Genome-wide detection of novel non-coding RNAs in S. cerevisiae by modulating an RNase P pathway through the depletion of a component RPP1. Nearly 400,000 36-mer oligonucleotide probes tiling the entire yeast genome including the mitochondrial chromosome with an average gap of 10 bases between two consecutive probes were synthesized on glass slides using a mask-less array synthesizer. RNA samples for hybridizing to the arrays were extracted from a conditional lethal allele of S. cerevisiae created by placing the RPP1 gene under control of GAL10 promoter. It allowed the expression of RPP1 in galactose-containing culture medium but suppressed its expression in glucose-containing medium. A wild-type isogenic strain was used as a control. Both strains were initially grown in galactose-containing medium and subsequently transferred and resuspended into glucose-containing medium. Eight arrays were hybridized with RNA extracted from the Rpp1-depleted cells at 0 4 7 12 16 21 30h and the control cell at 30h after initial transfer to glucose-containing medium.

Background: The germ cell nuclear factor (GCNF, also known as retinoid acid receptor-related testis-associated receptor, neuronal cell nuclear receptor or NR6A1) is an orphan receptor in the nuclear receptor superfamily found in mammals, amphibians and fish. The mouse Gcnf gene is expressed in the placenta and the developing nervous system and germ cells, and responds to retinoic acid. Results: We have defined the intron-exon structure of the mouse Gcnf gene and found that it contains 11 exons. Exons 1-4 encode the 75 amino acid amino-terminal domain and exon 4 also encodes the core DNA-binding domain. The carboxy-terminal extension is encoded by exon 5, exons 6 and 7 encode the hinge region, and exons 7-11 encode the putative ligand-binding domain. Unusually, the two zinc-finger motifs in the DNA-binding domain are encoded by separate exons. Conclusions: The protein-coding region of GCNF is contained in 11 exons. The genomic structure of this nuclear receptor gene will be useful for further studies.

Background: mRNA interactions with each other and other signaling molecules define different biological pathways and functions. Researchers have been investigating various tools to analyze these types of interactions. In particular gene co-expression network methods have proved useful in finding and analyzing these molecular interactions. Many different analytical pipelines to identify these interactions networks have been proposed with the aim of identifying an optimal partition of the network where the individual modules are neither too small to make any general inference or too large to be biologically interpretable. Results: In this study we propose a new pipeline to perform gene co-expression network analysis. The proposed pipeline uses WGCNA a widely used software to perform different aspects of gene co-expression network analysis and modularity maximization algorithm to analyze novel RNA-Seq data to understand the effects of low-dose 56Fe ion irradiation on the formation of hepatocellular carcinoma in mice. The network results along with experimental validation show that using WGCNA combined with Modularity provide a more biologically interpretable network in our dataset. Our pipeline showed better performance than the existing clustering algorithm in WGCNA in finding modules and identified a module with mitochondrial subunits that are supported by mitochondrial complex assay. Conclusions: We present a pipeline that can reduce the problem of parameter selection with the existing algorithm in WGCNA for comparable RNA-Seq datasets which may assist in future research to discover novel mRNA interactions and their downstream molecular effects. C57BL16 males were placed into 2 treatment groups and received the following irradiation treatments at Brookhaven National Laboratories (Long Island NY): 600 MeV/n 56Fe (0.2 Gy) and no irradiation. Left liver lobes were collected at 30 60 120 270 and 360 days post-irradiation flash frozen and stored at -80 xc2 xb0C until they could be processed for RNA-Seq. Livers were sampled by taking two 40-micron thick slices using a cryotome at -20 xc2 xb0C. This allowed multiple sampling of the tissue without the tissue going through multiple freeze/thaw cycles. Total RNA was isolated from the liver slices using RNAqueousTM Total RNA Isolation Kit (ThermoFisher Scientific Waltham MA) and rRNA was removed via Ribo-ZeroTM rRNA Removal Kit (Illumina San Diego CA) prior to library preparation with the Illumina TruSeq RNA Library kit. Samples were sequenced in a paired-end 50 base format on an Illumina HiSeq 1500. Reads were aligned to the mouse GRCm38 reference genome using the STAR alignment program version 2.5.3a with the recommended ENCODE options. The -quantMode GeneCounts option was used to obtain read counts per gene based on the Gencode release M14 annotation file. Total number of reads used in analysis varies between 23-35 millions of reads.

Background: Germ-cell nuclear factor (GCNF, NR6AI) is an orphan nuclear receptor. Its expression pattern suggests it functions during embryogenesis, in the placenta and in germ-cell development. Mouse GCNF cDNA codes for a protein of 495 amino acids, whereas the four reported human cDNA variants code for proteins of 454 to 480 amino acids. Apart from this size difference, there is sequence conservation of up to 98.7%. To elucidate the genomic structure that gives rise to the different human GCNF mRNAs, the sequence information of the human GCNF locus is compared to the previously reported structure of the mouse locus. Results: The genomic structures of the mouse and human GCNF genes are highly conserved. The comparison reveals that the shorter human protein results from skipping the 45 base-pair third exon. Three different human isoforms - GCNF-1, GCNF-2a and GCNF-2b - are generated by differential usage of alternative splice acceptor sites of the fourth and the seventh exon. Conclusion: By homology with the mouse gene, 11 GCNF coding exons can be defined on human chromosome 9. All human GCNF cDNAs identified so far are, however, derived from mRNAs generated by splicing the fourth to the second exon. Although the genomic sequence is highly conserved, the analysis suggests that alternative splicing generates a higher complexity of human GCNF isoforms compared with the situation in the mouse.

Background The availability of complete genome sequences enables all the members of a gene family to be identified without limitations imposed by temporal, spatial or quantitative aspects of mRNA expression. Using the nearly completed human genome sequence, we combined in silico and experimental approaches to define the complete human nuclear receptor (NR) set. This information was used to carry out a comparative genomic study of the NR superfamily. Results Our analysis of the human genome identified two novel NR sequences. Both these contained stop codons within the coding regions, indicating that both are pseudogenes. One (HNF4 γ-related) contained no introns and expressed no detectable mRNA, whereas the other (FXR-related) produced mRNA at relatively high levels in testis. If translated, the latter is predicted to encode a short, non-functional protein. Our analysis indicates that there are fewer than 50 functional human NRs, dramatically fewer than in Caenorhabditis elegans and about twice as many as in Drosophila. Using the complete human NR set we made comparisons with the NR sets of C. elegans and Drosophila. Searches for the >200 NRs unique to C. elegans revealed no human homologs. The comparative analysis also revealed a Drosophila member of NR subfamily NR3, confirming an ancient metazoan origin for this subfamily. Conclusions This work provides the basis for new insights into the evolution and functional relationships of NR superfamily members.

The 5'-terminal cap structure plays an important role in many aspects of mRNA metabolism. Capping enzymes encoded by viruses and pathogenic fungi are attractive targets for specific inhibitors. There is a large body of experimental data on viral and cellular methyltransferases (MTases) that carry out guanine-N7 (cap 0) methylation, including results of extensive mutagenesis. However, a crystal structure is not available and cap 0 MTases are too diverged from other MTases of known structure to allow straightforward homology-based interpretation of these data.

Background Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date. Results Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein. Conclusions We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed.

Background Alternative DNA conformations are of particular interest as potential signals to mark important sites on the genome. The structural variability of CA microsatellites is particularly pronounced; these are repetitive poly(CA) · poly(TG) DNA sequences spread in all eukaryotic genomes as tracts of up to 60 base pairs long. Many in vitro studies have shown that the structure of poly(CA) · poly(TG) can vary markedly from the classical right handed DNA double helix and adopt diverse alternative conformations. Here we have studied the mechanism of formation and the structure of an alternative DNA structure, named Form X, which was observed previously by polyacrylamide gel electrophoresis of DNA fragments containing a tract of the CA microsatellite poly(CA) · poly(TG) but had not yet been characterized. Results Formation of Form X was found to occur upon reassociation of the strands of a DNA fragment containing a tract of poly(CA) · poly(TG), in a process strongly stimulated by the nuclear proteins HMG1 and HMG2. By inserting Form X into DNA minicircles, we show that the DNA strands do not run fully side by side but instead form a DNA knot. When present in a closed DNA molecule, Form X becomes resistant to heating to 100°C and to alkaline pH. Conclusions Our data strongly support a model of Form X consisting in a DNA loop at the base of which the two DNA duplexes cross, with one of the strands of one duplex passing between the strands of the other duplex, and reciprocally, to form a semicatenated DNA junction also called a DNA hemicatenane.

Background: Programmed DNA-reorganization and DNA-elimination events take place frequently during cellular differentiation. An extreme form of such processes, involving DNA reorganization, DNA elimination and DNA fragmentation, is found during macronuclear differentiation in hypotrichous ciliates. Ciliated protozoa can therefore serve as a model system to analyze the molecular basis of these processes during cellular differentiation in eukaryotic cells. Results: Using a biological approach to identify cis-acting sequences involved in DNA fragmentation, we show that in the hypotrichous ciliate Stylonychia lemnae sequences required for specific DNA processing are localized in the 3'- and the 5'-subtelomeric regions of the macronuclear precursor sequence. They can be present at various positions in the two subtelomeric regions, and an interaction between the two regions seems to occur. Sequence comparison revealed a consensus inverted repeat in both subtelomeric regions that is almost identical to the putative Euplotes chromosome breakage sequence (E-Cbs), also identified by sequence comparison. When this sequence was mutagenized, a processed product could no longer be detected, demonstrating that the sequence plays a crucial role in DNA processing. By injecting a construct into the developing macronucleus, which exclusively contains the subtelomeric regions of the Stylonychia αl-tubulin gene, we show that subtelomeric regions are not only required but are also sufficient for DNA processing in Stylonychia. Conclusions: Our results indicate that an inverted repeat with the core sequence 5'-TGAA present in both subtelomeric regions acts as a Cbs in Stylonychia. The results allow us to propose a mechanistic model for DNA processing in this ciliate.

Background Ribose 2'-O-methylation, the most common nucleotide modification in mammalian rRNA, is directed by the C/D box small nucleolar RNAs (snoRNAs). Thus far, more than fifty putative human rRNA methylation guide snoRNAs have been identified. For nine of these snoRNAs, the respective ribose methylations in human 28S rRNA have been only presumptive. Results In this study, the methylation state of human 28S rRNA in the positions predicted by the snoRNAs U21, U26, U31, U48, U50, U73, U74, U80 and U81 was assessed using reverse transcription-based methods and several novel 2'-O-methylations were localized. Conclusions Seven novel ribose 2'-O-methylated residues (Am389, Am391, Gm1604, Gm1739, Gm2853, Cm3810, Gm4156, predicted by snoRNAs U26, U81, U80, U73, U50, U74 and U31, respectively) have been localized in human 28S rRNA. The total number of 2'-O-methylations in human rRNA is not yet known.