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. 2015 Apr 30;11(4):e1005091.
doi: 10.1371/journal.pgen.1005091. eCollection 2015 Apr.

Synergistic and independent actions of multiple terminal nucleotidyl transferases in the 3' tailing of small RNAs in Arabidopsis

Xiaoyan Wang  1 , Shuxin Zhang  2 , Yongchao Dou  2 , Chi Zhang  2 , Xuemei Chen  3 , Bin Yu  2 , Guodong Ren  4
Affiliations

Synergistic and independent actions of multiple terminal nucleotidyl transferases in the 3' tailing of small RNAs in Arabidopsis

Xiaoyan Wang et al. PLoS Genet. .

Abstract

All types of small RNAs in plants, piwi-interacting RNAs (piRNAs) in animals and a subset of siRNAs in Drosophila and C. elegans are subject to HEN1 mediated 3' terminal 2'-O-methylation. This modification plays a pivotal role in protecting small RNAs from 3' uridylation, trimming and degradation. In Arabidopsis, HESO1 is a major enzyme that uridylates small RNAs to trigger their degradation. However, U-tail is still present in null hen1 heso1 mutants, suggesting the existence of (an) enzymatic activities redundant with HESO1. Here, we report that UTP: RNA uridylyltransferase (URT1) is a functional paralog of HESO1. URT1 interacts with AGO1 and plays a predominant role in miRNA uridylation when HESO1 is absent. Uridylation of miRNA is globally abolished in a hen1 heso1 urt1 triple mutant, accompanied by an extensive increase of 3'-to-5' trimming. In contrast, disruption of URT1 appears not to affect the heterochromatic siRNA uridylation. This indicates the involvement of additional nucleotidyl transferases in the siRNA pathway. Analysis of miRNA tailings in the hen1 heso1 urt1 triple mutant also reveals the existence of previously unknown enzymatic activities that can add non-uridine nucleotides. Importantly, we show HESO1 may also act redundantly with URT1 in miRNA uridylation when HEN1 is fully competent. Taken together, our data not only reveal a synergistic action of HESO1 and URT1 in the 3' uridylation of miRNAs, but also independent activities of multiple terminal nucleotidyl transferases in the 3' tailing of small RNAs and an antagonistic relationship between uridylation and trimming. Our results may provide further insight into the mechanisms of small RNA 3' end modification and stability control.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. urt1-3 increases the silique length in hen1-2 heso1-2 and enhances miRNA function in hen1-1 heso1-2.
(A) Fully-expanded siliques from plants of the indicated genotypes. Scale bar, 1cm. (B) Average silique length in indicated genotypes. 40 siliques from at least 6 individual plants for each genotype were analyzed. (C) Schematic structure of URT1 protein. PRO_Rich: Proline rich domain; GLN_Rich: Glutamine rich domain; PAP/25A: Poly A polymerase domain; Core: Core regions of PAP/25A; PAP/25A associated: Poly A polymerase associated domain. (D) Conservation of URT1 P618 across species. At, A. thaliana; Hs, H. sapiens; Sp, S. pombe; Dm, D. melanogaster; Ce, C.elegans; Cr, C. reinhardtii. (E) urt1-3 enhances miRNA function in hen1-1 heso1-2. The transcript levels of five miRNA targets in indicated genotypes. Target mRNA accumulation in each genotype was quantified by qPCR using primers spanning the cleavage site and compared with those of wild type (Wt). The expression level of each gene in Wt was arbitrarily set to 1. Quantifications are normalized with GAPDH transcript. ACT2 was served as an internal control. Values are means of three biological replicates ±SD.
Fig 2
Fig 2. Small RNA northern blot analysis of miR167, miR166, miR171 and miR172 in various genotypes.
U6 was served as a loading control.
Fig 3
Fig 3. Global changes of miRNA profiles in hen1-1 heso1-2 urt1-3.
(A) Size distribution of miRNAs in indicated genotypes. (B) Size distribution of 5GMC of miRNAs in indicated genotypes. 5GMC, 5’ genome matched component, see text for details. (C) Overall 3’ end signatures (including tail length, nucleotides addition composition and trimming extent) of miRNAs in indicated genotypes. The tailing and trimming extent was calculated as % (number of reads with indicated modification divided by total analyzed reads); the nucleotides addition composition was calculated as number of a nucleotide in the tail divided by tail length. Data from biological replicate 1 were shown. See S3 Fig online for data from biological replicate 2.
Fig 4
Fig 4. urt1-3 affects miRNAs but not siRNAs uridylation in hen1-2 heso1-2.
The X-axis represents the degree of trimming and the Y-axis represents the degree of tailing. The annotated miRNA sequences from miRBase v17.0 were served as standard sequences (i.e. these sequences are considered as non-tailed and non-trimmed.) For simplicity, we only analyzed reads started from the annotated 5’ ends. Thus, reads at coordinate position (0, 0) are exactly the same as annotated sequences and all reads from the same coordinate position are of same length. The relative abundance of each small RNA species is proportional to the diameter of the circles. Data from biological replicate 1 were shown. See S4 Fig online for more small RNAs and data from biological replicate 2.
Fig 5
Fig 5. The tailing and trimming profiles of miR158a in Ler, heso1-2, heso1-2 urt1-3 resemble those in hen1-2, hen1-2 heso1-2, hen1-2 heso1-2 urt1-3.
For Ler, heso1-2, heso1-2 urt1-3, reads same as annotated miR158a sequence were removed and the rest of miR158a variants were renormalized to 100%. More interpretations of the matrix are depicted in Fig 4.
Fig 6
Fig 6. The P618L substitution impairs the URT1 activity, affects its subcellular localization and diminishes its interaction with AGO1.
(A) Expression and purification of GST, GST-URT1 and GST-URT1P618L in E.coli BL21(DE3). After purification, proteins were subject to SDS-polyacrylamide gel analysis and monitored by the comassie brillant blue staining. (B) Terminal nucleotidyl transferase activity of URT1 and URT1P618L. 5’ end P32 labeled miR165a were incubated with GST, GST-URT1 or GST-URT1P618L in the presence of various nucleotide triphosphates for 30 minutes. After the reaction, RNAs were extracted and analyzed on a denaturing polyacrylamide gel. (C) Sub-cellular localization of YFP-AGO1, HESO1-YFP, URT1-GFP and URT1P618L-GFP. Note that only about 20% of YFP-AGO1 showed discrete cytoplasmic foci (C) while the remaining ones had a relatively even distribution[26]. For each construct, 40–50 cells were analyzed and a similar nucleus localization pattern was observed. n, nucleus. Scale bar, 20 μm. (D) URT1-GFP colocalized with HESO1-RFP. Paired constructs were coinfiltrated into N. benthamiana leaves. RFP and GFP fluorescence signals were monitored 40–48 h after infiltration by confocal microscopy. Scale bar, 20 μm. (E, F) Interactions between AGO1 and URT1 by the co-immunoprecipitation assay. 10xMYC-AGO1 and respective GFP tagged proteins were co-expressed in N. benthamiana leaves in the presence of P19 for 3 days and protein extracts were incubated with either anti-MYC-protein G-agarose beads or anti-GFP-protein G-agarose beads for 4 hrs to overnight. After reaction, proteins were resolved by the SDS-polyacrylamide gel electrophoresis and detected with respective antibodies. Input = 1%. (E) 10xMYC-AGO1 co-immunoprecipitates with URT1-GFP and URT1P618L-GFP. (F) URT1-GFP and URT1P618L-GFP co-immunoprecipitates with 10xMYC-AGO1.
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