The transmission of information from DNA to RNA is a critical process. We compared RNA sequences from human B cells of 27 individuals to the corresponding DNA sequences from the same individuals and uncovered more than 10,000 exonic sites where the RNA sequences do not match that of the DNA. All 12 possible categories of discordances were observed. These differences were nonrandom as many sites were found in multiple individuals and in different cell types, including primary skin cells and brain tissues. Using mass spectrometry, we detected peptides that are translated from the discordant RNA sequences and thus do not correspond exactly to the DNA sequences. These widespread RNA-DNA differences in the human transcriptome provide a yet unexplored aspect of genome variation.

SUMMARY Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine, which is then recognized as guanosine. To study the role of ADAR proteins in RNA editing and gene regulation, we sequenced and compared the DNA and RNA of human B cells. Then, we followed up the findings experimentally with siRNA knockdown and RNA and protein immunoprecipitations. The results uncovered over 60,000 A-to-G editing sites and several thousand genes whose expression levels are influenced by ADARs. Of these ADAR targets, 90% were identified. Our results also reveal that ADAR regulates transcript stability and gene expression through interaction with HuR (ELAVL1). These findings extend the role of ADAR and show that it cooperates with other RNA-processing proteins to regulate the sequence and expression of transcripts in human cells.

Complex regulation of gene expression in mammals has evolved from simpler eukaryotic systems, yet the mechanistic features of this evolution remain elusive. Here, we compared the transcriptional landscapes of the distantly related budding and fission yeast. We adapted the Precision Run-On sequencing (PRO-seq) approach to map the positions of RNA polymerase active sites genome-wide in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Additionally, we mapped preferred sites of transcription initiation in each organism using PRO-cap. Unexpectedly, we identify a pause in early elongation, specific to S. pombe, that requires the conserved elongation factor subunit Spt4 and resembles promoter-proximal pausing in metazoans. PRO-seq profiles in strains lacking Spt4 reveal globally elevated levels of transcribing RNA Polymerase II (Pol II) within genes in both species. Messenger RNA abundance, however, does not reflect the increases in Pol II density, indicating a global reduction in elongation rate. Together, our results provide the first base-pair resolution map of transcription elongation in S. pombe and identify divergent roles for Spt4 in controlling elongation in budding and fission yeast.

Using genetic and molecular analyses, we identified over 1,000 polymorphic regulators that regulate expression levels of human genes.

R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor β (TGF-β), is reduced; that then leads to the activation of the TGF-β pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins.

RNA/DNA hybrids form when RNA hybridizes with its template DNA generating a three-stranded structure known as the R-loop. Knowledge of how they form and resolve, as well as their functional roles, is limited. Here, by pull-down assays followed by mass spectrometry, we identified 803 proteins that bind to RNA/DNA hybrids. Because these proteins were identified using in vitro assays, we confirmed that they bind to R-loops in vivo. They include proteins that are involved in a variety of functions, including most steps of RNA processing. The proteins are enriched for K homology (KH) and helicase domains. Among them, more than 300 proteins preferred binding to hybrids than double-stranded DNA. These proteins serve as starting points for mechanistic studies to elucidate what RNA/DNA hybrids regulate and how they are regulated.

A yeast gene has been identified that encodes a novel, evolutionarily conserved N ␣ -acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A. The gene has been named NAT4. Recombinant Nat4 protein acetylated a peptide corresponding to the N-terminal tail of H4, but not an H3 peptide nor the peptide adrenocorticotropin. H4 and H2A are N-terminally acetylated in all species from yeast to mammals and hence blocked from sequencing by Edman degradation. In contrast, H4 and H2A purified from a nat4 mutant were unacetylated and could be sequenced. Analysis of yeast histones by acid-urea gel electrophoresis showed that all the H4 and H2A from the mutant migrated more rapidly than the same histones from a wild type strain, consistent with the histones from the mutant having one extra positive charge due to one less acetylated amino group. A comparison of yeast proteins from wild type and a nat4 mutant by two-dimensional gel electrophoresis showed no evidence that other yeast proteins are substrates of this acetyltransferase. Thus, Nat4 may be dedicated specifically to the N-terminal acetylation of histones H4 and H2A. Surprisingly, nat4 mutants grow at a normal rate and have no readily observable phenotypes.Eukaryotic proteins are subject to two cotranslational modifications as the nascent polypeptides emerge from the ribosome, methionine cleavage by methionine aminopeptidase, and acetylation of the ␣-amino group of the N-terminal amino acid (1). Removal of the methionine occurs only if the second amino acid is a small one such as serine, alanine, or glycine. Acetylation of the ␣-amino group on either methionine, or on the N-terminal residue resulting from methionine cleavage, occurs in the great majority of (but not all) eukaryotic proteins, and is accomplished by one of several N ␣ -acetyltransferases (NATs) 1 (1). A number of years ago we identified the genes for a yeast NAT, now called NatA, that consists of two subunits, Ard1 and Nat1 (2). Subsequent work showed that NatA was responsible for the acetylation of many, but not all, yeast proteins beginning with small residues such as serine, alanine, or glycine (2-5). Yeast ard1 and nat1 mutants are viable and the many proteins that are no longer N-terminally acetylated in the mutant strains are as stable as they are in a wild type strain (2). Thus, there is no evidence that N-terminal acetylation serves to protect proteins from degradation in yeast.Ard1 protein has an acetyl-CoA binding motif found in members of the GNAT superfamily, enzymes that acetylate amino groups on proteins and other molecules (6). Thus, Ard1 is very likely to be the catalytic subunit of NatA. Two other NATs have been identified in yeast, called NatB and NatC, with catalytic subunits Nat3 and Mak3, respectively (4, 7). NatB and NatC both acetylate proteins with N-terminal methionine residues, although they have different specificities dictated by the nature of the subsequent amino acids (4).Histone H4 is a highly conserved protein with an N-terminal serin...

Summary RNA sequences are expected to be identical to their corresponding DNA sequences. Here, we found all 12 types of RNA-DNA sequence differences (RDDs) in nascent RNA. Our results show that RDDs begin to occur in RNA chains about 55 nucleotides from the RNA polymerase II (Pol II) active site. These RDDs occur so soon after transcription that they are incompatible with known deaminase-mediated RNA editing mechanisms. Moreover, the 55-nucleotide delay in appearance indicates they do not arise during RNA synthesis by Pol II or as a direct consequence of modified base incorporation. Preliminary data suggest that RDD and R-loop formations may be coupled. These findings identify sequence substitution as an early step in co-transcriptional RNA processing.