Biology subjects

Nir, R.

Publications and source records attributed to Nir, R..

5 recordsLinked to original sources

bioRxiv2024

A sequence-specific RNA acetylation catalyst

N4-acetylcytidine (ac4C) is a ubiquitous RNA modification incorporated by cytidine acetyltransferase enzymes. Here, we report the biochemical characterization of Thermococcus kodakarensis Nat10 (TkNat10), an RNA acetyltransferase involved in archaeal thermotolerance. We demonstrate that TkNat10s catalytic activity is critical for T. kodakarensis fitness at elevated temperatures. Unlike eukaryotic homologs, TkNat10 exhibits robust stand-alone activity, modifying diverse RNA substrates in a temperature, ATP, and acetyl-CoA-dependent manner. Transcriptome-wide analysis reveals TkNat10 preferentially modifies unstructured RNAs containing a 5-CCG-3 consensus sequence. Using a high-throughput mutagenesis approach, we define sequence and structural determinants of TkNat10 substrate recognition. We find TkNat10 can be engineered to use non-native acyl-CoA donors, providing insight into its cofactor specificity. Finally, we demonstrate TkNat10s utility for site-specific acetylation of RNA oligonucleotides, enabling analysis of ac4C-dependent RNA-protein interactions. Our findings establish a framework for understanding archaeal RNA acetylation and a new tool for studying the functional consequences of ac4C in diverse RNA contexts.

biochemistry↗

bioRxiv2024

Passive shaping of intra- and intercellular m6A dynamics via mRNA metabolism

m6A is the most widespread mRNA modification and is primarily implicated in controlling mRNA stability. Fundamental questions pertaining to m6A are the extent to which it is dynamically modulated within cells and across stimuli, and the forces underlying such modulation. Prior work has focused on investigating active mechanisms governing m6A levels, such as recruitment of m6A writers or erasers leading to either global or site-specific modulation. Here, we propose that changes in m6A levels across subcellular compartments and biological trajectories may result from passive changes in gene-level mRNA metabolism. To predict the intricate interdependencies between m6A levels, mRNA localization, and mRNA decay, we establish a differential model m6ADyn encompassing mRNA transcription, methylation, export, and m6A-dependent and independent degradation. We validate the predictions of m6ADyn in the context of intracellular m6A dynamics, where m6ADyn predicts associations between relative mRNA localization and m6A levels, which we experimentally confirm. We further explore m6ADyn predictions pertaining to changes in m6A levels upon controlled perturbations of mRNA metabolism, which we also experimentally confirm. Finally, we demonstrate the relevance of m6ADyn in the context of cellular heat stress response, where genes subjected to altered mRNA product and export also display predictable changes in m6A levels, consistent with m6ADyn predictions. Our findings establish a framework for dissecting m6A dynamics and suggest the role of passive dynamics in shaping m6A levels in mammalian systems.

systems biology↗

bioRxiv2023

Dissecting the basis for differential substrate specificity of ADAR1 and ADAR2

Millions of adenosines are deaminated throughout the transcriptome by ADAR1 and ADAR2, modulating double-stranded RNA (dsRNA) immunogenicity and recoding mRNA. The high variability in the susceptibility of different adenosines to editing begs the question of what are the determinants of substrate specificity. Here, we systematically monitor how secondary structure modulates ADAR2 vs ADAR1 substrate selectivity, on the basis of systematic probing of thousands of synthetic sequences transfected into ADAR1-deleted cell lines exogenously expressing either ADAR2 or ADAR1. In both cases, structural disruptions gave rise to symmetric, strand-specific induced editing at a fixed offset, but of varying length: -26 nt for ADAR2, and -35 nt for ADAR1. We dissect the basis for the differences in offset between ADAR1 and ADAR2 via diverse mutants, domain-swaps, and ADAR evolutionary homologs, and reveal that it is encoded by the differential RNA binding domain architecture. We demonstrate that this offset-enhanced editing can allow an improved design of ADAR2-recruiting therapeutics, with proof-of-concept experiments suggestive of increased on-target and potentially decreased off-target editing. Our findings provide novel insight into the determinants guiding ADAR2 substrate selectivity and into the roles of the RNA binding domains of ADAR1 and ADAR2 in mediating differential targeting, and should facilitate the design of improved ADAR-recruiting therapeutics.

biochemistry↗

bioRxiv2019

Deciphering the ‘m6A code’ via quantitative profiling of m6A at single-nucleotide resolution

N6-methyladenosine (m6A) is the most abundant modification on mRNA, and is implicated in critical roles in development, physiology and disease. The ability to map m6A using immunoprecipitation-based approaches has played a critical role in dissecting m6A functions and mechanisms of action. Yet, these approaches are of limited specificity, unknown sensitivity, and unable to quantify m6A stoichiometry. These limitations have severely hampered our ability to unravel the factors determining where m6A will be deposited, to which levels (the m6A code), and to quantitatively profile m6A dynamics across biological systems. Here, we used the RNase MazF, which cleaves specifically at unmethylated RNA sites, to develop MASTER-seq for systematic quantitative profiling of m6A sites at 16-25% of all m6A sites at single nucleotide resolution. We established MASTER-seq for orthogonal validation and de novo detection of m6A sites, and for tracking of m6A dynamics in yeast gametogenesis and in early mammalian differentiation. We discover that antibody-based approaches severely underestimate the number of m6A sites, and that both the presence of m6A and its stoichiometry are hard-coded via a simple and predictable code within the extended sequence composition at the methylation sites. This code accounts for ~50% of the variability in methylation levels across sites, allows excellent de novo prediction of methylation sites, and predicts methylation acquisition and loss across evolution. We anticipate that MASTER-seq will pave the path towards a more quantitative investigation of m6A biogenesis and regulation in a wide variety of systems, including diverse cell types, stimuli, subcellular components, and disease states.

molecular biology↗

bioRxiv2017

Deciphering Transcriptional Regulation of Human Core Promoters

Despite its pivotal role in regulating transcription, our understanding of core promoter function, architecture, and cis-regulatory elements is lacking. Here, we devised a highthroughput assay to quantify the activity of [~]15,000 fully designed core promoters that we integrated and expressed from a fixed location within the human genome. We find that core promoters drive transcription unidirectionally, and that sequences originating from promoters exhibit stronger activity than sequences originating from enhancers. Testing multiple combinations and distances of core promoter elements, we observe a positive effect of TATA and Initiator, a negative effect of BREu and BREd, and a 10bp periodicity in the optimal distance between the TATA and the Initiator. By comprehensively screening TF binding-sites, we show that site orientation has little effect, that the effect of binding site number on expression is factor-specific, and that there is a striking agreement between the effect of binding site multiplicity in our assay and the tendency of the TF to appear in homotypic clusters throughout the genome. Overall, our results systematically assay the elements that drive expression in core- and proximal-promoter regions and shed light on organization principles of regulatory regions in the human genome.

genomics↗