Direct Nanopore Sequencing of Individual Full Length tRNA Strands

We describe a method for direct tRNA sequencing using the Oxford Nanopore MinION. The principal technical advance is custom adapters that facilitate end-to-end sequencing of individual tRNA molecules. A second advance is a Nanopore sequencing pipeline optimized for tRNA. We tested this method using purified E. coli tRNAfMet, tRNALys, and tRNAPhe samples. 76-to-92% of individual aligned tRNA sequence reads were full length. As proof of concept, we showed that Nanopore sequencing detected all 42 expected tRNA isoacceptors in total E. coli tRNA. Alignment-based comparison between the three purified tRNAs and their synthetic controls revealed systematic miscalls at or adjacent to the positions of known nucleotide modifications. Systematic miscalls were also observed proximal to known modifications in total E. coli tRNA alignments.

Nucleotide resolution profiling of m3C RNA modification by HAC-seq

Cellular RNAs are subject to a myriad of different chemical modifications that play important roles in controlling RNA expression and function. Dysregulation of certain RNA modifications, the so-called ‘epitranscriptome’, contributes to human disease. One limitation in studying the functional, physiological, and pathological roles of the epitranscriptome is the availability of methods for the precise mapping of individual RNA modifications throughout the transcriptome. 3-Methylcytidine (m3C) modification of certain tRNAs is well established and was also recently detected in mRNA. However, methods for the specific mapping of m3C throughout the transcriptome are lacking. Here, we developed a m3C-specific technique, Hydrazine-Aniline Cleavage sequencing (HAC-seq), to profile the m3C methylome at single-nucleotide resolution. We applied HAC-seq to analyze ribosomal RNA (rRNA)-depleted total RNAs in human cells. We found that tRNAs are the predominant m3C-modified RNA species, with 17 m3C modification sites on 11 cytoplasmic and 2 mitochondrial tRNA isoacceptors in MCF7 cells. We found no evidence for m3C-modification of mRNA or other non-coding RNAs at comparable levels to tRNAs in these cells. HAC-seq provides a novel method for the unbiased, transcriptome-wide identification of m3C RNA modification at single-nucleotide resolution, and could be widely applied to reveal the m3C methylome in different cells and tissues.

METTL2 forms a complex with the DALRD3 anticodon-domain binding protein to catalyze formation of 3-methylcytosine in specific arginine tRNA isoacceptors

In mammals, a subset of arginine tRNA isoacceptors are methylated in the anticodon loop by the METTL2 methyltransferase to form the 3-methylcytosine (m3C) modification. However, the mechanism by which METTL2 identifies specific arginine tRNAs for m3C formation as well as the biological role of m3C in mammals is unknown. Here, we show that human METTL2 forms a complex with DALR anticodon binding domain containing 3 (DALRD3) protein in order to recognize particular arginine tRNAs destined for m3C modification. Using biochemical reconstitution, we find that METTL2-DALDR3 complexes catalyze m3C formation in vitro that is dependent upon sequence elements specific to certain arginine tRNAs. Notably, DALRD3-deficient human cells exhibit nearly complete loss of the m3C modification in arginine tRNAs. These findings uncover an unexpected function for the DALRD3 protein in the targeting of distinct arginine tRNAs for m3C modification.

A tRNA-mimic Strategy to Explore the Role of G34 of tRNAGly in Translation and Codon Frameshifting

Decoding of the 61 sense codons of the genetic code requires a variable number of tRNAs that establish codon-anticodon interactions. Thanks to the wobble base pairing at the third codon position, less than 61 different tRNA isoacceptors are needed to decode the whole set of codons. On the tRNA, a subtle distribution of nucleoside modifications shapes the anticodon loop structure and participates to accurate decoding and reading frame maintenance. Interestingly, although the 61 anticodons should exist in tRNAs, a strict absence of some tRNAs decoders is found in several codon families. For instance, in Eukaryotes, G34-containing tRNAs translating 3-, 4- and 6-codon boxes are absent. This includes tRNA specific for Ala, Arg, Ile, Leu, Pro, Ser, Thr, and Val. tRNAGly is the only exception for which in the three kingdoms, a G34-containing tRNA exists to decode C3 and U3-ending codons. To understand why G34-tRNAGly exists, we analysed at the genome wide level the codon distribution in codon +1 relative to the four GGN Gly codons. When considering codon GGU, a bias was found towards an unusual high usage of codons starting with a G whatever the amino acid at +1 codon. It is expected that GGU codons are decoded by G34-containing tRNAGly, decoding also GGC codons. Translation studies revealed that the presence of a G at the first position of the downstream codon reduces the +1 frameshift by stabilizing the G34•U3 wobble interaction. This result partially explains why G34-containing tRNAGly exists in Eukaryotes whereas all the other G34-containing tRNAs for multiple codon boxes are absent.

A Decrease in Serine Levels during Growth Transition Triggers Biofilm Formation inBacillus subtilis

ABSTRACTBiofilm development inBacillus subtilisis regulated at multiple levels. While a number of known signals that trigger biofilm formation do so through the activation of one or more sensory histidine kinases, it was discovered that biofilm activation is also coordinated by sensing intracellular metabolic signals, including serine starvation. Serine starvation causes ribosomes to pause on specific serine codons, leading to a decrease in the translation rate ofsinR, which encodes a master repressor for biofilm matrix genes and ultimately triggers biofilm induction. How serine levels change in different growth stages, howB. subtilisregulates intracellular serine levels, and how serine starvation triggers ribosomes to pause on selective serine codons remain unknown. Here, we show that serine levels decrease as cells enter stationary phase and that unlike most other amino acid biosynthesis genes, expression of serine biosynthesis genes decreases upon the transition into stationary phase. The deletion of the gene for a serine deaminase responsible for converting serine to pyruvate led to a delay in biofilm formation, further supporting the idea that serine levels are a critical intracellular signal for biofilm activation. Finally, we show that levels of all five serine tRNA isoacceptors are decreased in stationary phase compared with exponential phase. However, the three isoacceptors recognizing UCN serine codons are reduced to a much greater extent than the two that recognize AGC and AGU serine codons. Our findings provide evidence for a link between serine homeostasis and biofilm development inB. subtilis.IMPORTANCEInBacillus subtilis, biofilm formation is triggered in response to environmental and cellular signals. It was proposed that serine limitation acts as a proxy for nutrient status and triggers biofilm formation at the onset of biofilm entry through a novel signaling mechanism caused by global ribosome pausing on selective serine codons. In this study, we reveal that serine levels decrease at the biofilm entry due to catabolite control and a serine shunt mechanism. We also show that levels of five serine tRNA isoacceptors are differentially decreased in stationary phase compared with exponential phase; three isoacceptors recognizing UCN serine codons are reduced much more than the two recognizing AGC and AGU codons. This finding indicates a possible mechanism for selective ribosome pausing.

Decreasing Serine Levels During Growth Transition Triggers Biofilm Formation inBacillus subtilis

ABSTRACTBiofilm development inBacillus subtilisis regulated at multiple levels. While a number of known signals that trigger biofilm formation do so through the activation of one or more sensory histidine kinases, it was recently discovered that biofilm activation is also coordinated by sensing intracellular metabolic signals, including serine starvation. Serine starvation causes ribosomes to pause on specific serine codons, leading to a decrease in the translation rate ofsinR, which encodes a master repressor for biofilm matrix genes, and ultimately biofilm induction. How serine levels change in different growth stages, howB. subtilisregulates intracellular serine levels in response to metabolic status, and how serine starvation triggers ribosomes to pause on selective serine codons remain unknown. Here we show that serine levels decrease as cells enter stationary phase and that unlike most other amino acid biosynthesis genes, expression of serine biosynthesis genes decreases upon the transition into stationary phase. Deletion of the gene for a serine deaminase responsible for converting serine to pyruvate led to a delay in biofilm formation, further supporting the idea that serine levels are a critical intracellular signal for biofilm activation. Finally, we show that levels of all five serine tRNA isoacceptors are decreased in stationary phase compared to exponential phase. Interestingly, the three isoacceptors recognizing UCN serine codons are reduced to a much greater extent than the two that recognize AGC and AGU serine codons. Our findings provide evidence for a link between serine homeostasis and biofilm development inB. subtilis.IMPORTANCEInBacillus subtilis, biofilm formation is triggered in response to various environmental and cellular signals. It was previously proposed that serine limitation acts as a proxy for nutrient status and triggers biofilm formation at the onset of biofilm entry through a novel signaling mechanism caused by global ribosome pausing on selective serine codons. In this study, we revealed that serine levels decrease at the biofilm entry due to catabolite control and a shunt mechanism. We also show that levels of five serine tRNA isoacceptors are differentially decreased in stationary phase compared to exponential phase; three isoacceptors recognizing UCN serine codons are reduced much greater than the two recognizing AGC and AGU codons. This indicates a possible mechanism for selective ribosome pausing.