The RNAmute web server for the mutational analysis of RNA secondary structures

RNA mutational analysis at the secondary-structure level can be useful to a wide-range of biological applications. It can be used to predict an optimal site for performing a nucleotide mutation at the single molecular level, as well as to analyze basic phenomena at the systems level. For the former, as more sequence modification experiments are performed that include site-directed mutagenesis to find and explore functional motifs in RNAs, a pre-processing step that helps guide in planning the experiment becomes vital. For the latter, mutations are generally accepted as a central mechanism by which evolution occurs, and mutational analysis relating to structure should gain a better understanding of system functionality and evolution. In the past several years, the program RNAmute that is structure based and relies on RNA secondary-structure prediction has been developed for assisting in RNA mutational analysis. It has been extended from single-point mutations to treat multiple-point mutations efficiently by initially calculating all suboptimal solutions, after which only the mutations that stabilize the suboptimal solutions and destabilize the optimal one are considered as candidates for being deleterious. The RNAmute web server for mutational analysis is available at http://www.cs.bgu.ac.il/~xrnamute/XRNAmute

Multi-tissue integrative analysis of personal epigenomes

Evaluating the impact of genetic variants on transcriptional regulation is a central goal in biological science that has been constrained by reliance on a single reference genome. To address this, we constructed phased, diploid genomes for four cadaveric donors (using long-read sequencing) and systematically charted noncoding regulatory elements and transcriptional activity across more than 25 tissues from these donors. Integrative analysis revealed over a million variants with allele-specific activity, coordinated, locus-scale allelic imbalances, and structural variants impacting proximal chromatin structure. We relate the personal genome analysis to the ENCODE encyclopedia, annotating allele- and tissue-specific elements that are strongly enriched for variants impacting expression and disease phenotypes. These experimental and statistical approaches, and the corresponding EN-TEx resource, provide a framework for personalized functional genomics

Additional file 3: Figure S3. of A streamlined tethered chromosome conformation capture protocol

Comparison between RTCC experimental data (this work; black lines), Hi-C data from Crane et al. (2015; magenta lines); and relative representation in anti-LEM2 ChIP-chip data [28]. The curves were obtained by running the ICE pipeline [38] on our N2 dataset (N2 DpnII GSM2041038- SRR3105476) and on Crane et al. dataset (GSM1556154 - SRR1665087), as implemented in [ https://bitbucket.org/mirnylab/hiclib ], downloaded on Dec 1, 2015. To obtain the first Eigen Vector values, representing compartments along the chromosome axis, we have followed the tutorial from [ https://bitbucket.org/mirnylab/hiclib ], using the binnedData class function doEig(numPCs = 1). To inspect the correlation to LEM-2 binding compartments we added LEM-2 binding data [28] (MA2C normalized log2 ratio of ChIP signal over control), lifted from the ce4 genome assembly to the ce10 assembly, and averaged in 50KB bins. (PDF 46 kb

Additional file 2: Figure S2. of A streamlined tethered chromosome conformation capture protocol

Correlation between N2 DpnII experimental data and data from Crane et al. [22]. The 50KB chromatin contact matrix constructed using N2 young adults treated with DpnII restriction enzyme data (GSM2041038- SRR3105476) was compared with 50KB resolution chromatin contacts matrix constructed using the Crane et al. data (GSM1556154 - SRR1665087) from [22]. The total number of paired-ended 37X2 reads in our dataset was 88,466,514, while Crane et al. provide a total of 115,983,178 paired-ended 100X2 reads was. In order to build the chromatin contacts matrix we used the ICE pipeline [38] iterative mapping implementation from [ https://bitbucket.org/mirnylab/hiclib ], starting from 21nt up to 37nt in increments of 8. The number of detected Hi-C valid pairs in our dataset was 18,779,498 , consisting of 4,542,078 inter-chromosomal contacts and 14,237,420 intra-chromosomal contacts. In Crane’s dataset the number of valid Hi-C pairs was 59,200,047, consisting of 6,457,271 inter-chromosomal contacts and 52,742,776 intra-chromosomal contacts. Similarly to Additional file 1: Figure S1, any contacts between any location on chromosome I and region containing rRNA on chromosome I (the bin 15,050,000-end of chromosome I) are colored in green. Contacts between genomic loci in adjacent regions (up to 100KB apart are colored in purple). Versions of software used for analysis are as follows: Bowtie2-2.2.6 [39], and mirnylib/hiclib [ https://bitbucket.org/mirnylab/hiclib ] downloaded on December 1, 2015. Slight differences in aligned read counts from Tables 2 and 3 reflect updates in alignment software in the concerted package compared to the legacy versions used in Tables 2 and 3. (PDF 1132 kb

Preferential translation of Hsp83 in Leishmania requires a thermosensitive polypyrimidine-rich element in the 3′ UTR and involves scanning of the 5′ UTR

Heat shock proteins (HSPs) provide a useful system for studying developmental patterns in the digenetic Leishmania parasites, since their expression is induced in the mammalian life form. Translation regulation plays a key role in control of protein coding genes in trypanosomatids, and is directed exclusively by elements in the 3′ untranslated region (UTR). Using sequential deletions of the Leishmania Hsp83 3′ UTR (888 nucleotides [nt]), we mapped a region of 150 nt that was required, but not sufficient for preferential translation of a reporter gene at mammalian-like temperatures, suggesting that changes in RNA structure could be involved. An advanced bioinformatics package for prediction of RNA folding (UNAfold) marked the regulatory region on a highly probable structural arm that includes a polypyrimidine tract (PPT). Mutagenesis of this PPT abrogated completely preferential translation of the fused reporter gene. Furthermore, temperature elevation caused the regulatory region to melt more extensively than the same region that lacked the PPT. We propose that at elevated temperatures the regulatory element in the 3′ UTR is more accessible to mediators that promote its interaction with the basal translation components at the 5′ end during mRNA circularization. Translation initiation of Hsp83 at all temperatures appears to proceed via scanning of the 5′ UTR, since a hairpin structure abolishes expression of a fused reporter gene

Intricate and Cell Type-Specific Populations of Endogenous Circular DNA (eccDNA) in Caenorhabditis elegans and Homo sapiens

Investigations aimed at defining the 3D configuration of eukaryotic chromosomes have consistently encountered an endogenous population of chromosome-derived circular genomic DNA, referred to as extrachromosomal circular DNA (eccDNA). While the production, distribution, and activities of eccDNAs remain understudied, eccDNA formation from specific regions of the linear genome has profound consequences on the regulatory and coding capabilities for these regions. Here, we define eccDNA distributions in Caenorhabditis elegans and in three human cell types, utilizing a set of DNA topology-dependent approaches for enrichment and characterization. The use of parallel biophysical, enzymatic, and informatic approaches provides a comprehensive profiling of eccDNA robust to isolation and analysis methodology. Results in human and nematode systems provide quantitative analysis of the eccDNA loci at both unique and repetitive regions. Our studies converge on and support a consistent picture, in which endogenous genomic DNA circles are present in normal physiological states, and in which the circles come from both coding and noncoding genomic regions. Prominent among the coding regions generating DNA circles are several genes known to produce a diversity of protein isoforms, with mucin proteins and titin as specific examples