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. 2009 Oct 27;4(10):e7566.
doi: 10.1371/journal.pone.0007566.

Alternative processing of primary microRNA transcripts by Drosha generates 5' end variation of mature microRNA

Haoquan Wu  1 , Chunting Ye, Danielle Ramirez, N Manjunath
Affiliations

Alternative processing of primary microRNA transcripts by Drosha generates 5' end variation of mature microRNA

Haoquan Wu et al. PLoS One. .

Abstract

Background: It is generally believed that the miRNA processing machinery ensures the generation of a mature miRNA with a fixed sequence, particularly at its 5' end. However, we and others have recently noted that the ends of a given mature miRNA are not absolutely fixed, but subject to variation. Neither the significance nor the mechanism behind the generation of such miRNA polymorphism is understood. miR-142 is an abundantly expressed miRNA in hematopoietic cells and exhibits a high frequency of 5' end polymorphism.

Methodology/principal findings: Here we show that a shift in the Drosha processing of pri-miRNA generates multiple forms of miR-142s in vivo with differing 5' ends that might target different genes. Sequence analysis of several pre-miRNA ends cloned from T cells reveals that unlike many other pri-miRNAs that are processed into a single pre-miRNA, pri-miR-142 is processed into 3 distinct pre-miR-142s. Dicer processing studies suggest that each of the 3 pre-miR-142s is processed into a distinct double-stranded miRNA, giving rise to 4 mature miRNA variants that might regulate different target gene pools.

Conclusions/significance: Thus, alternative Drosha processing might be a novel mechanism for diversification of the miRNA target gene pool.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. Drosha-DGCR8 process most pri-miRNAs precisely into one pre-miRNA.
(A) Schematic of pre-miRNA cloning (see experimental procedures for details). (B) The end cloned and assembled pre-miRNAs are shown. The Drosha cleavage site (deduced from the cloning frequency analysis shown in Table 2) is indicated by arrows. The annotated mature miRNA sequence in miRBase is marked in red.
Figure 2
Figure 2. Alternative processing by Drosha-DGCR8 generates three pre-miR-142s from one pri-miR-142.
(A). Frequency analysis of the different pre-miR-142 ends cloned. Sequences exhibiting one or two nt internal mismatches were included in the list. (B). Schematic of the generation of 3 pre-miR-142s from one pri-miR142. The alternative Drosha cleavage sites deduced from the cloning data are indicated by arrows. The numeric values represent variations with respect to the predicted cleavage site (0) according to the model proposed by Han et al. The bottom panel shows how alternative processing of pri-miR142 generates 3 pre-miR-142s. (C). Full length pre-miR-142s were pulled down with biotinylated antisense oligos and cloned to visualize the entire sequence. The mature miRNA sequence designated in miRBase is marked in red.
Figure 3
Figure 3. Dicer cuts precisely 22 nt from the end set by Drosha.
(A) Synthesized pre-miR-142-2 and pre-miR-142-3 were digested with Dicer, the products separated on 15% urea gel and stained with SybrGold. 10 bp DNA ladder and 21, 22, and 23 nt synthetic RNA oligos were used as molecular weight markers. Note that the marker 21–23 nt RNA oligos lack the 5′ phosphate that Dicer-processed products posses and thus the 22 nt long miR-142s appear to migrate a little faster. The markings F1+F2+L, F1 or F2+L, F1 or F2 and L refers to the undigested, partially digested, fully digested fragments and the terminal loop respectively in lanes 3 and 4. (B). Dicer-processed pre-miR-142-3 fragments were cloned and sequenced. F1 or F2+L represents incompletely digested product (nicked at either 5′ or 3′end), F1 or F2 represents Dicer processed miRNA fragments and L represents the terminal loop. Only the sequences cloned more than twice are listed.
Figure 4
Figure 4. Pri-miR-342 is also alternatively processed by Drosha to generate 2 pre-miRNAs.
(A). Cloned pre-miR-342 end frequencies. (B). Schematic showing the processing of Pri-miR-342 into 2 pre-miR-342s. The cleavage sites are indicated with arrows and the mature miRNA sequence designated in the miRBase marked in red.
Figure 5
Figure 5. A model for the generation of mature miRNA end polymorphism.
(A). The biogenesis of class I miRNA (represented by miR-150): here the Drosha cleavage is precise and generates only one pre-miRNA from the pri-miRNA and hence the end is fixed and does not show polymorphism. Since the end of pre-miRNA is homogenous and Dicer processing is also precise, the 3′ end polymorphism seen in the mature miRNA is more likely generated subsequent to Drosha/Dicer processing. (B). The biogenesis of class II miRNAs. Here, because alternative processing of pri-miRNA by Drosha generates multiple pre-miRNAs that are each processed precisely by Dicer, the 5′ end of mature miRNA exhibits polymorphism. The 3′ end polymorphism arises after Drosha/Dicer processing as explained in (A).
Figure 6
Figure 6. Different models for the selection of both strands of a miRNA.
Two mechanisms might operate for both strand selection: Model A as proposed by Schwarz et al , where the termini of ds-miRNA duplex have similar thermodynamic stability and both strands in the ds-miRNA are selected. Model B might operate in cases where the two strands are derived from different ds-miRNAs, one with thermodynamic feature favoring selection of 5p and the other favoring selection of 3p. This mechanism might be responsible when the two selected strands do not form a perfect ds-miRNA with 2 nt overhangs.
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