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Unbiased screen of RNA tailing activities reveals a poly(UG) polymerase

Nature Methods volume 16, pages 437–445 (2019)Cite this article

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Abstract

Ribonucleotidyl transferases (rNTases) add untemplated ribonucleotides to diverse RNAs. We have developed TRAID-seq, a screening strategy in Saccharomyces cerevisiae to identify sequences added to a reporter RNA at single-nucleotide resolution by overexpressed candidate enzymes from different organisms. The rNTase activities of 22 previously unexplored enzymes were determined. In addition to poly(A)- and poly(U)-adding enzymes, we identified a cytidine-adding enzyme that is likely to be 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, Caenorhabditis elegans MUT-2, that adds alternating uridine and guanosine nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutants of the enzyme that result in defective silencing did not add poly(UG) tails in our assay. We propose that MUT-2 poly(UG) polymerase activity is required to promote genome integrity and RNA silencing.

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Fig. 1: The TRAID-seq assay measures nucleotide-addition activity in cellulo.
Fig. 2: Analyses of the nucleotide-addition activities of 40 noncanonical rNTases from seven species.
Fig. 3: Nucleotide-addition activities of S. pombe SPAC1093.04 and S. cerevisiae Cca1.
Fig. 4: CeMUT-2 is a poly(UG) polymerase.
Fig. 5: CeMUT-2 mutants resulting in RNAi deficiency lack poly(UG) polymerase activity.

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Data availability

All sequencing data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE123478.

Code availability

All custom scripts have been made available at https://github.com/melanieapreston/PuppyTails.

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Acknowledgements

We thank members of the Wickens, Kimble, Kennedy, and Anderson laboratories for advice throughout the work. We are particularly grateful to S. Kennedy for discussions concerning MUT-2, and A. Hopper and E. Phizicky for advice on CCA-adding enzymes. We thank A. Shukla and J. Yan for comments on the manuscript. We acknowledge the University of Wisconsin Biotechnology Center DNA Sequencing Facility, especially M. Adams and M. Sussmann, for providing Illumina sequencing facilities and services, and M. Harte for assistance with cloning S. pombe rNTases. We also thank S. Sasaki for collecting Xenopus oocytes and advice on injections. We are grateful to L. Vanderploeg of the UW Biochemistry Media Laboratory for help with figures. This work was supported by a Ruth Kirschstein National Research Service Award (1F32GM103130-01A1) to M.A.P. and NIH grants GM50942 to M.W. and HG003747 to S.K. J.K. is an Investigator of the Howard Hughes Medical Institute.

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  1. Melanie A. Preston & Natascha Buter

    Present address: Promega Corporation, Madison, WI, USA

  2. Douglas F. Porter

    Present address: Program in Epithelial Biology, Stanford University Medical School, Stanford, CA, USA

  3. Christopher P. Lapointe

    Present address: Department of Structural Biology, Stanford University, Stanford, CA, USA

Authors and Affiliations

  1. Department of Biochemistry, University of Wisconsin–Madison, Madison, WI, USA

    Melanie A. Preston, Douglas F. Porter, Natascha Buter, Christopher P. Lapointe, Judith Kimble & Marvin Wickens

  2. Department of Statistics, University of Wisconsin–Madison, Madison, WI, USA

    Fan Chen & Sunduz Keles

  3. Department of Biostatistics and Medical Informatics, University of Wisconsin–Madison, Madison, WI, USA

    Sunduz Keles

  4. Howard Hughes Medical Institute, University of Wisconsin–Madison, Madison, WI, USA

    Judith Kimble

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Contributions

M.A.P., J.K., and M.W. designed the experiments; M.A.P. performed the experiments and analyzed data unless otherwise noted. D.F.P. wrote the PuppyTails program used to analyze TRAID-seq data, including tail-o-grams. F.C. and S.K. performed statistical analyses of tail sequence motifs. N.B. prepared N. crassa and C. albicans TRAID-seq samples. C.P.L. wrote the script used to calculate total nucleotide incorporation. M.A.P. and M.W. wrote the manuscript, with contributions from all other authors.

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Correspondence to Marvin Wickens.

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Integrated supplementary information

Supplementary Figure 1 rNTase assay using RNase P-derived reporter RNA.

a, RNase P reporter RNA is co-expressed with MS2 coat protein-rNTase fusion in yeast. The tethered rNTase adds nucleotides to the 3ʹ end of the reporter RNA. b, Schematic of sample processing to detect adenosine tails or uridine tails added to the reporter RNA and example data identifying PUP or PAP activity. This tail-specific approach was also used to produce RT-PCR data in Fig. 1a. c, RT-PCR analysis to detect adenosine tails or uridine tails added to the RNase P reporter RNA by control rNTases, relative to empty vector or to no-reporter control. Lanes marked with a minus sign indicate reactions performed without reverse transcriptase. Representative gel image from 3 independent experiments.

Supplementary Figure 2 S. pombe rNTases with secondary nucleotide preferences.

a, Tail-o-gram depicting nucleotide composition of tails added by SpCid1. b, Left, tail-o-gram depicting nucleotide composition of tails added by SpCid13. Right, representative tail sequences added to tRNA reporter. c, Left, tail-o-gram depicting nucleotide composition of tails added by SpCid14. Right, representative tail sequences added to tRNA reporter. For all tail-o-grams: Percentage of each nucleotide at each tail length is plotted on the left y axis. Each nucleotide is indicated by a different color: green, uridine; yellow, cytidine; purple, guanosine; brown, adenosine. The number of tails detected per million heptamers (TPMH) is indicated by black diamonds, and corresponds to the log scale on the right y axis.

Supplementary Figure 3 Sequence motif effect analysis of S. pombe SPAC1093.04 and S. cerevisiae Cca1 enzymes.

Effect analysis of tails added in cells expressing SpSPAC1093.04 (red, n = 5) and ScCca1 (black, n = 3). Oligonucleotides with a significant effect after multiplicity correction with the Bonferroni procedure at significance level 0.05 (dashed line near baseline) are displayed.

Supplementary Figure 4 Expression of both SPAC1093.04 and SPCC645.10 rescues cca1-1 temperature sensitivity: second biological replicate.

cca1-1 mutant strains containing CEN plasmids expressing indicated plasmids were serially diluted, spotted on SD-Ura-Leu media and grown at 37 °C for 3 d or 23 °C for 4 d. This experiment was repeated twice with similar results.

Supplementary Figure 5 CeF31C3.2 (NPOL-1) adds tails that are random in sequence.

a, Left, tail-o-gram depicting nucleotide composition of tails added by CeF31C3.2 in TRAID-seq. The percentage of each nucleotide at each tail length is plotted on the left y axis. Each nucleotide is indicated by a different color: green, uridine; yellow, cytidine; purple, guanosine; brown, adenosine. The number of tails detected per million heptamers (TPMH) is indicated by black diamonds, and corresponds to the log scale on the right y axis. Right, example tail sequences randomly selected from tails of the same length added by CeF31C3.2. b, CeF31C3.2 tail composition parallels intracellular rNTP concentrations. CeF31C3.2 addition of each nucleotide ± s.d. (n = 5) versus measured rNTP concentrations in yeast (Proc. Natl. Acad. Sci. USA 107, 4949–4954; 2010). c, Statistical analysis of all possible dinucleotides in tails added by CeF31C3.2. Heat map of –log10 P values for all possible dinucleotides. No dinucleotides had a statistically significant effect after multiplicity correction with the Bonferroni procedure at significance level 0.05. Five independent biological replicates were evaluated. Details of statistical analyses are provided in the Methods (“Computational analyses of sequence motifs”).

Supplementary Figure 6 CeMUT-2 adds UG repeats to a non-tRNA substrate in yeast.

a, Schematic of experiment to test CeMUT-2 activity on RNase P RNA reporter in yeast. b, CeMUT-2 adds UG repeats to RNase P RNA reporter in three biological replicates, regardless of the 3ʹ end sequence of the RNA reporter. Randomly selected sequences of tails added to different 3ʹ ends are shown.

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Preston, M.A., Porter, D.F., Chen, F. et al. Unbiased screen of RNA tailing activities reveals a poly(UG) polymerase. Nat Methods 16, 437–445 (2019). https://doi.org/10.1038/s41592-019-0370-6

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