Dissecting the basis for differential substrate specificity of ADAR1 and ADAR2.

Millions of adenosines are deaminated throughout the transcriptome by ADAR1 and/or ADAR2 at varying levels, raising the question of what are the determinants guiding substrate specificity and how these differ between the two enzymes. We monitor how secondary structure modulates ADAR2 vs ADAR1 substrate selectivity, on the basis of systematic probing of thousands of synthetic sequences transfected into cell lines expressing exclusively ADAR1 or ADAR2. Both enzymes induce symmetric, strand-specific editing, yet with distinct offsets with respect to structural disruptions: -26 nt for ADAR2 and -35 nt for ADAR1.

Exclusion of m6A from splice-site proximal regions by the exon junction complex dictates m6A topologies and mRNA stability.

N6-methyladenosine (m6A), a widespread destabilizing mark on mRNA, is non-uniformly distributed across the transcriptome, yet the basis for its selective deposition is unknown. Here, we propose that m6A deposition is not selective. Instead, it is exclusion based: m6A consensus motifs are methylated by default, unless they are within a window of ∼100 nt from a splice junction.

Cloning of DNA oligo pools for expression.

Oligo library pools are powerful tools for systematic investigation of genetic and transcriptomic machinery such as promoter function and gene regulation, non-coding RNAs, or RNA modifications. Here, we provide a detailed protocol for cloning DNA oligo pools made up of tens of thousands of different constructs, aiming to preserve the complexity of the pools. This system would be suitable for expression in cell lines and can be followed up by next-generation sequencing analysis.

Deciphering the principles of the RNA editing code via large-scale systematic probing.

Adenosine-to-inosine editing is catalyzed by ADAR1 at thousands of sites transcriptome-wide. Despite intense interest in ADAR1 from physiological, bioengineering, and therapeutic perspectives, the rules of ADAR1 substrate selection are poorly understood. Here, we used large-scale systematic probing of ∼2,000 synthetic constructs to explore the structure and sequence context determining editability.

Systematic interrogation of human promoters.

Despite much research, our understanding of the architecture and -regulatory elements of human promoters is still lacking. Here, we devised a high-throughput assay to quantify the activity of approximately 15,000 fully designed sequences that we integrated and expressed from a fixed location within the human genome. We used this method to investigate thousands of native promoters and preinitiation complex (PIC) binding regions followed by in-depth characterization of the sequence motifs underlying promoter activity, including core promoter elements and TF binding sites.

Unraveling the determinants of microRNA mediated regulation using a massively parallel reporter assay.

Despite extensive research, the sequence features affecting microRNA-mediated regulation are not well understood, limiting our ability to predict gene expression levels in both native and synthetic sequences. Here we employed a massively parallel reporter assay to investigate the effect of over 14,000 rationally designed 3' UTR sequences on reporter construct repression. We found that multiple factors, including microRNA identity, hybridization energy, target accessibility, and target multiplicity, can be manipulated to achieve a predictable, up to 57-fold, change in protein repression.

Sequence features of viral and human Internal Ribosome Entry Sites predictive of their activity.

Translation of mRNAs through Internal Ribosome Entry Sites (IRESs) has emerged as a prominent mechanism of cellular and viral initiation. It supports cap-independent translation of select cellular genes under normal conditions, and in conditions when cap-dependent translation is inhibited. IRES structure and sequence are believed to be involved in this process.

TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA via a predictable and conserved code.

Following synthesis, RNA can be modified with over 100 chemically distinct modifications, which can potentially regulate RNA expression post-transcriptionally. Pseudouridine (Ψ) was recently established to be widespread and dynamically regulated on yeast mRNA, but less is known about Ψ presence, regulation, and biogenesis in mammalian mRNA. Here, we sought to characterize the Ψ landscape on mammalian mRNA, to identify the main Ψ-synthases (PUSs) catalyzing Ψ formation, and to understand the factors governing their specificity toward selected targets.

Comparative genetics. Systematic discovery of cap-independent translation sequences in human and viral genomes.

To investigate gene specificity at the level of translation in both the human genome and viruses, we devised a high-throughput bicistronic assay to quantify cap-independent translation. We uncovered thousands of novel cap-independent translation sequences, and we provide insights on the landscape of translational regulation in both humans and viruses. We find extensive translational elements in the 3' untranslated region of human transcripts and the polyprotein region of uncapped RNA viruses.

Phosphorylation of the Drosophila melanogaster RNA-binding protein HOW by MAPK/ERK enhances its dimerization and activity.

Drosophila melanogaster Held Out Wings (HOW) is a conserved RNA-binding protein (RBP) belonging to the STAR family, whose closest mammalian ortholog Quaking (QKI) has been implicated in embryonic development and nervous system myelination. The HOW RBP modulates a variety of developmental processes by controlling mRNA levels and the splicing profile of multiple key regulatory genes; however, mechanisms regulating its activity in tissues have yet to be elucidated. Here, we link receptor tyrosine kinase (RTK) signaling to the regulation of QKI subfamily of STAR proteins, by showing that HOW undergoes phosphorylation by MAPK/ERK.

Tissue development and RNA control: "HOW" is it coordinated?

The regulation of developmental processes at the RNA level enables selective and rapid modulation of gene expression. Studies in model organisms revealed the essential contribution of the signal transduction and activation of RNA (STAR) family of RNA binding proteins to developmental processes. STAR proteins coordinate the proper timing of developmental events by delaying expression or altering the mRNA or protein levels of essential genes.

Post-transcriptional repression of the Drosophila midkine and pleiotrophin homolog miple by HOW is essential for correct mesoderm spreading.

The even spreading of mesoderm cells in the Drosophila embryo is essential for its proper patterning by ectodermally derived signals. In how germline clone embryos, defects in mesoderm spreading lead to a partial loss of dorsal mesoderm derivatives. HOW is an RNA-binding protein that is thought to regulate diverse mRNA targets.

Dissection of the target specificity of the RNA-binding protein HOW reveals dpp mRNA as a novel HOW target.

Regulation of RNA metabolism plays a major role in controlling gene expression during developmental processes. The Drosophila RNA-binding protein Held out wing (HOW), regulates an array of developmental processes in embryonic and adult growth. We have characterized the primary sequence and secondary structural requirements for the HOW response element (HRE), and show that this site is necessary and sufficient for HOW binding.

DCX's phosphorylation by not just another kinase (JNK).

The mammalian cortex is generally subdivided into six organized layers, which are formed during development in an organized fashion. This organized cortical layering is disrupted in case of mutations in the doublecortin (DCX) gene. DCX is a Microtubule Associated Protein (MAP).