated by TFIIS, which rescues complexes that have arrested and backtracked. How TFIIS affects transcription under normal conditions is poorly understood. We identified backtracking sites in human cells using a dominant-negative TFIIS (TFIIS DN ) that inhibits RNA cleavage and stabilizes backtracked complexes. Backtracking is most frequent within 2 kb of start sites, consistent with slow elongation early in transcription, and in 3' flanking regions where termination is enhanced by TFIIS DN , suggesting that backtracked pol II is a favorable substrate for termination. Rescue from backtracking by RNA cleavage also promotes escape from 5' pause sites, prevents premature termination of long transcripts, and enhances activation of stress-inducible genes. TFIIS DN slowed elongation rates genome-wide by half, suggesting that rescue of backtracked pol II by TFIIS is a major stimulus of elongation under normal conditions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A polymerase ribozyme can be used to label the 3' end of RNA or DNA molecules by incorporating a variety of functionalized nucleotide analogs. Guided by a complementary template, the ribozyme adds a single nucleotide that may contain a fluorophore, biotin, azide or alkyne moiety, thus enabling the detection and/or capture of selectively labeled materials. Employing a variety of commercially available nucleotide analogs, efficient labeling was demonstrated for model RNAs and DNAs, human microRNAs and natural tRNA.

The association studies of several immune-diseases with the 3' Regulatory Region 1 (3'RR1) increased interest on the role that the region plays in the immune-regulation. The 3'RR1 is a polymorphic region on human chromosome 14q32, acting as a cis-regulative element on the Immunoglobulin constant-gene locus recently considered as super-enhancer. The human 3'RR1 share large sequences with its paralogous 3'RR2, at high level of similarity. Thus, a focused investigation was necessary to discriminate each one of the duplicated components of the two regions and its specific contribution to the immunologic phenotype. One of the duplicated elements is the hs1.2 enhancer. The 3'RR1 alleles of this enhancer were demonstrated to play a role in autoimmune diseases, including Psoriasis. We sequenced a specific region internal to the 3'RR1 in hs1.2 homozygous subjects, to detect SNPs associated to the main alleles of the enhancer. We identified two alternative nine-SNPs haplotypes strictly linked to the allele *1 and *2 of hs1.2, that could be used as markers to further investigate the region and associations to pathology. Finally, we identified two haplotypes, namely E2A1 and E2A2, that strongly support the hypothesis of a relevant effect of the rs35216181 in the onset of Psoriasis when the *2 allele is present., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We examined 20,648 prokaryotic unique taxids with respect to the annotation of the 3' end of the 16S rRNA, which contains the anti-Shine-Dalgarno sequence. We used the sequence of highly conserved helix 45 of the 16S rRNA as a guide. By this criterion, 8,153 annotated 3' ends correctly included the anti-Shine-Dalgarno sequence, but 12,495 were foreshortened or otherwise mis-annotated, missing part or all of the anti-Shine-Dalgarno sequence, which immediately follows helix 45. We re-annotated, giving a total of 20,648 16S rRNA 3' ends. The vast majority indeed contained a consensus anti-Shine-Dalgarno sequence, embedded in a highly conserved 13 base "tail". However, 128 exceptional organisms had either a variant anti-Shine-Dalgarno, or no recognizable anti-Shine-Dalgarno, in their 16S rRNA(s). For organisms both with and without an anti-Shine-Dalgarno, we identified the Shine-Dalgarno motifs actually enriched in front of each organism's open reading frames. This showed to what extent the Shine-Dalgarno motifs correlated with anti-Shine Dalgarno motifs. In general, organisms whose rRNAs lacked a perfect anti-Shine-Dalgarno motif also lacked a recognizable Shine-Dalgarno. For organisms whose 16S rRNAs contained a perfect anti-Shine-Dalgarno motif, a variety of results were obtained. We found one genus, Alteromonas, where several taxids apparently maintain two different types of 16S rRNA genes, with different, but conserved, antiSDs. The fact that some organisms do not seem to have or use Shine-Dalgarno motifs supports the idea that prokaryotes have other robust mechanisms for recognizing start codons for translation., Competing Interests: The authors have declared that no competing interests exist.

The Shine-Dalgarno motif occurs in front of prokaryotic start codons, and is complementary to the 3' end of the 16S ribosomal RNA. Hybridization between the Shine-Dalgarno sequence and the anti-Shine-Dalgarno region of the16S rRNA (CCUCCU) directs the ribosome to the start AUG of the mRNA for translation. Shine-Dalgarno-like motifs (AGGAGG in E. coli) are depleted from open reading frames of most prokaryotes. This may be because hybridization of the 16S rRNA at Shine-Dalgarnos inside genes would slow translation or induce internal initiation. However, we analyzed 128 species from diverse phyla where the 16S rRNA gene(s) lack the anti-Shine-Dalgarno sequence, and so the 16S rRNA is incapable of interacting with Shine-Dalgarno-like sequences. Despite this lack of an anti-Shine-Dalgarno, half of these species still displayed depletion of Shine-Dalgarno-like sequences when analyzed by previous methods. Depletion of the same G-rich sequences was seen by these methods even in eukaryotes, which do not use the Shine-Dalgarno mechanism. We suggest previous methods are partly detecting a non-specific depletion of G-rich sequences. Alternative informatics approaches show that most prokaryotes have only slight, if any, specific depletion of Shine-Dalgarno-like sequences from open reading frames. Together with recent evidence that ribosomes do not pause at ORF-internal Shine-Dalgarno motifs, these results suggest the presence of ORF-internal Shine-Dalgarno-like motifs may be inconsequential, perhaps because internal regions of prokaryotic mRNAs may be structurally "shielded" from translation initiation., Competing Interests: The authors have declared that no competing interests exist.

Post-translational modifications of the transcription elongation complex provide mechanisms to fine-tune gene expression, yet their specific impacts on RNA polymerase II regulation remain difficult to ascertain. Here, in Schizosaccharomyces pombe, we examine the role of Cdk9, and related Mcs6/Cdk7 and Lsk1/Cdk12 kinases, on transcription at base-pair resolution with Precision Run-On sequencing (PRO-seq). Within a minute of Cdk9 inhibition, phosphorylation of Pol II-associated factor, Spt5 is undetectable. The effects of Cdk9 inhibition are more severe than inhibition of Cdk7 and Cdk12, resulting in a shift of Pol II toward the transcription start site (TSS). A time course of Cdk9 inhibition reveals that early transcribing Pol II can escape promoter-proximal regions, but with a severely reduced elongation rate of only ~400 bp/min. Our results in fission yeast suggest the existence of a conserved global regulatory checkpoint that requires Cdk9 kinase activity.

Bemisia tabaci (Gennadius; Hempitera: Aleyrodidae) is considered to be a cryptic (sibling) species complex, the members of which exhibit morphological invariability while being genetically and behaviorally distinct. Members of the complex are agricultural pests that cause direct damage by feeding on plants, and indirectly by transmitting viruses that cause diseases leading to reduced crop yield and quality. In Pakistan, cotton leaf curl disease, caused by multiple begomovirus species, is the most economically important viral disease of cotton. In the study outlined here, the diversity and geographic distribution of B. tabaci cryptic species was investigated by analyzing a taxonomically informative fragment of the mitochondrial cytochrome c oxidase 1 gene (mtCOI-3'). The mtCOI-3' sequence was determined for 285 adult whiteflies and found to represent six cryptic species, the most numerous being Asia II-1 and Middle East Asia Minor 1 (MEAM-1), the later also referred to as the B-biotype, which was previously thought to be confined to Sindh province but herein, was also found to be present in the Punjab province. The endemic Asia I was restricted to Sindh province, while an individual in the Asia II-8 was identified in Pakistan for the first time. Also for the first time, samples were collected from northwestern Pakistan and Asia II-1 was identified. Results indicate that in Pakistan the overall diversity of B. tabaci cryptic species is high and, based on comparisons with findings from previous studies, the distribution is dynamic., (© The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The regulation of active microRNAs (miRNAs) and maturation of messenger RNAs (mRNAs) that are competent for translation is a crucial point in the control of all cellular processes, with established roles in development and differentiation. Terminal nucleotidyltransferases (TNTases) are potent regulators of RNA metabolism. TNTases promote the addition of single or multiple nucleotides to an RNA transcript that can rapidly alter transcript stability. The well-known polyadenylation promotes transcript stability while the newly discovered but ubiquitious 3'-end polyuridylation marks RNA for degradation. Monoadenylation and uridylation are essential control mechanisms balancing mRNA and miRNA homeostasis., Scope of Review: This review discusses the multiple functions of non-canonical TNTases, focusing on their substrate range, biological functions, and evolution. TNTases directly control mRNA and miRNA levels, with diverse roles in transcriptome stabilization, maturation, silencing, or degradation. We will summarize the current state of knowledge on non-canonical nucleotidyltransferases and their function in regulating miRNA and mRNA metabolism. We will review the discovery of uridylation as an RNA degradation pathway and discuss the evolution of nucleotidyltransferases along with their use in RNA labeling and future applications as therapeutic targets., Major Conclusions: The biochemically and evolutionarily highly related adenylyl- and uridylyltransferases play antagonizing roles in the cell. In general, RNA adenylation promotes stability, while uridylation marks RNA for degradation. Uridylyltransferases evolved from adenylyltransferases in multiple independent evolutionary events by the insertion of a histidine residue into the active site, altering nucleotide, but not RNA specificity., General Significance: Understanding the mechanisms regulating RNA stability in the cell and controlling the transcriptome is essential for efforts aiming to influence cellular fate. Selectively enhancing or reducing RNA stability allows for alterations in the transcriptome, proteome, and downstream cellular processes. Genetic, biochemical, and clinical data suggest TNTases are potent targets for chemotherapeutics and have been exploited for RNA labeling applications. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The dog is considered to be a useful biomedical model for human diseases and disorders, including obesity. One of the numerous genes associated with human polygenic obesity is MC4R, encoding the melanocortin 4 receptor. The aim of our study was to analyze polymorphisms and methylation of the canine MC4R in relation to adiposity. Altogether 270 dogs representing four breeds predisposed to obesity: Labrador Retriever (n = 187), Golden Retriever (n = 38), Beagle (n = 28) and Cocker Spaniel (n = 17), were studied. The dogs were classified into three groups: lean, overweight and obese, according to the 5-point Body Condition Score (BCS) scale. In the cohort of Labradors a complete phenotypic data (age, sex, neutering status, body weight and BCS) were collected for 127 dogs. The entire coding sequence as well as 5' and 3'-flanking regions of the studied gene were sequenced and six polymorphic sites were reported. Genotype frequencies differed considerably between breeds and Labrador Retrievers appeared to be the less polymorphic. Moreover, distribution of some polymorphic variants differed significantly (P < 0.05) between small cohorts with diverse BCS in Golden Retrievers (c.777T>C, c.868C>T and c.*33C>G) and Beagles (c.-435T>C and c.637G>T). On the contrary, in Labradors no association between the studied polymorphisms and BCS or body weight was observed. Methylation analysis, using bisulfite DNA conversion followed by Sanger sequencing, was carried out for 12 dogs with BCS = 3 and 12 dogs with BCS = 5. Two intragenic CpG islands, containing 19 cytosines, were analyzed and the methylation profile did not differ significantly between lean and obese animals. We conclude that an association of the MC4R gene polymorphism with dog obesity or body weight is unlikely, in spite of the fact that some associations were found in small cohorts of Beagles and Golden Retrievers. Also methylation level of this gene is not related with dog adiposity.

Cells regulate biological responses in part through changes in transcription start sites (TSS) or cleavage and polyadenylation sites (PAS). To fully understand gene regulatory networks, it is therefore critical to accurately annotate cell type-specific TSS and PAS. Here we present a simple and straightforward approach for genome-wide annotation of 5΄- and 3΄-RNA ends. Our approach reliably discerns bona fide PAS from false PAS that arise due to internal poly(A) tracts, a common problem with current PAS annotation methods. We applied our methodology to study the impact of temperature on the Drosophila melanogaster head transcriptome. We found hundreds of previously unidentified TSS and PAS which revealed two interesting phenomena: first, genes with multiple PASs tend to harbor a motif near the most proximal PAS, which likely represents a new cleavage and polyadenylation signal. Second, motif analysis of promoters of genes affected by temperature suggested that boundary element association factor of 32 kDa (BEAF-32) and DREF mediates a transcriptional program at warm temperatures, a result we validated in a fly line where beaf-32 is downregulated. These results demonstrate the utility of a high-throughput platform for complete experimental and computational analysis of mRNA-ends to improve gene annotation., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The detailed analysis of the impact of deletions on proteins or nucleic acids can reveal important functional regions and lead to variants with improved macromolecular properties. We present a method to generate large libraries of mutants with deletions of varying length that are randomly distributed throughout a given gene. This technique facilitates the identification of crucial sequence regions in nucleic acids or proteins. The approach utilizes in vitro transposition to generate 5΄ and 3΄ fragment sub-libraries of a given gene, which are then randomly recombined to yield a final library comprising both terminal and internal deletions. The method is easy to implement and can generate libraries in three to four days. We used this approach to produce a library of >9000 random deletion mutants of an artificial RNA ligase enzyme representing 32% of all possible deletions. The quality of the library was assessed by next-generation sequencing and detailed bioinformatics analysis. Finally, we subjected this library to in vitro selection and obtained fully functional variants with deletions of up to 18 amino acids of the parental enzyme that had been 95 amino acids in length., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

MicroRNAs have emerged as important regulators of osteoclast differentiation in recent years. Of these, miR-34c has been reported to play an important role in bone development. However, its role and the underlying mechanism in osteoclast differentiation remains poorly understood. In this study, we aimed to investigate the precise role and molecular mechanism of miR-34c in osteoclast differentiation. We found an obvious increase in miR-34c expression during osteoclast differentiation in osteoclast precursors induced by receptor activator of nuclear factor κB (NF-κB) ligand and macrophage colony-stimulating factor in vitro. Further experiments showed that overexpression of miR-34c significantly promoted osteoclast differentiation while suppression of miR-34c showed the opposite effect. Interestingly, bioinformatics analysis and dual-luciferase reporter assays showed that miR-34c targets the 3'-untranslated region of leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4). The expression of LGR4 was regulated by miR-34c in osteoclasts. Moreover, miR-34c regulated NF-κB and glycogen synthase kinase 3-β signaling during osteoclast differentiation. Overexpression of LGR4 partially reversed the promoting effect of miR-34c overexpression on osteoclast differentiation. Taken together, our study suggests that miR-34c contributes to osteoclast differentiation by targeting LGR4, providing novel insights into understanding the molecular mechanism underlying osteoclast differentiation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

ATP-dependent chromatin remodeling complexes are important regulators of gene expression in Eukaryotes. In plants, SWI/SNF-type complexes have been shown critical for transcriptional control of key developmental processes, growth and stress responses. To gain insight into mechanisms underlying these roles, we performed whole genome mapping of the SWI/SNF catalytic subunit BRM in Arabidopsis thaliana, combined with transcript profiling experiments. Our data show that BRM occupies thousands of sites in Arabidopsis genome, most of which located within or close to genes. Among identified direct BRM transcriptional targets almost equal numbers were up- and downregulated upon BRM depletion, suggesting that BRM can act as both activator and repressor of gene expression. Interestingly, in addition to genes showing canonical pattern of BRM enrichment near transcription start site, many other genes showed a transcription termination site-centred BRM occupancy profile. We found that BRM-bound 3΄ gene regions have promoter-like features, including presence of TATA boxes and high H3K4me3 levels, and possess high antisense transcriptional activity which is subjected to both activation and repression by SWI/SNF complex. Our data suggest that binding to gene terminators and controlling transcription of non-coding RNAs is another way through which SWI/SNF complex regulates expression of its targets., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The complete genomic sequence of a new virus from cherry trees was determined. Its genome is 5857 nt long and resembles that of members of the genus Luteovirus in its genomic organization and nucleotide sequence. Based on the species demarcation criteria for luteoviruses, the virus represents a new luteovirus species. Furthermore, a 47-nt-long inverted repeat was found at the 3' end of its genome. The virus has been provisionally named cherry-associated luteovirus (ChALV) and is the fourth member of the family Luteoviridae reported to naturally infect woody plants.

Background: Although the pathogenicity of the prominin-1 (PROM1) gene has already been described as associated with autosomal dominant Stargardt disease, little is known about sequence variations in this gene., Purpose: The aim of this study was to evaluate PROM1 gene sequence variations in patients with macular dystrophy., Material and Methods: This retrospective study evaluated variations in the PROM1 gene detected by next-generation sequencing test in patients with macular dystrophy and Stargardt disease., Results: Of 25 medical records of patients with Stargardt disease, three records of patients with PROM1 gene sequence variations were selected for the study. The p.Asp776Val and p.Asp829Asn variants were detected in cases 1 and 2, respectively, and predicted to be pathogenic; they were probably responsible for macular dystrophy in these patients. Case 3 showed a p.Ala643Gly variant in the PROM1 gene and a single variation in the ABCA4 gene, but molecular testing results were inconclusive., Conclusions: In cases of Stargardt disease, where molecular testing results are inconclusive for pathogenic variations in the ABCA4 gene, variations in the PROM1 gene may occur and be considered responsible for the disease in the molecular analysis. This study described three cases in which variations in PROM1 gene may play a role in the pathogenesis of macular dystrophy or be associated with both autosomal recessive and autosomal dominant inheritance.

Short interfering RNA (siRNA)-triggered gene knockdown through the RNA interference (RNAi) pathway is widely used to study gene function, and siRNA-based therapeutics are in development. However, as the guide strand of an siRNA can function like a natural microRNA (miRNA), siRNAs often repress hundreds of off-target transcripts with complementarity only to the seed region (nucleotides 2-8) of the guide strand. Here, we describe novel guide strand 3'-end modifications derived from 1-ethynylribose (1-ER) and copper-catalyzed azide-alkyne cycloaddition reactions and evaluate their impact on target versus miRNA-like off-target knockdown. Surprisingly, when positioned at the guide strand 3'-end, the parent 1-ER modification substantially reduced off-target knockdown while having no measurable effect on on-target knockdown potency. In addition, these modifications were shown to modulate siRNA affinity for the hAgo2 PAZ domain. However, the change in PAZ domain binding affinity was not sufficient to predict the modification's effect on miRNA-like off targeting., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report an infant with sickle cell disease phenotype by biochemical analysis whose β-globin gene (HBB) sequencing showed sickle cell mutation (HBB S ) heterozygosity. The proband has a unique head-to-tail duplication of the β-globin gene cluster having wild-type (HBB A ) and HBB S alleles inherited from her father; constituting her HBB S /HBB S -HBB A genotype. Further analyses revealed that proband's duplicated β-globin gene cluster (∼650 kb) encompassing HBB A does not include the immediate upstream locus control region (LCR) or 3' DNase I hypersensitivity (HS) element. The LCR interacts with β-globin gene cluster involving long range DNA interactions mediated by various transcription factors to drive the regulation of globin genes expression. However, a low level of HBB A transcript was clearly detected by digital PCR. In this patient, the observed transcription from the duplicated, distally displaced HBB A cluster demonstrates that the loss of LCR and flanking 3'HS sites do not lead to complete silencing of HBB transcription., (© 2016 WILEY PERIODICALS, INC.)

ATRX is a SWI/SNF chromatin remodeler proposed to govern genomic stability through the regulation of repetitive sequences, such as rDNA, retrotransposons, and pericentromeric and telomeric repeats. However, few direct ATRX target genes have been identified and high-throughput genomic approaches are currently lacking for ATRX. Here we present a comprehensive ChIP-sequencing study of ATRX in multiple human cell lines, in which we identify the 3' exons of zinc finger genes (ZNFs) as a new class of ATRX targets. These 3' exonic regions encode the zinc finger motifs, which can range from 1-40 copies per ZNF gene and share large stretches of sequence similarity. These regions often contain an atypical chromatin signature: they are transcriptionally active, contain high levels of H3K36me3, and are paradoxically enriched in H3K9me3. We find that these ZNF 3' exons are co-occupied by SETDB1, TRIM28, and ZNF274, which form a complex with ATRX. CRISPR/Cas9-mediated loss-of-function studies demonstrate (i) a reduction of H3K9me3 at the ZNF 3' exons in the absence of ATRX and ZNF274 and, (ii) H3K9me3 levels at atypical chromatin regions are particularly sensitive to ATRX loss compared to other H3K9me3-occupied regions. As a consequence of ATRX or ZNF274 depletion, cells with reduced levels of H3K9me3 show increased levels of DNA damage, suggesting that ATRX binds to the 3' exons of ZNFs to maintain their genomic stability through preservation of H3K9me3.

Objective: LNK is an adapter protein negatively regulating the JAK/STAT cell signaling pathway. In this study, we observed the correlation between variation in LNK gene and the clinical type of myeloproliferative neoplasms (MPN)., Methods: A total of 285 MPN cases were recruited, including essential thrombocythemia (ET) 154 cases, polycythemia vera (PV) 76 cases, primary myelofibrosis (PMF) 19 cases, and chronic myeloid leukemia (CML) 36 cases. Ninety-three healthy individuals were used as normal controls. V617F mutation in JAK2 was identified by allele-specific PCR method, RT-PCR was used for the detection of BCR/ABL1 fusion gene, and mutations and variations in coding exons and their flanking sequences of LNK gene were examined by PCR-sequencing., Results: Missense mutations of A300V, V402M, and R415H in LNK were found in 8 patients including ET (4 cases, all combined with JAK2-V617F mutation), PV (2 cases, one combined with JAK2-V617F mutation), PMF (one case, combined with JAK2-V617F mutation) and CML (one case, combined with BCR/ABL1 fusion gene). The genotype and allele frequencies of the three SNPs (rs3184504, rs111340708 and rs78894077) in LNK were significantly different between MPN patients and controls. For rs3184504 (T/C, in exon2), the T allele (p.262W) and TT genotype were frequently seen in ET, PV and PMF (P<0.01), and C allele (p.262R) and CC genotype were frequently seen in CML (P<0.01). For rs78894077 (T/C, in exon1), the T allele (p.242S) was frequently found in ET (P<0.05). For rs111340708 (TGGGGx5/TGGGGx4, in intron 5), the TGGGG x4 allele was infrequently found in ET, PMF and CML(P<0.01)., Conclusion: Mutations in LNK could be found in some of MPN patients in the presence or absence of JAK2-V617F mutation. Several polymorphisms in LNK gene may affect the clinical type or the genetic predisposition of MPN.

Non-homologous end joining (NHEJ) is the main repair pathway for DNA double-strand breaks (DSBs) in cells with limited 5' resection. To better understand how overhang polarity of chromosomal DSBs affects NHEJ, we made site-specific 5'-overhanging DSBs (5' DSBs) in yeast using an optimized zinc finger nuclease at an efficiency that approached HO-induced 3' DSB formation. When controlled for the extent of DSB formation, repair monitoring suggested that chromosomal 5' DSBs were rejoined more efficiently than 3' DSBs, consistent with a robust recruitment of NHEJ proteins to 5' DSBs. Ligation-mediated qPCR revealed that Mre11-Rad50-Xrs2 rapidly modified 5' DSBs and facilitated protection of 3' DSBs, likely through recognition of overhang polarity by the Mre11 nuclease. Next-generation sequencing revealed that NHEJ at 5' DSBs had a higher mutation frequency, and validated the differential requirement of Pol4 polymerase at 3' and 5' DSBs. The end processing enzyme Tdp1 did not impact joining fidelity at chromosomal 5' DSBs as in previous plasmid studies, although Tdp1 was recruited to only 5' DSBs in a Ku-independent manner. These results suggest distinct DSB handling based on overhang polarity that impacts NHEJ kinetics and fidelity through differential recruitment and action of DSB modifying enzymes., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Converting single-stranded viral RNA into double stranded DNA for integration is an essential step in HIV-1 replication. Initial polymerization of minus-strand DNA is primed from a host derived tRNA, whereas subsequent plus-strand synthesis requires viral primers derived from the 3' and central polypurine tracts (3' and cPPTs). The 5' and 3' termini of these conserved RNA sequence elements are precisely cleaved by RT-associated RNase H to generate specific primers that are used to initiate plus-strand DNA synthesis. In this study, siRNA wad used to produce a replicative HIV-1 variant contained G(-1)A and T(-16)A substitutions within/adjacent to the 3'PPT sequence. Introducing either or both mutations into the 3'PPT region or only the G(-1)A substitution in the cPPT region of NL4-3 produced infectious virus with decreased fitness relative to the wild-type virus. In contrast, introducing the T(-16)A or both mutations into the cPPT rendered the virus(es) incapable of replication, most likely due to the F185L integrase mutation produced by this nucleotide substitution. Finally, the effects of G(-1)A and T(-16)A mutations on cleavage of the 3'PPT were examined using an in vitro RNase H cleavage assay. Substrate containing both mutations was mis-cleaved to a greater extent than either wild-type substrate or substrate containing the T(-16)A mutation alone, which is consistent with the observed effects of the equivalent nucleotide substitutions on the replication fitness of NL4-3 virus. In conclusion, siRNA targeting of the HIV-1 3'PPT region can substantially suppress virus replication, and this selective pressure can be used to generate infectious virus containing mutations within or near the HIV-1 PPT. Moreover, in-depth analysis of the resistance mutations demonstrates that although virus containing a G(-1)A mutation within the 3'PPT is capable of replication, this nucleotide substitution shifts the 3'-terminal cleavage site in the 3'PPT by one nucleotide (nt) and significantly reduces viral fitness.

Differentiation of the protozoan parasite Toxoplasma gondii into its latent bradyzoite stage is a key event in the parasite's life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/GRA24 knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in T. gondii. A better understanding of the proteins and mechanisms regulating T. gondii differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen.

The phenotypic consequences of expression quantitative trait loci (eQTLs) are presumably due to their effects on protein expression levels. Yet the impact of genetic variation, including eQTLs, on protein levels remains poorly understood. To address this, we mapped genetic variants that are associated with eQTLs, ribosome occupancy (rQTLs), or protein abundance (pQTLs). We found that most QTLs are associated with transcript expression levels, with consequent effects on ribosome and protein levels. However, eQTLs tend to have significantly reduced effect sizes on protein levels, which suggests that their potential impact on downstream phenotypes is often attenuated or buffered. Additionally, we identified a class of cis QTLs that affect protein abundance with little or no effect on messenger RNA or ribosome levels, which suggests that they may arise from differences in posttranslational regulation., (Copyright © 2015, American Association for the Advancement of Science.)

Polycystic ovary syndrome (PCOS) is the most common gynecological endocrine disorder. The genetic background is believed to play a crucial role in the pathogenesis of PCOS. In recent years, the role of insulin receptor (INSR) polymorphisms in PCOS predisposition has attracted much attention. We performed a meta-analysis to investigate the association between the single nucleotide polymorphisms (SNPs) of INSR and PCOS. Published literature from Pubmed, Embase, and Cochrane CENTRAL was retrieved up until 7 August 2014. A total of 20 case-control studies including 23,845 controls and 17,460 PCOS cases with an average Newcastle-Ottawa quality assessment scale (NOS) score of 6.75 were analyzed. Ninety-eight SNPs distributed in 23 exons and the flanking regions of INSR were investigated, among which 17 SNPs were found to be associated with PCOS. Three SNPs detected in more than three studies were selected for further analyses. Twelve studies including 1158 controls and 1264 PCOS cases entered the analysis of rs1799817, but no significant association was found for every genotype (p > 0.05). Further subgroup stratification by ethnicity and weight did not lead to discovery of significant correlation (p > 0.05). For rs2059806, four studies including 442 controls and 524 PCOS cases were qualified for meta-analysis, and no significant association with PCOS was found for any genotype (p > 0.05). Four studies including 12,830 controls and 11,683 PCOS cases investigated the correlation between rs2059807 and PCOS, and five of the six cohorts indicated a significant impact. Our current meta-analysis suggests no significant correlation between rs1799817/rs2059806 SNPs and susceptibility of PCOS, while rs2059807 could be a promising candidate SNP that might be involved in the susceptibility of PCOS.

The activity and dynamic nature of TATA-binding protein (TBP) crucial to RNA polymerase II-mediated transcription is under control of the Mot1p and NC2 complexes. Here we show that both TBP regulatory factors play 'hidden' roles in ensuring transcription fidelity by restricting anti-sense non-coding RNA (ncRNA) synthesis. Production of anti-sense ncRNA transcripts is suppressed by Mot1p- and NC2-mediated release of TBP from binding sites at the 3'-end of genes. In this, Mot1p and NC2 collaborate with the Nrd1p-Nab3p-Sen1p (NNS) complex that terminates the synthesis of anti-sense ncRNAs. In several cases anti-sense ncRNA expression interferes with expression of the cognate sense transcript. Our data reveal a novel regulatory mechanism to suppress anti-sense ncRNA expression and pre-initiation complex (PIC) formation at spurious sites., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase coding region. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on enzyme function and susceptibility to integrase inhibitors. Accordingly, we cloned and purified integrase proteins from each of HIV-1 group O clades A and B, an HIV-O divergent strain, and HIV-1 group M (HIV-M, subtype B), used as a reference. To assess enzymatic function of HIV-O integrase, we carried out strand transfer and 3' processing assays with various concentrations of substrate (DNA target and long terminal repeats [LTR], respectively) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of various concentrations of several INSTIs. The inhibition constant (Ki) and 50% effective concentration (EC50) values were calculated for HIV-O integrases and HIV-O viruses, respectively, and compared with those of HIV-M. The results showed that HIV-O integrase displayed lower activity in strand transfer assays than did HIV-M enzyme, whereas 3' processing activities were similar to those of HIV-M. HIV-O integrases were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. Molecular modeling suggests that two key polymorphic residues that are close to the integrase catalytic site, 74I and 153A, may play a role in these differences., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The THSC/TREX-2 complex of Saccharomyces cerevisiae mediates the anchoring of transcribed genes to the nuclear pore, linking transcription elongation with mRNA export and genome stability, as shown for specific reporters. However, it is still unknown whether the function of TREX-2 is global and the reason for its relevant role in genome integrity. Here, by studying two TREX-2 representative subunits, Thp1 and Sac3, we show that TREX-2 has a genome-wide role in gene expression. Both proteins show similar distributions along the genome, with a gradient disposition at active genes that increases towards the 3' end. Thp1 and Sac3 have a relevant impact on the expression of long, G+C-rich and highly transcribed genes. Interestingly, replication impairment detected by the genome-wide accumulation of the replicative Rrm3 helicase is increased preferentially at highly expressed genes in the thp1Δ and sac3Δ mutants analyzed. Therefore, our work provides evidence of a function of TREX-2 at the genome-wide level and suggests a role for TREX-2 in preventing transcription-replication conflicts, as a source of genome instability derived from a defective messenger ribonucleoprotein particle (mRNP) biogenesis., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Introduction: The rearrangement of the mixed-lineage leukemia (MLL) gene occurs through translocations and insertions involving a variety of partner chromosome genes. However, there are few studies on aberrant MLL signal patterns such as concurrent 3' MLL deletion., Methods: A total of 84 patients with acute leukemia (AL) who had MLL rearrangements detected by florescence in situ hybridization (FISH) were enrolled in the study. The distribution of MLL fusion partner genes was analyzed, and aberrant MLL signals were evaluated., Results: Seventy-seven (91.7%) patients had MLL rearrangements, involving previously described translocation partner genes (TPGs). Among these TPGs, the frequencies of MLLT3, AFF1, MLLT4, and ELL were 29.8%, 17.9%, 15.5%, and 13.1%, respectively. A high frequency of MLLT4 in our study was due to the high proportion of acute myeloid leukemia cases in pediatric and adult patients. Aberrant MLL signals were found in 18 patients: 11 (61.1%) with 3' MLL signal loss and 7 with 3' MLL signal gain. All cases with 3' MLL signal gain were due to an extra derivative partner chromosome. The median overall survival period of patients with 3' MLL gain was shorter than that in patients without aberrant MLL signal patterns., Conclusion: Aberrant MLL signals were frequently detected by FISH analysis. The 3' MLL gain was associated with poor prognosis in patients with AL. Therefore, it is important to detect aberrant MLL signal patterns using FISH analysis., (© 2014 John Wiley & Sons Ltd.)

Rationale: The evolutionary conserved Tbx3/Tbx5 gene cluster encodes T-box transcription factors that play crucial roles in the development and homeostasis of the cardiac conduction system in human and mouse. Both genes are expressed in overlapping patterns and function in strictly tissue-specific and dose-dependent manners, yet, their regulation is poorly understood., Objective: To analyze the mechanism underlying the complex regulation of the Tbx3/Tbx5 cluster., Methods and Results: By probing the 3-dimensional architecture of the Tbx3/Tbx5 cluster using high-resolution circular chromosome conformation capture sequencing in vivo, we found that its regulatory landscape is in a preformed conformation similar in embryonic heart, limbs, and brain. Tbx3 and its flanking gene desert form a 1 Mbp loop between CCCTC-binding factor (CTCF)-binding sites that is separated from the neighboring Tbx5 loop. However, Ctcf inactivation did not result in transcriptional regulatory interaction between Tbx3 and Tbx5. Multiple sites within the Tbx3 locus contact the promoter, including sites corresponding to regions known to contain variations in the human genome influencing conduction. We identified an atrioventricular-specific enhancer and a pan-cardiac enhancer that contact the promoter and each other and synergize to activate transcription in the atrioventricular conduction system., Conclusions: We provide a high-resolution model of the 3-dimensional structure and function of the Tbx3/Tbx5 locus and show that the locus is organized in a preformed, permissive structure. The Tbx3 locus forms a CTCF-independent autonomous regulatory domain with multiple combinatorial regulatory elements that control the precise pattern of Tbx3 in the cardiac conduction system., (© 2014 American Heart Association, Inc.)

Sexual reproduction and breeding systems are driving forces for genetic diversity. The mating-type (MAT) locus represents a mutation and chromosome rearrangement hotspot in yeasts. Zygosaccharomyces rouxii complex yeasts are naturally faced with hostile low water activity (aw) environments and are characterized by gene copy number variation, genome instability, and aneuploidy/allodiploidy. Here, we investigated sex-determination system in Zygosaccharomyces sapae diploid strain ABT301(T), a member of the Z. rouxii complex. We cloned three divergent mating type-like (MTL) α-idiomorph sequences and designated them as ZsMTLα copies 1, 2, and 3. They encode homologs of Z. rouxii CBS 732(T) MATα2 (amino acid sequence identities spanning from 67.0 to 99.5%) and MATα1 (identity range 81.5-99.5%). ABT301(T) possesses two divergent HO genes encoding distinct endonucleases 100% and 92.3% identical to Z. rouxii HO. Cloning of MATA: -idiomorph resulted in a single ZsMTLA: locus encoding two Z. rouxii-like proteins MATA: 1 and MATA: 2. To assign the cloned ZsMTLα and ZsMTLA: idiomorphs as MAT, HML, and HMR cassettes, we analyzed their flanking regions. Three ZsMTLα loci exhibited the DIC1-MAT-SLA2 gene order canonical for MAT expression loci. Furthermore, four putative HML cassettes were identified, two containing the ZsMTLα copy 1 and the remaining harboring ZsMTLα copies 2 and 3. Finally, the ZsMTLA: locus was 3'-flanked by SLA2, suggesting the status of MAT expression locus. In conclusion, Z. sapae ABT301(T) displays an aααα genotype missing of the HMR silent cassette. Our results demonstrated that mating-type switching is a hypermutagenic process in Z. rouxii complex that generates genetic diversity de novo. This error-prone mechanism could be suitable to generate progenies more rapidly adaptable to hostile environments., (Copyright © 2014 Solieri et al.)

DNA repair mechanisms are essential for preservation of genome integrity. However, it is not clear how DNA are selected and processed at broken ends by exonucleases during repair pathways. Here we show that the DnaQ-like exonuclease RNase T is critical for Escherichia coli resistance to various DNA-damaging agents and UV radiation. RNase T specifically trims the 3' end of structured DNA, including bulge, bubble, and Y-structured DNA, and it can work with Endonuclease V to restore the deaminated base in an inosine-containing heteroduplex DNA. Crystal structure analyses further reveal how RNase T recognizes the bulge DNA by inserting a phenylalanine into the bulge, and as a result the 3' end of blunt-end bulge DNA can be digested by RNase T. In contrast, the homodimeric RNase T interacts with the Y-structured DNA by a different binding mode via a single protomer so that the 3' overhang of the Y-structured DNA can be trimmed closely to the duplex region. Our data suggest that RNase T likely processes bulge and bubble DNA in the Endonuclease V-dependent DNA repair, whereas it processes Y-structured DNA in UV-induced and various other DNA repair pathways. This study thus provides mechanistic insights for RNase T and thousands of DnaQ-like exonucleases in DNA 3'-end processing., Competing Interests: The authors have declared that no competing interests exist.

CGβ subunits comprise a unique carboxyl-terminal peptide (CTP) that has multiple O-linked glycans and extends serum half-life of the protein. It has evolved by incorporating a previously untranslated region of the LHβ gene into the reading frame. Although CTP-like sequences are encrypted in the LHβ genes of several mammals, the CGβ subunit developed only in primates and equids. To study this restriction in evolution, we examined whether the cryptic CTP decoded from the bovine LHβ gene (boCTP) possesses key characteristics of the human (h) CGβ-CTP. The boCTP does not impede several crucial aspects of hormone biosynthesis, but compared to the hCGβ-CTP, the stretch lacks O-glycans and determinants for circulatory survival. O-glycan deficiency and the associated incapacity to extend serum half-life is a major drawback of the boCTP. This may explain why LH did not evolve into CG in ruminants and consequently alternative mechanisms evolved to delay luteolysis early in gestation., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Statistical positioning, the localization of nucleosomes packed against a fixed barrier, is conjectured to explain the array of well-positioned nucleosomes at the 5' end of genes, but the extent and precise implications of statistical positioning in vivo are unclear. We examine this hypothesis quantitatively and generalize the idea to include moving barriers as well as nucleosomes actively packed against a barrier. Early experiments noted a similarity between the nucleosome profile aligned and averaged across genes and that predicted by statistical positioning; however, we demonstrate that aligning random nucleosomes also generates the same profile, calling the previous interpretation into question. New rigorous results reformulate statistical positioning as predictions on the variance structure of nucleosome locations in individual genes. In particular, a quantity termed the variance gradient, describing the change in variance between adjacent nucleosomes, is tested against recent high-throughput nucleosome sequencing data. Constant variance gradients provide support for generalized statistical positioning in ∼ 50% of long genes. Genes that deviate from predictions have high nucleosome turnover and cell-to-cell gene expression variability. The observed variance gradient suggests an effective nucleosome size of 158 bp, instead of the commonly perceived 147 bp. Our analyses thus clarify the role of statistical positioning in vivo.

Although studies with the ciliate Tetrahymena thermophila have played a central role in advancing our understanding of telomere biology and telomerase mechanisms and composition, the full complement of Tetrahymena telomere proteins has not yet been identified. Previously, we demonstrated that in Tetrahymena, the telomeric 3' overhang is protected by a three-protein complex composed of Pot1a, Tpt1, and Pat1. Here we show that Tpt1 and Pat1 associate with a fourth protein, Pat2 (Pot1 associated Tetrahymena 2). Mass spectrometry of proteins copurifying with Pat1 or Tpt1 identified peptides from Pat2, Pot1a, Tpt1, and Pat1. The lack of other proteins copurifying with Pat1 or Tpt1 implies that the overhang is protected by a four-protein Pot1a-Tpt1-Pat1-Pat2 complex. We verified that Pat2 localizes to telomeres, but we were unable to detect direct binding to telomeric DNA. Cells depleted of Pat2 continue to divide, but the telomeres exhibit gradual shortening. The lack of growth arrest indicates that, in contrast to Pot1a and Tpt1, Pat2 is not required for the sequestration of the telomere from the DNA repair machinery. Instead, Pat2 is needed to regulate telomere length, most likely by acting in conjunction with Pat1 to allow telomerase access to the telomere.

Background: Genetic variation associated with human leukocyte antigen (HLA) genes has immunological functions and is associated with autoimmune diseases. To date, large-scale studies involving classical HLA genes have been limited by time-consuming and expensive HLA-typing technologies. To reduce these costs, single-nucleotide polymorphisms (SNPs) have been used to predict HLA-allele types. Although HLA allelic distributions differ among populations, most prediction model of HLA genes are based on Caucasian samples, with few reported studies involving non-Caucasians., Results: Our sample consisted of 437 Han Chinese with Affymetrix 5.0 and Illumina 550 K SNPs, of whom 214 also had data on Affymetrix 6.0 SNPs. All individuals had HLA typings at a 4-digit resolution. Using these data, we have built prediction model of HLA genes that are specific for a Han Chinese population. To optimize our prediction model of HLA genes, we analyzed a number of critical parameters, including flanking-region size, genotyping platform, and imputation. Predictive accuracies generally increased both with sample size and SNP density., Conclusions: SNP data from the HapMap Project are about five times more dense than commercially available genotype chip data. Using chips to genotype our samples, however, only reduced the accuracy of our HLA predictions by only ~3%, while saving a great deal of time and expense. We demonstrated that classical HLA alleles can be predicted from SNP genotype data with a high level of accuracy (80.37% (HLA-B) ~95.79% (HLA-DQB1)) in a Han Chinese population. This finding offers new opportunities for researchers in obtaining HLA genotypes via prediction using their already existing chip datasets. Since the genetic variation structure (e.g. SNP, HLA, Linkage disequilibrium) is different between Han Chinese and Caucasians, and has strong impact in building prediction models for HLA genes, our findings emphasize the importance of building ethnic-specific models when analyzing human populations.

Genetic variants in human microRNA (miRNA) genes may alter mature miRNA processing and/or target selection, and likely contribute to cancer susceptibility and disease progression. Previous studies have suggested that miR-101 may play important roles in the development of cancer by regulating key tumor-associated genes. However, the role of single nucleotide polymorphisms (SNPs) of miR-101 in breast cancer susceptibility remains unclear. In this study, we genotyped 11 SNPs of the miR-101 genes (including miR-101-1 and miR-101-2) in a case-control study of 1064 breast cancer cases and 1073 cancer-free controls. The results revealed that rs462480 and rs1053872 in the flank regions of pre-miR-101-2 were significantly associated with increased risk of breast cancer (rs462480 AC/CC vs AA: adjusted OR = 1.182, 95% CI: 1.030-1.357, P = 0.017; rs1053872 CG/GG vs CC: adjusted OR = 1.179, 95% CI: 1.040-1.337, P = 0.010). However, the remaining 9 SNPs were not significantly associated with risk of breast cancer. Additionally, combined analysis of the two high-risk SNPs revealed that subjects carrying the variant genotypes of rs462480 and rs1053872 had increased risk of breast cancer in a dose-response manner (P(trend) = 0.002). Compared with individuals with "0-1" risk allele, those carrying "2-4" risk alleles had 1.29-fold risk of breast cancer. In conclusion, these findings suggested that the SNPs rs462480 and rs1053872 residing in miR-101-2 gene may have a solid impact on genetic susceptibility to breast cancer, which may improve our understanding of the potential contribution of miRNA SNPs to cancer pathogenesis.

Metazoan replication-dependent histone mRNAs are the only known eukaryotic mRNAs that lack a poly(A) tail, ending instead in a conserved stem-loop sequence, which is bound to the stem-loop binding protein (SLBP) on the histone mRNP. Histone mRNAs are rapidly degraded when DNA synthesis is inhibited in S phase in mammalian cells. Rapid degradation of histone mRNAs is initiated by oligouridylation of the 3' end of histone mRNAs and requires the cytoplasmic Lsm1-7 complex, which can bind to the oligo(U) tail. An exonuclease, 3'hExo, forms a ternary complex with SLBP and the stem-loop and is required for the initiation of histone mRNA degradation. The Lsm1-7 complex is also involved in degradation of polyadenylated mRNAs. It binds to the oligo(A) tail remaining after deadenylation, inhibiting translation and recruiting the enzymes required for decapping. Whether the Lsm1-7 complex interacts directly with other components of the mRNP is not known. We report here that the C-terminal extension of Lsm4 interacts directly with the histone mRNP, contacting both SLBP and 3'hExo. Mutants in the C-terminal tail of Lsm4 that prevent SLBP and 3'hExo binding reduce the rate of histone mRNA degradation when DNA synthesis is inhibited.

Drug-resistant derivatives of serovar-specific virulence plasmids, such as pSLT, in clinically-relevant Salmonella enterica serovar Typhimurium strains, represent a threat for human health. We have analysed 14 S. Typhimurium isolates recovered in Italy and the United Kingdom from swine and from cases of human infection for the presence of virulence-resistance (VR) plasmids. They were negative for the multidrug resistance (MDR) region of the Salmonella genomic island 1 (SGI1), but expressed resistance to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfamethoxazole, and tetracyclines. The isolates were characterised by XbaI pulsed-field gel electrophoresis, multilocus sequence typing, and detection of resistance and virulence determinants (PCR/sequencing). Identification of VR plasmids was accomplished by PCR detection of bla genes (encoding ampicillin resistance), class 1 integrons and the pSLT virulence gene spvC. Plasmid analyses were performed by alkaline lysis, S1-nuclease digestion, replicon typing, conjugation, restriction analyses, and Southern blot/hybridization. Two blaOXA-1 positive isolates contained pSLT-derived plasmids related to pUO-StVR2. In nine isolates, eight from swine and one from a patient, MDR-conferring-IncFII-VR plasmids were detected. They contained the blaTEM-1 gene as well as a nonconventional class 1 integron with dfrA12-aadA2 gene cassettes in its variable region, and a sul3 gene in the 3' conserved segment. Restriction analysis suggested a novel pSLT variant. The results obtained underline the role of swine as a potential reservoir for the blaTEM-1-IncFII-plasmids. The occurrence and spread of virulence- and MDR-conferring plasmids should be considered as a potential public health problem.