RNA sequencing (RNA-seq) offers a snapshot of cellular RNA populations, but not temporal information about the sequenced RNA. Here we report TimeLapse-seq, a chemical method that uses oxidative-nucleophilic-aromatic-substitution to convert 4-thiouridine into cytidine analogues, yielding apparent U-to-C mutations that mark new transcripts upon sequencing. TimeLapse-seq is a single molecule approach that is adaptable to many applications, and reveals RNA dynamics and induced differential expression concealed in traditional RNA-seq.

Highlights d Pvt1b is a p53-dependent lncRNA isoform, induced by genotoxic and oncogenic stress d Production of Pvt1b RNA represses Myc transcription in cis d Pvt1b suppresses Myc transcriptional program and cellular proliferation d Pvt1b limits tumor growth, but not tumor progression, in a mouse lung tumor model

Decapping is the first committed step in 5′-to-3′ RNA decay, and in the cytoplasm of human cells, multiple decapping enzymes regulate the stabilities of distinct subsets of cellular transcripts. However, the complete set of RNAs regulated by any individual decapping enzyme remain incompletely mapped, and no consensus sequence or property is currently known to unambiguously predict decapping enzyme substrates. Dcp2 was the first-identified and beststudied eukaryotic decapping enzyme, but it has been shown to regulate the stability of <400 transcripts in mammalian cells to date. Here, we globally profile changes in stability of the human transcriptome in Dcp2 knockout cells via TimeLapse-seq. We find that P-body enrichment is the strongest correlate of Dcp2-dependent decay, and that modification with m 6 A exhibits an additive effect with P-body enrichment for Dcp2 targeting. These results are consistent with a model in which P-bodies represent sites where translationally repressed transcripts are sorted for decay by soluble cytoplasmic decay complexes through additional molecular marks.

Proteogenomic identification of translated small open reading frames in humans has revealed thousands of microproteins, or polypeptides of fewer than 100 amino acids, that were previously invisible to geneticists. Hundreds of microproteins have been shown to be essential for cell growth and proliferation, and many regulate macromolecular complexes. One such regulatory microprotein is NBDY, a 68-amino acid component of the human cytoplasmic RNA decapping complex. Heterologously expressed NBDY was previously reported to regulate cytoplasmic ribonucleoprotein granules known as P-bodies and reporter gene stability, but the global effect of endogenous NBDY on the cellular transcriptome remained undefined. In this work, we demonstrate that endogenous NBDY directly interacts with the human RNA decapping complex through EDC4 and DCP1A and localizes to P-bodies. Global profiling of RNA stability changes in NBDY knockout (KO) cells reveals dysregulated stability of more than 1400 transcripts. DCP2 substrate transcript half-lives are both increased and decreased in NBDY KO cells, which correlates with 5′ UTR length. NBDY deletion additionally alters the stability of non-DCP2 target transcripts, possibly as a result of downregulated expression of nonsense-mediated decay factors in NBDY KO cells. We present a comprehensive model of the regulation of RNA stability by NBDY.

Cellular RNA levels are the result of a juggling act between RNA transcription, processing, and degradation. By tuning one or more of these parameters, cells can rapidly alter the available pool of transcripts in response to stimuli. While RNA sequencing (RNA-seq) is a vital method to quantify RNA levels genome-wide, it is unable to capture the dynamics of different RNA populations at steady-state or distinguish between different mechanisms that induce changes to the steady-state (i.e. altered rate of transcription versus degradation). The dynamics of different RNA populations can be studied by targeted incorporation of non-canonical nucleosides. 4-thiouridine (s4U) is a commonly used and versatile RNA metabolic label that allows the study of many properties of RNA metabolism from synthesis to degradation. Numerous experimental strategies have been developed that leverage the power of s4U to label newly transcribed RNA in whole cells, followed by enrichment with activated disulfides or chemistry to induce C mutations at sites of s4U during sequencing. This review presents existing methods to study RNA population dynamics genome-wide using s4U metabolic labeling, as well as a discussion of considerations and challenges when designing s4U metabolic labeling experiments.

RNA-sequencing (RNA-seq) measures RNA abundance in a biological sample but does not provide temporal information about the sequenced RNAs. Metabolic labeling can be used to distinguish newly made RNAs from pre-existing RNAs. Mutations induced from chemical recoding of the hydrogen bonding pattern of the metabolic label can reveal which RNAs are new in the context of a sequencing experiment. These nucleotide recoding strategies have been developed for a single uridine analogue, 4-thiouridine (s4U), limiting the scope of these experiments. Here we report the first use of nucleoside recoding with a guanosine analogue, 6-thioguanosine (s6G). Using TimeLapse sequencing (TimeLapse-seq), s6G can be recoded under RNA-friendly oxidative nucleophilic-aromatic substitution conditions to produce adenine analogues (substituted 2-aminoadenosines). We demonstrate the first use of s6G recoding experiments to reveal transcriptome-wide RNA population dynamics.

Metal complexes incorporating the tris(3,5-diphenylpyrazolyl)borate ligand (Tp(Ph2)) and ortho-dihalophenolates were synthesized and characterized in order to explore metal-halogen secondary bonding in biorelevant model complexes. The complexes Tp(Ph2)ML were synthesized and structurally characterized, where M was Fe(II), Co(II), or Ni(II) and L was either 2,6-dichloro- or 2,6-dibromophenolate. All six complexes exhibited metal-halogen secondary bonds in the solid state, with distances ranging from 2.56 Å for the Tp(Ph2)Ni(2,6-dichlorophenolate) complex to 2.88 Å for the Tp(Ph2)Fe(2,6-dibromophenolate) complex. Variable temperature NMR spectra of the Tp(Ph2)Co(2,6-dichlorophenolate) and Tp(Ph2)Ni(2,6-dichlorophenolate) complexes showed that rotation of the phenolate, which requires loss of the secondary bond, has an activation barrier of ~30 and ~37 kJ/mol, respectively. Density functional theory calculations support the presence of a barrier for disruption of the metal-halogen interaction during rotation of the phenolate. On the other hand, calculations using the spectroscopically calibrated angular overlap method suggest essentially no contribution of the halogen to the ligand-field splitting. Overall, these results provide the first quantitative measure of the strength of a metal-halogen secondary bond and demonstrate that it is a weak noncovalent interaction comparable in strength to a hydrogen bond. These results provide insight into the origin of the specificity of the enzyme 2,6-dichlorohydroquinone 1,2-dioxygenase (PcpA), which is specific for ortho-dihalohydroquinone substrates and phenol inhibitors.

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