Pre-miRNA loop nucleotides control the distinct activities of mir-181a-1 and mir-181c in early T cell development - PubMed (original) (raw)

Pre-miRNA loop nucleotides control the distinct activities of mir-181a-1 and mir-181c in early T cell development

Gwen Liu et al. PLoS One. 2008.

Abstract

Background: Mature miRNAs can often be classified into large families, consisting of members with identical seeds (nucleotides 2 through 7 of the mature miRNAs) and highly homologous approximately 21-nucleotide (nt) mature miRNA sequences. However, it is unclear whether members of a miRNA gene family, which encode identical or nearly identical mature miRNAs, are functionally interchangeable in vivo.

Methods and findings: We show that mir-181a-1, but not mir-181c, can promote CD4 and CD8 double-positive (DP) T cell development when ectopically expressed in thymic progenitor cells. The distinct activities of mir-181a-1 and mir-181c are largely determined by their unique pre-miRNA loop nucleotides-not by the one-nucleotide difference in their mature miRNA sequences. Moreover, the activity of mir-181a-1 on DP cell development can be quantitatively influenced by nucleotide changes in its pre-miRNA loop region. We find that both the strength and the functional specificity of miRNA genes can be controlled by the pre-miRNA loop nucleotides. Intriguingly, we note that mutations in the pre-miRNA loop regions affect pre-miRNA and mature miRNA processing, but find no consistent correlation between the effects of pre-miRNA loop mutations on the levels of mature miRNAs and the activities of the mir-181a-1/c genes.

Conclusions: These results demonstrate that pre-miRNA loop nucleotides play a critical role in controlling the activity of miRNA genes and that members of the same miRNA gene families could have evolved to achieve different activities via alterations in their pre-miRNA loop sequences, while maintaining identical or nearly identical mature miRNA sequences.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. An in vitro assay for measuring the activities of miRNA genes on T cell differentiation.

(A) Schematics depicting the OP9-DL1 stromal co-culture assay for T cell differentiation. (B) Box-plots to summarize the effects of mir-181a-1 on the percentage of DP cells differentiated from DN progenitor cells. The results of a representative OP9-DL1 stromal co-culture assay (12 independent replicates for each construct) are shown. (C) Normalized box-plots. The activities of mir-181a-1 in DP cell development were normalized so that the empty vector (negative control) had a median activity of “0” and mir-181a-1 expressing vector (positive control) had a median activity of “1.” (D) Representative FACS plots showing the effects of mir-181a-1 on DP cell development (gated on infected GFP cells).

Figure 2

Figure 2. Effects of the mutations in the stem region on mir-181a-1 activity in promoting DP cell development.

(A) Scanning mutations in the stem region of the mir-181a-1 gene. Two nucleotides (2-nt mutants, shaded yellow) or a stretch of nucleotides (segment mutants, green) in the mature miRNA region are altered (shown in red). Nucleotides are altered to disrupt the potential base pairing to target genes. Compensatory mutations are also generated on the miR* strand to maintain the secondary structure of the pre-miRNAs. (B) Expression and processing of wild-type mir-181a-1 and stem mutants. Specific probes that perfectly match the mature miR-181a and each of its mutant forms were used in hybridization to determine the expression of mature miR-181a and its stem mutant forms. (C) The effects of mir-181a-1 and its stem mutants on DP cell development. Normalized data from 3–5 independent T cell assays (each with 12 independent replicates, total 36–60 replicates) are pooled and graphed in the distribution box plots to summarize the distribution of the relative activities of mir-181a-1 (shaded grey), the 2-nt mutants (shaded yellow), and the segment mutants (shaded green) in DP cell development. Mann-Whitney Rank Sum Tests were performed to determine whether the activities of individual 2-nt mutants were statistically different from those of the control vector (red *, p<0.0001) and the mir-181a-1 vector (black *, p<0.0001; **, p<0.05) (Table S1 for statistics). A representative OP9-DL1 stromal co-culture assay without normalization (12 independent replicates for each constructs) is also shown (Fig. S5).

Figure 3

Figure 3. The pre-miRNA loop nucleotides control the distinct activities of mir-181a-1 and mir-181c on DP cell development.

(A) Nucleotide sequences of mature miR-181a and miR-181c. (B) Schematics and nucleotide sequences depicting the wild-type mir-181a-1 and mir-181c genes and corresponding precursors (shaded grey). Also shown are the chimeric miRNA genes, with the mature miRNAs, pre-miRNAs, and pre-miRNA loops swapped between mir-181a-1 and mir-181c, termed “mature-chimeric” (shaded yellow), “pre-chimeric” (shaded orange), and “loop-chimeric” (shaded blue), respectively. These mutant genes are designated as 181a(c stem), 181c(a stem), 181a (pre-181c), 181c (pre-181a), 181a (c-loop), and 181c (a-loop). Similar color codes are used in all figures. (C) The effects of the chimeric mir-181a-1/c genes on DP cell development. Normalized data from 3–7 independent T cell assays (each with 12 independent replicates for a total of 36–84 replicates) are pooled and graphed in the distribution box plots. Mann-Whitney Rank Sum Tests were performed (Table S2 for statistics) to determine whether the activities of the chimeric miRNA genes are statistically different from those of the negative control vector (red *, p<0.0001) and/or mir-181a-1 positive control (black *, p<0.0001). A representative OP9-DL1 stromal co-culture assay without normalization is also shown (Fig. S6).

Figure 4

Figure 4. The activity of mir-181a-1 is sensitive to nucleotide changes in its pre-miRNA loop.

(A) Schematics of the pre-miR-181a-1 loop mutants. (B) The effects of pre-miR-181a-1 loop mutants on DP cell development. Normalized data from six independent T cell assays (each with 12 independent replicates for a total of 72 replicates) is shown. Mann-Whitney Rank Sum Tests were performed (Table S3 for statistics) to determine whether the activities of the loop mutants were statistically different from those of the negative control vector (red *, p<0.0001) and/or the mir-181a-1 positive control vector (black *, p<0.0001). A representative OP9-DL1 stromal co-culture assay (12 independent replicates) without normalization is also shown (Fig. S7).

Figure 5

Figure 5. Mature miRNAs produced from the mir-181a-1/c mutants have the same 5′ ends.

The 5′ ends of mature miR-181a (A) and miR-181c (B) produced from mir-181a-1/c domain swapping mutant genes, and the 5′ ends of mature miR-181a (C) produced from mir-181a-1 loop mutant genes were determined by primer extension analyses. Synthetic miR-181a or miR-181c oligo nucleotides in single nucleotide increments (15 nt–22/23-nt) were radio-labeled and used as size ladders. The upper band represents the major cDNA product of miR-181a (23-nt) or miR-181c (22-nt), and the lower band represents radio-labeled probes for the mature miRNAs.

Figure 6

Figure 6. The effects of mir-181a-1/c mutants on mature miRNA expression in BOSC 23 and DP cells.

(A, B) The copy numbers of mature miR-181a (A) and miR-181c (B) expressed in BOSC 23 cells transfected with the same amounts of vectors expressing different mir-181a-1/c mutants determined by quantitative Northern blot analyses (Fig. S8 for standard curves and quantification, and Table S4 for statistics). (C, D) The copy numbers of mature miR-181a (C) and miR-181c (D) expressed in DP thymocytes transduced with viral vectors expressing mir-181a-1/c mutants determined by miRNA qPCR analyses (Tables S5 and S6 for statistics). (E) The copy numbers of mature miR-181a expressed in BOSC 23 cells transfected with the same amounts of viral vectors expressing various mir-181a-1 loop mutants determined by quantitative Northern blot analyses (Fig. S9 for standard curves, and Table S7 for statistics). (F). The copy numbers of mature miR-181a expressed in DP thymocytes transduced with viral vectors expressing various mir-181a-1 loop mutants, determined by miRNA qPCR analyses (Table S8 for statistics). Statistical significance was determined by an unpaired two-tailed student's t test (compared to the control vector, red *, p<0.05). Representative blots of four or more independent quantitative Northern blot analyses are shown (A, B, E).

Figure 7

Figure 7. Phylogenetic comparison of pre-miR-181a-1 and pre-miR-181c loop sequences.

The full genus and species names and their abbreviations are as follows: Danio rerio, dre; Fugu rubripes, fru; Homo sapiens, hsa; Gallus gallus, gga; Gorilla gorilla, ggo; Lagothrix lagotricha, lla; Macaca mulatta, mml; Mus musculus, mmu; Macaca nemestrina, mne; Pan paniscus, ppa; Pongo pygmaeus, ppy; Pan troglodytes, ptr; Rattus norvegicus, rno; Sus scrofa, ssc; Tetraodon nigroviridis, tni.

Figure 8

Figure 8. A “heat map” of the functionally important nucleotides in the pre-miR-181a-1 region according to mutagenesis analyses.

Color-code was used to illustrate the importance of the pre-miRNA nucleotides to the activity of the miR-181a-1 gene. Possible mechanisms by which pre-miRNA loop nucleotides control the activities of miRNA genes are listed.

References

    1. Bushati N, Cohen SM. microRNA Functions. Annu Rev Cell Dev Biol. 2007;23:175–205. -PubMed
    1. Kim VN. MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol. 2005;6:376–385. -PubMed
    1. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993;75:843–854. -PubMed
    1. Reinhart BJ, Slack FJ, Basson M, Bettinger JC, Pasquinelli AE, et al. The 21 nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature. 2000;403:901–906. -PubMed
    1. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian microRNA targets. Cell. 2003;115:787–798. -PubMed

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