Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jul 16;59(2):217-28.
doi: 10.1016/j.molcel.2015.05.034. Epub 2015 Jul 2.

Selective Suppression of the Splicing-Mediated MicroRNA Pathway by the Terminal Uridyltransferase Tailor

Diane Bortolamiol-Becet  1 , Fuqu Hu  1 , David Jee  2 , Jiayu Wen  1 , Katsutomo Okamura  3 , Ching-Jung Lin  2 , Stefan L Ameres  4 , Eric C Lai  5
Affiliations

Selective Suppression of the Splicing-Mediated MicroRNA Pathway by the Terminal Uridyltransferase Tailor

Diane Bortolamiol-Becet et al. Mol Cell. .

Abstract

Several terminal uridyltransferases (TUTases) are known to modulate small RNA biogenesis and/or function via diverse mechanisms. Here, we demonstrate that Drosophila splicing-derived pre-miRNAs (mirtrons) are efficiently modified by the previously uncharacterized TUTase, Tailor. Tailor is necessary and sufficient for mirtron hairpin uridylation, and this modification inhibits mirtron biogenesis. Genome-wide analyses demonstrate that mirtrons are dominant Tailor substrates, and three features contribute to substrate specificity. First, reprogramming experiments show Tailor preferentially identifies splicing-derived miRNAs. Second, in vitro tests indicate Tailor prefers substrate hairpins over mature miRNAs. Third, Tailor exhibits sequence preference for 3'-terminal AG, a defining mirtron characteristic. Our work supports the notion that Tailor preferentially suppresses biogenesis of mirtrons, an evolutionarily adventitious pre-miRNA substrate class. Moreover, we detect preferential activity of Tailor on 3'-G canonical pre-miRNAs, and specific depletion of such loci from the pool of conserved miRNAs. Thus, Tailor activity may have had collateral impact on shaping populations of canonical miRNAs.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Preferred uridylation of hairpins from an alternative miRNA biogenesis pathway. (A) Pre-miRNA hairpins can be generated by Drosha-mediated cleavage, or by splicing of a short hairpin intron (mirtron). (B) In general, most miRNA reads do not carry untemplated additions. However, many mirtron-3p species exhibit high rates of terminal uridylation, in some cases comprising the dominant species. Shown are three mirtrons used as models in this study. The fraction of uridylated species are calculated with respect to the splicing-derived species. Their corresponding mirtron-5p species essentially lack untemplated modifications, reflecting that uridylation likely occurred at the hairpin stage.
Figure 2
Figure 2
Functional screening identifies CG1091 (Tailor) as the Drosophila mirtron uridylase. (A) Knockdown screening of a panel of terminal nucleotidyltransferases in S2 cells transfected with various mirtron plasmids; shown are Northern blots probed for the indicated small RNA species. Depletion of CG1091 selectively caused loss of tailed mirtron hairpins (brackets) and tailed mirtron-3p miRNAs (arrowheads). (B) Co-expression of CG1091-A or CG1091-B with the indicated mirtron constructs strongly enhanced accumulation of tailed hairpin and mature 3p species. (C) A transposon allele that disrupts the common open reading frame of CG1091 isoforms. (D) qPCR analyses show loss of CG1091 transcripts, especially distal to the insertion, confirming it as a knockout (KO). (E) Northern analysis of mature small RNAs from wildtype and KO ovaries shows loss of normally abundant tailed mirtron-3p miRNAs. In contrast, there are only modest CG1091-dependent species for the canonical miRNA-3p species of bantam and miR-184. (F) Loss of CG1091 reduces numbers of progeny per female fly, and is rescued by a genomic transgene. Error bars represent SEM and student’s t-tests were used to calculate significance. See also Figures S1 and S2.
Figure 3
Figure 3
Tailor is a uridyltransferase that inhibits mirtron biogenesis and activity. (A) Immunopurified wildtype and catalytically inactive myc-Tailor from S2 cells, detected with anti-myc, used for subsequent tailing assays. (B) Timecourse assay of myc-Tailor incubated with radiolabeled mature miR-1010 in the presence of uridine. Its robust tailing activity is dependent on its catalytic site. (C) Tailor only generates extensively tailed products using uridine; 5′ reaction time. (D) Tailor is dependent on Mg++ ions. (E) Tailor is highly efficient on a mirtron hairpin (pre-mir-1010) substrate. (F, G) Reporter assays in S2 cells transiently expressing ub-Gal4, UAS-mir-1003 (mirtron) and the indicated luciferase sensors. Tailor knockdown enhances mirtron activity (F) while Ectopic Tailor antagonizes mirtron activity (G). Error bars represent SD and unpaired T-tests were used to calculate significance. (H) In vitro dicing of unmodified and uridylated pre-mir-1010 hairpins by recombinant Dcr-1/Loqs-PB. Tailing inhibits (for 2U) or abolishes (for 4U) the accumulation of mature species. (I) Quantification of three dicing assays; standard deviations are shown. See also Figure S3.
Figure 4
Figure 4
Impact of biogenesis strategy on substrate recognition by Tailor. (A) The mature-3p species of mirtrons mir-1003 and mir-1008 were reprogrammed into the canonical mir-278 backbone. (B) Northern analysis of natural and reprogrammed mirtron constructs transfected into S2 cells, with or without Tailor expression construct. Robust tailing of mirtron hairpins and mature products is abrogated when their sequences are directed through the Drosha pathway. (C) Quantification of replicate experiments; SD is shown. The ratios of tailed to unmodified species (marked with arrowheads in B) in the presence of ectopic Tailor were normalized to parallel myc control conditions. The analysis shows mirtrons are preferred Tailor substrates, and their hairpins accumulate more tailed species than their mature miRNAs. (D, E) Reporter assays in S2 cells transiently expressing ub-Gal4, UAS-miRNA and the indicated luciferase sensors. (D) Activity of the reprogrammed mir-278/miR-1003 construct is less enhanced in Tailor-depleted cells than wildtype mir-1003 (Figure 3F). (E) Reprogrammed mir-278/miR-1003 construct is not inhibited by Tailor overexpression, as is wildtype mir-1003 (Figure 3G). Error bars represent SD and unpaired T-tests were used to calculate significance.
Figure 5
Figure 5
Genomewide analysis of Tailor substrates in cultured cells and ovaries. Genome-matching reads are represented in gray while different untemplated nucleotides are color-coded. (A) Comparison of control and Tailor-depleted S2R+ cells demonstrates that mirtron-3p species are heavily uridylated, that this is nearly completely dependent on Tailor, and that other substrates are little affected by Tailor. (B) Analysis of individual mirtron-3p species shows their uridylation depends on Tailor. The number of mirtron reads in each library is shown in parentheses. (C) Expression levels of miRNAs in the presence and absence of Tailor. Most mirtron-3p species increase in Tailor-knockdown cells, whereas canonical miRNAs are not directionally affected. (D) Comparisons of w[1118] and Tailor-KO ovaries similarly shows that mirtron-3p species are the dominant class of Tailor-dependent, uridylated substrate. (E) Analysis of individual mirtron-3p species shows their Tailor-dependent modification. (F) Most mirtron-3p species increase in Tailor-knockout ovaries, whereas canonical miRNA species are not directionally affected. For analyses in A and D, all reads mapped to each sRNA class were pooled. For analyses in B–C and E–F, we used only mirtrons with ≥10 3p reads in both datasets under comparison. The box plots in C and F follow Tukey’s standard convention; significance was measured by t-test. See also Figure S4.
Figure 6
Figure 6
Intrinsic activity of Tailor for 3′-G hairpin substrates. (A, B) Analysis of miRNAs with ≥10 reads and ≥1% untemplated uridylation in both paired datasets under comparison. In addition to the dominant uridylation of mirtron-3p reads, canonical miRNA-3p reads exhibit a greater degree of Tailor-dependent untemplated uridylation than do canonical miRNA-5p reads, in S2R+ cells (A) and ovaries (B). Wilcoxon rank-sum test (one-sided) was used to measure statistical significance; mean±SEM shown. (C–F) Analysis of miRNAs with ≥10 reads in the pooled cell line or ovary datasets (see Table S1). Wilcoxon rank-sum one-sided tests, incorporating multiple testing correction by Holm method, measured statistical significance of uridylation fraction enrichment between indicated small RNA classes. (C, D) Untemplated uridylation of mirtrons and canonical miRNA-3p species segregated by terminal nucleotide. While mirtrons exhibit far greater uridylation than canonical miRNAs, those canonical miRNA-3p species ending in G acquire greater uridylation than other miRNA subsets. (E, F) Terminal dinucleotide analyses of canonical miRNA-3p species and their uridylation. miRNAs ending in 3′-AG are more uridylated than other terminal G miRNAs (GG, CG, UG, i.e. “BG”), which in turn acquire greater uridylation than other terminal identities (A, U, C, i.e. “H”). This is true in both cell line (E) and ovary (F) data. (G–J) In vitro tailing assays of substrates bearing different 3′ nucleotides. To highlight substrate preferences, these assays utilize immunopurified Tailor-B material from stable cells, which is not as active as those in Figure 3. (G, H) Tailing assays on mature miR-1010 3′ variants. Tailor is most active on the normal 3′-G isoform, and also exhibits strong activity on the 3′-U variant. (I, J) Tailing assays on pre-mir-1010 3′ variants. Tailor maintains 3′-nt selectivity on hairpins, and tails them more effectively than mature miRNAs. See also Figure S5 and Tables S2 and S3.
Figure 7
Figure 7
Terminal sequence-specific activity of Tailor and correlation with miRNA evolution. (A–C) Analysis of mirtrons and their hybrids in a canonical mir-278 backbone. These include a straight reprogramming of mirtron-3p sequences into mir-278 (including its 3′-AG splice acceptor), and variants where the splice site was altered to AA or GG. Expression constructs were transfected into S2 cells that were manipulated for ldbr or Tailor as indicated, and analyzed by Northern blotting. (A) Dependency of mir-1008 constructs on ldbr. Only the maturation of the unmodified mirtron is sensitive to ldbr depletion, resulting in lariat accumulation (arrow), reduction of pre-miRNA and loss of mature miR-1008. Note that ldbr knockdown cells are relatively poorly transfected, leading to generally decreased products from the canonical miRNA constructs. (B, C) Tailing response of different mir-1008 variants (B) and mir-1003 variants (C) to ectopic Tailor. (D, E) “Wiggle” plots (D) and bar plots (E) comparing relative levels of tailing of mirtron and reprogrammed canonical miRNA substrates. Splicing-derived pre-miRNAs are preferred Tailor substrates, but 3′-G also enhances Tailor modification within the canonical pre-miRNA context. (F) 3′-terminal bias of canonical pre-miRNAs. We compared frequencies of 3′-terminal nucleotides against background frequencies of all bases in canonical miRNA hairpins, separately for young and old loci. There is significant depletion of 3′-G specifically amongst well-conserved canonical miRNA hairpins, as determined by binomial test. (G) Direct comparison of 3′-terminal nucleotide frequency between young and old canonical pre-miRNAs. There is specific depletion of 3′-G amongst well-conserved miRNA hairpins, as determined by Fisher’s exact test. See also Figure S6 and Table S4.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ameres SL, Horwich MD, Hung JH, Xu J, Ghildiyal M, Weng Z, Zamore PD. Target RNA-directed trimming and tailing of small silencing RNAs. Science. 2010;328:1534–1539. - PMC - PubMed
    1. Auyeung VC, Ulitsky I, McGeary SE, Bartel DP. Beyond Secondary Structure: Primary-Sequence Determinants License Pri-miRNA Hairpins for Processing. Cell. 2013;152:844–858. - PMC - PubMed
    1. Axtell MJ, Westholm JO, Lai EC. Vive la différence: biogenesis and evolution of microRNAs in plants and animals. Genome biology. 2011;12:221–221. 213. - PMC - PubMed
    1. Bartel DP, Chen CZ. Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs. Nature genetics. 2004;5:396–400. - PubMed
    1. Berezikov E, Chung WJ, Willis J, Cuppen E, Lai EC. Mammalian mirtron genes. Molecular cell. 2007;28:328–336. - PMC - PubMed

Publication types

MeSH terms

Associated data