TRAID-seq: Unbiased analysis of RNA tailing enzyme activity at single-nucleotide resolution
Ribonucleotidyl transferases (rNTases) add non-templated ribonucleotides to diverse RNAs. We developed a screening strategy in S. cerevisiae to identify sequences added by candidate enzymes from different organisms at single-nucleotide resolution. The assay, referred to as TRAID-Seq (tethered rNTase activity identified by high-throughput sequencing), circumvents the need for purified enzymes, which can be problematic with rNTases, and precisely identifies many thousands of independently captured tail sequences, enabling a sensitive determination of their sequences and relative abundances. The rNTase activities of 19 previously unexplored enzymes were determined using TRAID-Seq. In addition to poly(A)- and poly(U)-adding enzymes, we identified a C-adding enzyme that is likely part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase, C. elegans MUT-2, which adds alternating U and G nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutations in the enzyme that are defective in these functions also fail to add poly(UG) tails in our assay, thus implicating poly(UG) polymerase activity in genome integrity and RNA silencing.
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Posted 22 May, 2019
TRAID-seq: Unbiased analysis of RNA tailing enzyme activity at single-nucleotide resolution
Posted 22 May, 2019
Ribonucleotidyl transferases (rNTases) add non-templated ribonucleotides to diverse RNAs. We developed a screening strategy in S. cerevisiae to identify sequences added by candidate enzymes from different organisms at single-nucleotide resolution. The assay, referred to as TRAID-Seq (tethered rNTase activity identified by high-throughput sequencing), circumvents the need for purified enzymes, which can be problematic with rNTases, and precisely identifies many thousands of independently captured tail sequences, enabling a sensitive determination of their sequences and relative abundances. The rNTase activities of 19 previously unexplored enzymes were determined using TRAID-Seq. In addition to poly(A)- and poly(U)-adding enzymes, we identified a C-adding enzyme that is likely part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase, C. elegans MUT-2, which adds alternating U and G nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutations in the enzyme that are defective in these functions also fail to add poly(UG) tails in our assay, thus implicating poly(UG) polymerase activity in genome integrity and RNA silencing.
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