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. 2012 Jun 4:3:99.
doi: 10.3389/fgene.2012.00099. eCollection 2012.

Modulation of microRNA Activity by Semi-microRNAs

Isabelle Plante  1 , Hélène Plé, Patricia Landry, Preethi H Gunaratne, Patrick Provost
Affiliations

Modulation of microRNA Activity by Semi-microRNAs

Isabelle Plante et al. Front Genet. .

Abstract

The ribonuclease Dicer plays a central role in the microRNA pathway by catalyzing the formation of 19-24-nucleotide (nt) long microRNAs. Subsequently incorporated into Argonaute 2 (Ago2) effector complexes, microRNAs are known to regulate messenger RNA (mRNA) translation. Whether shorter RNA species derived from microRNAs exist and play a role in mRNA regulation remains unknown. Here, we report the serendipitous discovery of a 12-nt long RNA species corresponding to the 5' region of the microRNA let-7, and tentatively termed semi-microRNA, or smiRNA. Using a smiRNA derived from the precursor of miR-223 as a model, we show that 12-nt long smiRNA species are devoid of any direct mRNA regulatory activity, as assessed in a reporter gene activity assay in transfected cultured human cells. However, smiR-223 was found to modulate the ability of the microRNA from which it derives to mediate translational repression or cleavage of reporter mRNAs. Our findings suggest that the 12-nt RNA species, generated along the microRNA pathway, may participate to the control of gene expression by regulating the activity of the related full-length mature microRNA in vivo.

Keywords: Argonaute 2; Dicer; RNA silencing; gene regulation; microRNA; non-coding RNA.

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Figures

Figure 1
Figure 1
Detection of a 12-nt long RNA sequence derived from hsa-let-7 microRNA. (A) Sequences obtained upon analysis of primary human platelet small RNAs by high-throughput sequencing (HTS) were aligned to the human genome and mapped to microRNA genes (miRBase Release 18). The five most abundant RNA species mapping to the hsa-let-7c pre-microRNA are shown. More than 4004 sequences reads represented mature let-7c microRNA, whereas 63 sequences derived from the 5′ half of mature let-7c microRNA. (B) Secondary structure of the hsa-pre-let-7c, as predicted by MFOLD. The arrowheads indicate the Drosha and Dicer cleavage sites that yield mature hsa-let-7c microRNA, which may be further processed into a 12-nt hsa-let-7c RNA species half the size of the mature hsa-let-7c microRNA. Tentatively termed semi-microRNA (or smiRNA), these 12-nt hsa-let-7c RNAs may be generated upon direct processing of the 5′ end of the pre-microRNA species. (C) Sequences obtained upon analysis of primary human neutrophil small RNAs by HTS, as in A. RNA species of a length (14–16 nt) similar to smiRNAs, mapping to the hsa-miR-223 pre-microRNA, are shown.
Figure 2
Figure 2
smiRNAs may not directly mediate gene silencing through mRNA cleavage. (A) Predicted base pairing of smiR-223 or smiR-223 3′ to a binding site (BS) of perfect complementarity (PC) to miR-223. (B) HEK 293 cells were co-transfected with a synthetic smiR-223 (25–250 pmol; left) or smiR-223 3′ (25–250 pmol; right) and a psiCHECK reporter construct, in which the Rluc reporter gene is coupled with a BS of PC to miR-223 (50 ng). An unrelated smiRNA was used as a normalization control. The Dual Luciferase assays were performed as described previously (Boissonneault et al., 2009). Results are expressed as mean ± standard error of the mean (s.e.m.; n = 2, in duplicate).
Figure 3
Figure 3
smiRNAs may not directly mediate gene silencing through mRNA translational repression. (A) Predicted base pairing of smiR-223 or smiR-223 3′ to three copies of the wild-type (WT) or mutated (MUT) binding site (BS) to miR-223. (B) HEK 293 cells were co-transfected, at ∼50% confluence in 24-well plates, with a synthetic smiR-223 (25–250 pmol; left) or smiR-223 3′ (25–250 pmol; right) and a psiCHECK reporter construct in which the Rluc reporter gene is coupled with three copies of the WT or MUT BS for miR-223. An unrelated smiRNA was used as a normalization control. The Dual Luciferase assays were performed as described previously (Boissonneault et al., 2009). Results are expressed as mean ± standard error of the mean (SEM; n = 4, in duplicate).
Figure 4
Figure 4
smiRNAs may regulate the gene silencing properties of microRNAs. (A,B) HEK 293 cells were co-transfected with a synthetic smiR-223 (0, 25, or 50 pmol), a psiSTRIKE vector expressing pre-miR-223 (2.5 or 25 ng) and a psiCHECK reporter construct (50 ng), in which the Rluc reporter gene is coupled either (A) to a binding site (BS) of perfect complementarity (PC) to miR-223, or (B) three copies of the wild-type (WT) or mutated (MUT) BS for miR-223. An unrelated smiRNA and a construct expressing an unrelated pre-microRNA were used as normalization controls. The Dual Luciferase assays were performed as described previously (Boissonneault et al., 2009). Results are expressed as mean ± standard error of the mean (s.e.m.; n = 2–4, in duplicate). *p <  0.05 or **p < 0.01 versus 0 pmol smiR-223 (A) or versus the 3×BS MUT (B) (Student’s t test).
Figure 5
Figure 5
5′ Phosphorylation status of small RNAs. Small RNAs (<200 nt) extracted from cultured HEK 293 cells (left panel, lanes 1–3) or primary human platelets (right panel, lanes 4–6) were either dephosphorylated with calf intestine phosphatase (+; lanes 1 and 4), or not (−; lanes 2, 3, 5, and 6), and 5′ end labeled with 32P-ATP using T4 polynucleotide kinase (lanes 1, 2, 4, and 5). 32P-labeled ATP incorporation at the 5′ end of small RNAs was analyzed by denaturing PAGE and autoradiography.
Figure 6
Figure 6
Proposed model for the biogenesis and function of smiRNAs. In the canonical microRNA pathway of human cells, microRNAs are generated upon pre-microRNA processing by the complex formed of Dicer and its cofactor TRBP, and are subsequently incorporated into a microRNP effector complex containing Ago2 and active in regulating mRNA translation (right-hand side with black arrows). Twelve-nt long smiRNA species may be produced (1) by the sequential processing of the passenger-strand by Ago2 and removal of the loop by Dicer, or by Ago2 cleavage of the passenger-strand of microRNA duplexes (Diederichs and Haber, 2007) (left-hand side with dotted arrows). (2) smiRNA species may also be produced upon pre-microRNA processing by Dicer in the presence of a Dicer-interacting protein (IP), such as 5LO, which interacts with the C-terminal RNase III domains of Dicer and possibly modifies its enzymatic activity (Dincbas-Renqvist et al., 2009). smiRNAs may remain bound to its complementary microRNA to form an intermediate microRNA:smiRNA RNP complex, which may contribute to preserve the stability and functional integrity of microRNP complexes. (3) smiRNA species may derive either from the mature microRNA or the passenger-strand. (4–6) smiRNA-regulated microRNA activity. (4) By base pairing with microRNAs, smiRNAs may contribute to preserve the effector complex until it encounters its destined mRNA target, (5) which may displace the smiRNA and allow microRNA-regulated control of mRNA translation. Similarly, smiRNAs derived from the mature microRNA strand may regulate the function of the passenger-strand through partial base pairing complementarity (not shown in the Figure). (6) Albeit shorter, smiRNAs share nucleotide sequence identity and may compete with the microRNA from which it derives for the recognition of a binding site (BS) located in the 3′ UTR of a mRNA target, thereby modulating microRNA activity. Adapted from Ouellet et al. (2006).
Figure A1
Figure A1
Predicted secondary structure of the precursors of the let-7 12-nt RNA species. The mature microRNA species usually derive from the 5p (in red) and 3p (in green) arms of the microRNA precursor (pre-) stem and exhibit a relatively high degree of complementary. The putative smiRNA sequence derived from let-7 and obtained through HTS is as follows: 5′-UGAGGUAGUAGG-3′ (in bold red). This smiRNA is common to the let-7a, let-7b, and let-7c microRNAs, and originates from the 5′ half of the mature let-7 microRNA species.
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