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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., Graphical Abstract Graphical AbstractBy using a sequence designed to contain all possible NYYN sites, the effect of the dipyrimidine and its flanking sequence, as well as wavelength, and sensitizer on the relative frequency of cyclobutane pyrimidine dimer formation was revealed.

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., The miR‐9‐1 promoter is hypermethylated in nasopharyngeal carcinoma (NPC), thus decreasing its expression. Reduced miR‐9‐1 expression increases HK2 levels, thus increasing glycolysis. FISH score analysis showed that miR‐9‐1 is an independent prognostic factor in NPC.

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 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.

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.

Improving the quality of a siRNA-knockdown cloning vector requires simpler, shorter, and more effective flanking sequences. In this study, we designed such flanking sequences based on those found in zebrafish pre-miR3906, namely, internal element (IE) 1 and IE2. We engineered a vegf-shRNA fragment flanked by an 80-bp IE1/IE2 and then inserted into the 3′ UTR of GFP reporter cDNA driven by a cytomegalovirus promoter to obtain a plasmid containing gfp-IE-vegf-shRNA-polA. Upon microinjection of this plasmid into zebrafish embryos, we found that IE flanking sequences could effectively induce the production of vegf-shRNA fragment, which was then processed into a functional siRNA to silence the target vegf121 gene. Northern blot showed that the vegf-shRNA fragment was cleaved from gfp-IE-vegf-shRNA-polA, resulting in the loss of polyA tails, subsequently degrading the remaining RNA-containing GFP. Moreover, Western blot revealed that addition of IE-based vegf-shRNA fragment could markedly decrease the expression of VEGF. Finally, to facilitate a more versatile application of the IE-based knockdown vector, we generated an inducible expression vector in which IE-vegf-shRNA was constructed downstream in a Tet-on system to generate a Tet-on-IE-vegf-shRNA construct. After doxycycline induction, the protein level of VEGF in SW620 cells harboring the Tet-on-IE-vegf-shRNA construct was decreased 77%. Interestingly, when SW620 cells harboring Tet-on-IE-vegf-shRNA cells were induced and transplanted into zebrafish embryos, we found that abnormal branch of the sub-intestinal vessels was reduced in the recipient embryos, suggesting that vegf-shRNA cleaved from Tet-on-IE-vegf-shRNA-polA was processed into a functional vegf-siRNA in embryos suppressing endogenous VEGF and reducing tumor angiogenesis. Therefore, we conclude that fish-origin IEs are flanking sequences with short, simple, and effective DNA elements. This IE-based knockdown cloning vector provides a new alternative material to facilitate the generation of functional siRNA with which to perform loss-of-function experiments, both in vitro (mammalian cells) and in vivo (zebrafish embryos).

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.

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.

Summary Plants represent a promising platform for the highly scalable production of recombinant proteins. Previously, we identified the tobacco extensin terminator lacking its intron as an element that reduced transcript read‐through and improved recombinant protein production in a plant‐based system. In this study, we systematically compared nonreplicating plant expression vectors containing over 20 commonly used or newly identified terminators from diverse sources. We found that eight gene terminators enhance reporter gene expression significantly more than the commonly used 35S and NOS terminators. The intronless extensin terminator provided a 13.6‐fold increase compared with the NOS terminator. Combining terminators in tandem produced large synergistic effects, with many combinations providing a >25‐fold increase in expression. Addition of the tobacco Rb7 or TM6 matrix attachment region (MAR) strongly enhanced protein production when added to most terminators, with the Rb7 MAR providing the greatest enhancement. Using deletion analysis, the full activity of the 1193 bp Rb7 MAR was found to require only a 463‐bp region at its 3’ end. Combined terminators and MAR together provided a >60‐fold increase compared with the NOS terminator alone. These combinations were then placed in a replicating geminiviral vector, providing a total of >150‐fold enhancement over the original NOS vector, corresponding to an estimated yield of 3–5 g recombinant protein per kg leaf fresh weight or around 50% of the leaf total soluble protein. These results demonstrate the importance of 3’ flanking regions in optimizing gene expression and show great potential for 3’ flanking regions to improve DNA‐based recombinant protein production systems.

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., While post-translational modifications of the RNA polymerase II complex regulate gene expression, the exact mechanism remains unknown. Here, the authors use altered-specificity kinase mutations to examine the role of Cdk9, Mcs6/Cdk7, and Lsk1/Cdk12 on transcription at base-pair resolution in yeast.

Leptin is a protein involved in the regulation of feed intake, fat metabolism, whole body energy balance and hematopoiesis in cattle. The diversity of the Leptin (LEP) gene in Indonesian indigenous cattle can be used as molecular livestock selection to improve productivity. The objective of this study was to identify Single Nucleotide Polymorphisms (SNP) in the 3’flanking region of LEP gene from 31 heads of Sumba Ongole (Bos indicus) cattle. A total of 17 SNP’s in the 3’flanking region (3506 - 4019 bp) of the LEP gene were identified according to GenBank: U50365. Five SNP’s (g.C3558T; g.G3566T; g.A3567C; g.G3574A; g.C3575A) were found in all samples. The moderate Polymorphic Informative Content (PIC) values (0.25

Background/Aims Differences in the Helicobacter pylori infection rate are not sufficient to clarify the dissimilarity of gastric cancer incidence between Myanmar and its neighboring countries. To better understand this trend, the H. pylori virulence gene cagA was characterized in Myanmar. Methods Glutamate-proline-isoleucine-tyrosine-alanine (EPIYA) patterns and CagA multimerization (CM) motifs of cagA genotypes were examined by performing polymerase chain reactions and DNA sequencing. Results Of 69 tested H. pylori strains, cagA-positive patients had significantly more severe histological scores in their antrum than cagA-negative patients. Sequence analysis revealed that 94.1% of strains had Western-type cagA containing an EPIYA motif (92.6%) or EPIYT motif (6.4%). The intestinal metaplasia scores in the antral of patients infected with the ABC and ABCC types of cagA were significantly higher than those of patients with AB-type cagA. Interestingly, in patients infected with H. pylori, 46.3% of strains with three EPIYA motifs contained two identical Western-typical CM motifs, and these patients showed significantly higher antrum inflammation scores than patients infected with two identical nontypical-CM motif strains (p=0.02). Conclusions In Myanmarese strains, Western-type cagA was predominant. The presence of CM motifs and the proportion of multiple EPIYA-C segments might partially explain the intermediate gastric cancer risk found in Myanmar.

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 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. Electronic supplementary material The online version of this article (doi:10.1007/s11033-017-4114-3) contains supplementary material, which is available to authorized users.

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.

// Sandrine Le Noir 1,* , Brice Laffleur 1,* , Claire Carrion 1 , Armand Garot 1 , Sandrine Lecardeur 1 , Eric Pinaud 1 , Yves Denizot 1 , Jane Skok 2 and Michel Cogne 1 1 UMR 7276 CNRS and Universite de Limoges: Controle de la Reponse Immune B et Lymphoproliferation, Limoges, France 2 Department of Pathology, New York University School of Medicine, New York, NY, USA * These authors have contributed equally to this work Correspondence to: Michel Cogne, email: // Keywords : super-enhancer; alleles; transvection; nuclear positioning; gene regulation Received : December 20, 2016 Accepted : January 01, 2017 Published : Janaury 10, 2017 Abstract Immunoglobulin heavy chain ( IgH ) alleles have ambivalent relationships: they feature both allelic exclusion, ensuring monoallelic expression of a single immunoglobulin (Ig) allele, and frequent inter-allelic class-switch recombination (CSR) reassembling genes from both alleles. The IgH locus 3’ regulatory region (3’RR) includes several transcriptional cis -enhancers promoting activation-induced cytidine deaminase (AID)-dependent somatic hypermutation (SHM) and CSR, and altogether behaves as a strong super-enhancer. It can also promote deregulated expression of translocated oncogenes during lymphomagenesis. Besides these rare, illegitimate and pathogenic interactions, we now show that under physiological conditions, the 3’RR super-enhancer supports not only legitimate cis- , but also trans- recruitment of AID, contributing to IgH inter-allelic proximity and enabling the super-enhancer on one allele to stimulate biallelic SHM and CSR. Such inter-allelic activating interactions define transvection, a phenomenon well-known in drosophila but rarely observed in mammalian cells, now appearing as a unique feature of the IgH 3’RR super-enhancer.

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 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.

Short interspersed nuclear elements (SINEs) are small, non-autonomous and heterogeneous retrotransposons that are widespread in plants. To explore the amplification dynamics and evolutionary history of SINE populations in representative deciduous tree species, we analyzed the genomes of the six following Salicaceae species: Populus deltoides, Populus euphratica, Populus tremula, Populus tremuloides, Populus trichocarpa, and Salix purpurea. We identified 11 Salicaceae SINE families (SaliS-I to SaliS-XI), comprising 27 077 full-length copies. Most of these families harbor segmental similarities, providing evidence for SINE emergence by reshuffling or heterodimerization. We observed two SINE groups, differing in phylogenetic distribution pattern, similarity and 3' end structure. These groups probably emerged during the 'salicoid duplication' (~65 million years ago) in the Salix-Populus progenitor and during the separation of the genus Salix (45-65 million years ago), respectively. In contrast to conserved 5' start motifs across species and SINE families, the 3' ends are highly variable in sequence and length. This extraordinary 3'-end variability results from mutations in the poly(A) tail, which were fixed by subsequent amplificational bursts. We show that the dissemination of newly evolved 3' ends is accomplished by a displacement of older motifs, leading to various 3'-end subpopulations within the SaliS families.

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. Electronic supplementary material The online version of this article (10.1186/s13104-019-4247-7) contains supplementary material, which is available to authorized users.

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.

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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)-click-seq (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.

While the role of cytokine genes has been well documented in the context of Leishmania (Viannia) braziliensis infection, no studies have addressed the influence of human leukocyte antigen-G (HLA-G) in susceptibility/resistance to American Tegumentary Leishmaniasis (ATL). Here, we evaluated the influences of HLA-G, IL-10, TNF-A and IFN-G in the susceptibility and clinical manifestations of ATL. DNA of 114 ATL patients and 346 healthy individuals were sequenced for well-documented polymorphisms in HLA-G 3′ untranslated region (UTR), in IL-10 and TNF-A promoters and in IFN-G intron 1. Soluble HLA-G (sHLA-G) and cytokine levels were evaluated by ELISA and flow cytometry, respectively. Analyses were performed using GraphPad and R-package software. Individuals bearing HLA-G +3142G/G showed an association with increased risk for ATL, whereas those carrying the HLA-G +3142C/G and one copy of UTR6 haplotype, showed an association with decreased risk for ATL. sHLA-G was overexpressed in “susceptible” patients compared to the “resistant’’ one, and also in patients bearing +3142G/G genotype. From these results, HLA-G +3142G/G may be considered as genotype of susceptibility and UTR6 as marker of protection to ATL. Our findings showed a participation of HLA-G in the pathogenesis of the ATL.

This study aimed to determine the effect of different BRCA1 exonal mutations on the clinical course of epithelial ovarian cancer (EOC).Clinicopathological variables and survival outcomes were compared among 53 primary EOC patients with pathogenic BRCA1 mutations in exons 1-11 (5' mutations) and in exons 12-24 (3' mutations).BRCA1 5' exonal mutations were found in 35 (66.0%) patients. The median follow-up period was 40 months. Clinicopathological variables remained unchanged between the two groups. Patients with 5' mutations had a significantly longer progression-free survival than those with C-terminal mutations (p=0.034), better predicting progression-free survival [2.923 (1.402-6.093), p=0.004], but not overall survival in cases of multiple relapses (p=0.497).N-terminal BRCA1 mutations in EOC patients are associated with favourable primary progression-free survival, a trend observed only in primary progression-free survival, not in overall survival.

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.

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.

Summary 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 (TFIISDN) 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 TFIISDN, 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. TFIISDN 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.

In vitro run-off transcription by T7 RNA polymerase generates heterogeneous 3′-ends because the enzyme tends to add untemplated adenylates. To generate homogeneous 3′-termini, HDV ribozymes have been used widely. Their sequences are added to the 3′-terminus such that co-transcriptional self-cleavage generates homogeneous 3′-ends. A shorter HDV sequence that cleaves itself efficiently would be advantageous. Here we show that a recently discovered, small HDV ribozyme is a good alternative to the previously used HDV ribozyme. The new HDV ribozyme is more efficient in some sequence contexts, and less efficient in other sequence contexts than the previously used HDV ribozyme. The smaller size makes the new HDV ribozyme a good alternative for transcript 3′-end processing.

During an adaptive immune response, B cells can change their surface immunoglobulins from IgM to IgG, IgE or IgA through a process called class switch recombination (CSR). Switching is preceded by inducible non-coding germline transcription (GLT) of the selected constant gene(s), which is largely controlled by a super-enhancer called the 3′ regulatory region (3′RR). Despite intense efforts, the precise mechanisms that regulate GLT are still elusive. In order to gain additional insights into these mechanisms, we analyzed GLT and CSR in mutant B cells carrying a duplication of the promoter of the α constant gene (Iα) downstream of 3′RR. Duplication of the Iα promoter affected differently GLT and CSR. While for most isotypes a drop in GLT was accompanied by a decrease in CSR, that was not the case for switching to IgA, which diminished despite unchanged GLT. Unexpectedly, there was no obvious effect on GLT and CSR to IgG3. Remarkably, specific stimuli that normally induce switching to IgG2b had contrasting effects in mutant B cells; Iγ2b was now preferentially responsive to the stimulus that induced Iα promoter. We propose that one mechanism underlying the induced 3′RR-mediated activation of GL promoters involves, at least in part, specific transcription factories.

S-adenosyl-L-methionine-dependent 2′-O-methylati-on of the 3′-terminal nucleotide plays important roles in biogenesis of eukaryotic small non-coding RNAs, such as siRNAs, miRNAs and Piwi-interacting RNAs (piRNAs). Here we demonstrate that, in contrast to Mg2+/Mn2+-dependent plant and bacterial homologues, the Drosophila DmHen1 and human HsHEN1 piRNA methyltransferases require cobalt cations for their enzymatic activity in vitro. We also show for the first time the capacity of the animal Hen1 to catalyse the transfer of a variety of extended chemical groups from synthetic analogues of the AdoMet cofactor onto a wide range (22–80 nt) of single-stranded RNAs permitting their 3′-terminal functionalization and labelling. Moreover, we provide evidence that deletion of a small C-terminal region of the DmHen1 protein further increases its modification efficiency and abolishes a modest 3′-terminal nucleotide bias observed for the full-length protein. Finally, we show that fluorophore-tagged ssRNA molecules are successfully detected in fluorescence resonance energy transfer assays both individually and in a total RNA mixture. The presented DmHen1-assisted RNA labelling provides a solid basis for developing novel chemo-enzymatic approaches for in vitro studies and in vivo monitoring of single-stranded RNA pools.

Data obtained from Illumina resequencing of 63 apple cultivars were used to obtain full-length S-RNase sequences using a strategy based on both alignment and de novo assembly of reads. The reproductive biology of apple is regulated by the S-RNase-based gametophytic self-incompatibility system, that is genetically controlled by the single, multi-genic and multi-allelic S locus. Resequencing of apple cultivars provided a huge amount of genetic data, that can be aligned to the reference genome in order to characterize variation to a genome-wide level. However, this approach is not immediately adaptable to the S-locus, due to some peculiar features such as the high degree of polymorphism, lack of colinearity between haplotypes and extensive presence of repetitive elements. In this study we describe a dedicated procedure aimed at characterizing S-RNase alleles from resequenced cultivars. The S-genotype of 63 apple accessions is reported; the full length coding sequence was determined for the 25 S-RNase alleles present in the 63 resequenced cultivars; these included 10 previously incomplete sequences (S 5 , S 6a , S 6b , S 8 , S 11 , S 23 , S 39 , S 46 , S 50 and S 58 ). Moreover, sequence divergence clearly suggests that alleles S 6a and S 6b , proposed to be neutral variants of the same alleles, should be instead considered different specificities. The promoter sequences have also been analyzed, highlighting regions of homology conserved among all the alleles.

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Pseudoautosomal region 1 (PAR1) contains SHOX, in addition to seven highly conserved non-coding DNA elements (CNEs) with cis-regulatory activity. Microdeletions involving SHOX exons 1-6a and/or the CNEs result in idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). Here, we report six rare copy-number variations (CNVs) in PAR1 identified through copy-number analyzes of 245 ISS/LWD patients and 15 unaffected individuals. The six CNVs consisted of three microduplications encompassing SHOX and some of the CNEs, two microduplications in the SHOX 3'-region affecting one or four of the downstream CNEs, and a microdeletion involving SHOX exon 6b and its neighboring CNE. The amplified DNA fragments of two SHOX-containing duplications were detected at chromosomal regions adjacent to the original positions. The breakpoints of a SHOX-containing duplication resided within Alu repeats. A microduplication encompassing four downstream CNEs was identified in an unaffected father-daughter pair, whereas the other five CNVs were detected in ISS patients. These results suggest that microduplications involving SHOX cause ISS by disrupting the cis-regulatory machinery of this gene and that at least some of microduplications in PAR1 arise from Alu-mediated non-allelic homologous recombination. The pathogenicity of other rare PAR1-linked CNVs, such as CNE-containing microduplications and exon 6b-flanking microdeletions, merits further investigation.

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 &gt, 0.05). Further subgroup stratification by ethnicity and weight did not lead to discovery of significant correlation (p &gt, 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 &gt, 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.

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.

Leptin has a pleiotropic effect on regulating appetite, energy metabolism, growth, reproduction, body composition and immunity. This property supports leptin and its receptor as candidate genes for evaluating genetic polymorphisms to associate with growth, milk yield and other economic traits. The aim of this study is to characterize the leptin receptor gene in Bubalus bubalis, to identify single-nucleotide polymorphism (SNP) sites in different coding and non-coding regions and to analyse potential associations between SNPs identified and the body measurements traits of growing buffalo heifers. A group of 64 animals were genotyped by direct sequencing and twenty-eight SNPs were detected. A sequence analysis revealed the presence of nine interesting SNPs in gene sequence. The association analysis of polymorphisms with the body measurements traits of growing buffalo heifers shows significant statistical effects on chest depth and sacrum height. Therefore according to the results obtained from this study, the leptin receptor gene appears to have potential effects on the body measurement traits of Bubalus bubalis.

Bacterial translation initiation is influenced by base pairing between the Shine-Dalgarno (SD) sequence in the 5′ UTR of mRNA and the anti-SD (aSD) sequence at the free 3′ end of the 16S rRNA (3′ TAIL) due to: 1) the SD/aSD sequence binding location and 2) SD/aSD binding affinity. In order to understand what makes an SD/aSD interaction optimal, we must define: 1) terminus of the 3′ TAIL and 2) extent of the core aSD sequence within the 3′ TAIL. Our approach to characterize these components in Escherichia coli and Bacillus subtilis involves 1) mapping the 3′ boundary of the mature 16S rRNA using high-throughput RNA sequencing (RNA-Seq), and 2) identifying the segment within the 3′ TAIL that is strongly preferred in SD/aSD pairing. Using RNA-Seq data, we resolve previous discrepancies in the reported 3′ TAIL in B. subtilis and recovered the established 3′ TAIL in E. coli. Furthermore, we extend previous studies to suggest that both highly and lowly expressed genes favor SD sequences with intermediate binding affinity, but this trend is exclusive to SD sequences that complement the core aSD sequences defined herein.

Twelve nucleotides located at the 3' end of viral genomic RNA (vRNA) are conserved among influenza A viruses (IAV) and have a promoter function. Hoffmann's 8-plasmid reverse genetics vector system introduced mutations at position 4, C nucleotide (C4) to U nucleotide (U4), of the 3' ends of neuraminidase (NA) and matrix (M) vRNAs of wild-type A/PR/8/34 (PR8). This resulted in a constellation of C4 and U4 vRNAs coding for low (polymerases) and relatively high (all others) copy number proteins, respectively. U4 has been reported to increase promoter activity in comparison to C4, but the constellation effect on the replication efficiency and pathogenicity of reverse genetics PR8 (rgPR8) has not been fully elucidated. In the present study, we generated 3 recombinant viruses with C4 in the NA and/or M vRNAs and rgPR8 by using reverse genetics and compared their pathobiological traits. The mutant viruses showed lower replication efficiency than rgPR8 due to the low transcription levels of NA and/or M genes. Furthermore, C4 in the NA and/or M vRNAs induced lower PR8 virus pathogenicity in BALB/c mice. The results suggest that the constellation of C4 and U4 among vRNAs may be one of the multigenic determinants of IAV pathogenicity.

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.The aim of this study was to evaluate PROM1 gene sequence variations in patients with macular dystrophy.This retrospective study evaluated variations in the PROM1 gene detected by next-generation sequencing test in patients with macular dystrophy and Stargardt disease.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.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.

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.

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.

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.

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.