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Method Article
Marvin Wickens
Dept of Biochemistry, University of Wisconsin, Madison
Corresponding Author
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This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
Ribonucleotidyl transferase, RNA tailing enzymes, rNTases, poly(UG)polymerase, poly(A)polymerase, PAP, poly(U)polymerase, PUP, CCA-adding enzyme, TRAID-Seq
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The authors declare no competing financial interests.
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Associated Publications
Unbiased screen of RNA tailing activities reveals a poly(UG) polymerase
by Melanie A. Preston, Douglas F. Porter, Fan Chen, +5
Nature Methods (15 April, 2019)
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Marvin Wickens
Dept of Biochemistry, University of Wisconsin, Madison
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 22 May, 2019
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Molecular Biology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
Figure 13
Figure 14
The authors declare no competing financial interests.
Loading...
Associated Publications
Unbiased screen of RNA tailing activities reveals a poly(UG) polymerase
by Melanie A. Preston, Douglas F. Porter, Fan Chen, +5
Nature Methods (15 April, 2019)