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.
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.
The sodium hydrogen exchanger isoform one (NHE1) plays a critical role coordinating asymmetric events at the leading edge of migrating cells and is regulated by a number of phosphorylation events influencing both the ion transport and cytoskeletal anchoring required for directed migration. Lysophosphatidic acid (LPA) activation of RhoA kinase (Rock) and the Ras-ERK growth factor pathway induces cytoskeletal reorganization, activates NHE1 and induces an increase in cell motility. We report that both Rock I and II stoichiometrically phosphorylate NHE1 at threonine 653 in vitro using mass spectrometry and reconstituted kinase assays. In fibroblasts expressing NHE1 alanine mutants for either Rock (T653A) or ribosomal S6 kinase (Rsk; S703A) we show each site is partially responsible for the LPA-induced increase in transport activity while NHE1 phosphorylation by either Rock or Rsk at their respective site is sufficient for LPA stimulated stress fiber formation and migration. Furthermore, mutation of either T653 or S703 leads to a higher basal pH level and a significantly higher proliferation rate. Our results identify the direct phosphorylation of NHE1 by Rock and suggest that both RhoA and Ras pathways mediate NHE1-dependent ion transport and migration in fibroblasts.
RNA metabolic labeling using 4-thiouridine (s4U) captures the dynamics of RNA synthesis and decay. The power of this approach is dependent on appropriate quantification of labeled and un-labeled sequencing reads, which can be compromised by the apparent loss of s4U-labeled reads in a process we refer to as dropout. Here we show that s4U containing transcripts can be selectively lost when RNA samples are handled under sub-optimal conditions, but that this loss can be minimized using an optimized protocol. We demonstrate a second cause of dropout in nucleotide recoding and RNA sequencing (NR-seq) experiments that is computational and downstream of library preparation. NR seq experiments involve chemically converting s4U from a uridine analog to a cytidine analog and using the apparent T-to-C mutations to identify the populations of newly synthesized RNA. We show that high levels of T-to-C mutations can prevent read alignment with some computational pipelines, but that this bias can be overcome using improved alignment pipelines. Importantly, kinetic parameter estimates are affected by dropout independent of the NR chemistry employed, and all chemistries are practically indistinguishable in bulk, short-read RNA-seq experiments. Dropout is an avoidable problem that can be identified by including unlabeled controls, and mitigated through improved sample handing and read alignment that together improve the robustness and reproducibility of NR-seq experiments.
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