The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA.

Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N(6)-methyladenosine (m(6)A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N(1)-methyladenosine (m(1)A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m(1)A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m(1)A in promoting translation of methylated mRNA.

Genome-wide analysis of fitness data and its application to improve metabolic models.

BACKGROUND: Synthetic biology and related techniques enable genome scale high-throughput investigation of the effect on organism fitness of different gene knock-downs/outs and of other modifications of genomic sequence. RESULTS: We develop statistical and computational pipelines and frameworks for analyzing high throughput fitness data over a genome scale set of sequence variants. Analyzing data from a high-throughput knock-down/knock-out bacterial study, we investigate differences and determinants of the effect on fitness in different conditions. Comparing fitness vectors of genes, across tens of conditions, we observe that fitness consequences strongly depend on genomic location and more weakly depend on gene sequence similarity and on functional relationships. In analyzing promoter sequences, we identified motifs associated with conditions studied in bacterial media such as Casaminos, D-glucose, Sucrose, and other sugars and amino-acid sources. We also use fitness data to infer genes associated with orphan metabolic reactions in the iJO1366 E. coli metabolic model. To do this, we developed a new computational method that integrates gene fitness and gene expression profiles within a given reaction network neighborhood to associate this reaction with a set of genes that potentially encode the catalyzing proteins. We then apply this approach to predict candidate genes for 107 orphan reactions in iJO1366. Furthermore - we validate our methodology with known reactions using a leave-one-out approach. Specifically, using top-20 candidates selected based on combined fitness and expression datasets, we correctly reconstruct 39.7% of the reactions, as compared to 33% based on fitness and to 26% based on expression separately, and to 4.02% as a random baseline. Our model improvement results include a novel association of a gene to an orphan cytosine nucleosidation reaction. CONCLUSION: Our pipeline for metabolic modeling shows a clear benefit of using fitness data for predicting genes of orphan reactions. Along with the analysis pipelines we developed, it can be used to analyze similar high-throughput data.

e23D: database and visualization of A-to-I RNA editing sites mapped to 3D protein structures.

UNLABELLED: e23D, a database of A-to-I RNA editing sites from human, mouse and fly mapped to evolutionary related protein 3D structures, is presented. Genomic coordinates of A-to-I RNA editing sites are converted to protein coordinates and mapped onto 3D structures from PDB or theoretical models from ModBase. e23D allows visualization of the protein structure, modeling of recoding events and orientation of the editing with respect to nearby genomic functional sites from databases of disease causing mutations and genomic polymorphism. AVAILABILITY AND IMPLEMENTATION: http://www.sheba-cancer.org.il/e23D CONTACT: oz.solomon@live.biu.ac.il or Eran.Eyal@sheba.health.gov.il.

G23D: Online tool for mapping and visualization of genomic variants on 3D protein structures.

BACKGROUND: Evaluation of the possible implications of genomic variants is an increasingly important task in the current high throughput sequencing era. Structural information however is still not routinely exploited during this evaluation process. The main reasons can be attributed to the partial structural coverage of the human proteome and the lack of tools which conveniently convert genomic positions, which are the frequent output of genomic pipelines, to proteins and structure coordinates. RESULTS: We present G23D, a tool for conversion of human genomic coordinates to protein coordinates and protein structures. G23D allows mapping of genomic positions/variants on evolutionary related (and not only identical) protein three dimensional (3D) structures as well as on theoretical models. By doing so it significantly extends the space of variants for which structural insight is feasible. To facilitate interpretation of the variant consequence, pathogenic variants, functional sites and polymorphism sites are displayed on protein sequence and structure diagrams alongside the input variants. G23D also provides modeling of the mutant structure, analysis of intra-protein contacts and instant access to functional predictions and predictions of thermo-stability changes. G23D is available at http://www.sheba-cancer.org.il/G23D . CONCLUSIONS: G23D extends the fraction of variants for which structural analysis is applicable and provides better and faster accessibility for structural data to biologists and geneticists who routinely work with genomic information.

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR).

Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery.

Characterizing of functional human coding RNA editing from evolutionary, structural, and dynamic perspectives.

A-to-I RNA editing has been recently shown to be a widespread phenomenon with millions of sites spread in the human transcriptome. However, only few are known to be located in coding sequences and modify the amino acid sequence of the protein product. Here, we used high-throughput data, variant prediction tools, and protein structural information in order to find structural and functional preferences for coding RNA editing. We show that RNA editing has a unique pattern of amino acid changes characterized by enriched stop-to-tryptophan changes, positive-to-neutral and neutral-to-positive charge changes. RNA editing tends to have stronger structural effect than equivalent A-to-G SNPs but weaker effect than random A-to-G mutagenesis events. Sites edited at low level tend to be located at conserved positions with stronger predicted deleterious effect on proteins comparing to sites edited at high frequencies. Lowly edited sites tend to destabilize the protein structure and affect amino acids with larger number of intra-molecular contacts. Still, some highly edited sites are predicted also to prominently affect structure and tend to be located at critical positions of the protein matrix and are likely to be functionally important. Using our pipeline, we identify and discuss several novel putative functional coding changing editing sites in the genes COPA (I164V), GIPC1 (T62A), ZN358 (K382R), and CCNI (R75G).

AL101, a gamma-secretase inhibitor, has potent antitumor activity against adenoid cystic carcinoma with activated NOTCH signaling.

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland malignancy with limited treatment options for recurrent or metastatic disease. Due to chemotherapy resistance and lack of targeted therapeutic approaches, current treatment options for the localized disease are limited to surgery and radiation, which fails to prevent locoregional recurrences and distant metastases in over 50% of patients. Approximately 20% of patients with ACC carry NOTCH-activating mutations that are associated with a distinct phenotype, aggressive disease, and poor prognosis. Given the role of NOTCH signaling in regulating tumor cell behavior, NOTCH inhibitors represent an attractive potential therapeutic strategy for this subset of ACC. AL101 (osugacestat) is a potent γ-secretase inhibitor that prevents activation of all four NOTCH receptors. While this investigational new drug has demonstrated antineoplastic activity in several preclinical cancer models and in patients with advanced solid malignancies, we are the first to study the therapeutic benefit of AL101 in ACC. Here, we describe the antitumor activity of AL101 using ACC cell lines, organoids, and patient-derived xenograft models. Specifically, we find that AL101 has potent antitumor effects in in vitro and in vivo models of ACC with activating NOTCH1 mutations and constitutively upregulated NOTCH signaling pathway, providing a strong rationale for evaluation of AL101 in clinical trials for patients with NOTCH-driven relapsed/refractory ACC.

Molecular harvesting with electroporation for tissue profiling.

Recent developments in personalized medicine are based on molecular measurement steps that guide personally adjusted medical decisions. A central approach to molecular profiling consists of measuring DNA, RNA, and/or proteins in tissue samples, most notably in and around tumors. This measurement yields molecular biomarkers that are potentially predictive of response and of tumor type. Current methods in cancer therapy mostly use tissue biopsy as the starting point of molecular profiling. Tissue biopsies involve a physical resection of a small tissue sample, leading to localized tissue injury, bleeding, inflammation and stress, as well as to an increased risk of metastasis. Here we developed a technology for harvesting biomolecules from tissues using electroporation. We show that tissue electroporation, achieved using a combination of high-voltage short pulses, 50 pulses 500 V cm-1, 30 µs, 1 Hz, with low-voltage long pulses 50 pulses 50 V cm-1, 10 ms, delivered at 1 Hz, allows for tissue-specific extraction of RNA and proteins. We specifically tested RNA and protein extraction from excised kidney and liver samples and from excised HepG2 tumors in mice. Further in vivo development of extraction methods based on electroporation can drive novel approaches to the molecular profiling of tumors and of tumor environment and to related diagnosis practices.

Synthetic 5' UTRs Can Either Up- or Downregulate Expression upon RNA-Binding Protein Binding.

The construction of complex gene-regulatory networks requires both inhibitory and upregulatory modules. However, the vast majority of RNA-based regulatory "parts" are inhibitory. Using a synthetic biology approach combined with SHAPE-seq, we explored the regulatory effect of RNA-binding protein (RBP)-RNA interactions in bacterial 5' UTRs. By positioning a library of RNA hairpins upstream of a reporter gene and co-expressing them with the matching RBP, we observed a set of regulatory responses, including translational stimulation, translational repression, and cooperative behavior. Our combined approach revealed three distinct states in vivo: in the absence of RBPs, the RNA molecules can be found in either a molten state that is amenable to translation or a structured phase that inhibits translation. In the presence of RBPs, the RNA molecules are in a semi-structured phase with partial translational capacity. Our work provides new insight into RBP-based regulation and a blueprint for designing complete gene-regulatory circuits at the post-transcriptional level.

An in Vivo Binding Assay for RNA-Binding Proteins Based on Repression of a Reporter Gene.

We study translation repression in bacteria by engineering a regulatory circuit that functions as a binding assay for RNA binding proteins (RBP) in vivo. We do so by inducing expression of a fluorescent protein-RBP chimera, together with encoding its binding site at various positions within the ribosomal initiation region (+11-13 nt from the AUG) of a reporter module. We show that when bound by their cognate RBPs, the phage coat proteins for PP7 (PCP) and Qß (QCP), strong repression is observed for all hairpin positions within the initiation region. Yet, a sharp transition to no-effect is observed when positioned in the elongation region, at a single-nucleotide resolution. Employing in vivo Selective 2'-hydroxyl acylation analyzed by primer extension followed by sequencing (SHAPE-seq) for a representative construct, established that in the translationally active state the mRNA molecule is nonstructured, while in the repressed state a structured signature was detected. We then utilize this regulatory phenomena to quantify the binding affinity of the coat proteins of phages MS2, PP7, GA, and Qß to 14 cognate and noncognate binding sites in vivo. Using our circuit, we demonstrate qualitative differences between in vitro to in vivo binding characteristics for various variants when comparing to past studies. Furthermore, by introducing a simple mutation to the loop region for the Qß-wt site, MCP binding is abolished, creating the first high-affinity QCP site that is completely orthogonal to MCP. Consequently, we demonstrate that our hybrid transcriptional-post-transcriptional circuit can be utilized as a binding assay to quantify RNA-RBP interactions in vivo.

A Synthetic Oligo Library and Sequencing Approach Reveals an Insulation Mechanism Encoded within Bacterial σ54 Promoters.

We use an oligonucleotide library of >10,000 variants to identify an insulation mechanism encoded within a subset of σ54 promoters. Insulation manifests itself as reduced protein expression for a downstream gene that is expressed by transcriptional readthrough. It is strongly associated with the presence of short CT-rich motifs (3-5 bp), positioned within 25 bp upstream of the Shine-Dalgarno (SD) motif of the silenced gene. We provide evidence that insulation is triggered by binding of the ribosome binding site (RBS) to the upstream CT-rich motif. We also show that, in E. coli, insulator sequences are preferentially encoded within σ54 promoters, suggesting an important regulatory role for these sequences in natural contexts. Our findings imply that sequence-specific regulatory effects that are sparsely encoded by short motifs may not be easily detected by lower throughput studies. Such sequence-specific phenomena can be uncovered with a focused oligo library (OL) design that mitigates sequence-related variance, as exemplified herein.

RNA editing by ADAR1 leads to context-dependent transcriptome-wide changes in RNA secondary structure.

Adenosine deaminase acting on RNA 1 (ADAR1) is the master RNA editor, catalyzing the deamination of adenosine to inosine. RNA editing is vital for preventing abnormal activation of cytosolic nucleic acid sensing pathways by self-double-stranded RNAs. Here we determine, by parallel analysis of RNA secondary structure sequencing (PARS-seq), the global RNA secondary structure changes in ADAR1 deficient cells. Surprisingly, ADAR1 silencing resulted in a lower global double-stranded to single-stranded RNA ratio, suggesting that A-to-I editing can stabilize a large subset of imperfect RNA duplexes. The duplexes destabilized by editing are composed of vastly complementary inverted Alus found in untranslated regions of genes performing vital biological processes, including housekeeping functions and type-I interferon responses. They are predominantly cytoplasmic and generally demonstrate higher ribosomal occupancy. Our findings imply that the editing effect on RNA secondary structure is context dependent and underline the intricate regulatory role of ADAR1 on global RNA secondary structure.

Mutagen-specific mutation signature determines global microRNA binding.

Micro-RNAs (miRNAs) are small non-coding RNAs that regulate gene products at the post-transcriptional level. It is thought that loss of cell regulation by miRNAs supports cancer development. Based on whole genome sequencing of a melanoma tumor, we predict, using three different computational algorithms, that the melanoma somatic mutations globally reduce binding of miRNAs to the mutated 3'UTRs. This phenomenon reflects the nature of the characteristic UV-induced mutation, C-to-T. Furthermore, we show that seed regions are enriched with Guanine, thus rendering miRNAs prone to reduced binding to UV-mutated 3'UTRs. Accordingly, mutation patterns in non UV-induced malignancies e.g. lung cancer and leukemia do not yield similar predictions. It is suggested that UV-induced disruption of miRNA-mediated gene regulation plays a carcinogenic role. Remarkably, dark-skinned populations have significantly higher GC content in 3'UTR SNPs than light-skinned populations, which implies on evolutionary pressure to preserve regulation by trans-acting oligonucleotides under conditions with excess UV radiation.