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Uridine Addition After MicroRNA-Directed Cleavage

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Science  05 Nov 2004:
Vol. 306, Issue 5698, pp. 997
DOI: 10.1126/science.1103521

Abstract

One of the important roles of microRNA (miRNA) is to direct the cleavage of messenger RNA (mRNA). However, the mechanisms of decay of the cleaved mRNA products is not well understood. We show that miRNA-directed cleavage products in organisms as diverse as Arabidopsis, mouse, and Epstein-Barr virus have at their 3' ends a stretch (1 to 24 nucleotides) of oligouridine posttranscriptionally added downstream of the cleavage site. This 3' uridine addition, as shown for Arabidopsis, is correlated with decapping and 5' shortening of the cleaved products, suggesting a mechanistic step in the miRNA-directed mRNA decay mechanism.

MicroRNA (miRNA) directs mRNA cleavage, but the molecular mechanisms involved in the post-cleavage mRNA decay are poorly understood. We attempted to clone the decaying intermediates of miRNA-directed cleavage in Arabidopsis, using adapter-ligation reverse transcription–polymerase chain reaction (alRT-PCR). We found a stretch [1 to 9 nucleotides (nt)] of nonencoded uridines in the sequence of the cleaved mRNA targets, uniquely inserted downstream (3′) of the cleavage site (Fig. 1, A and B).

Fig. 1.

(A) Direct sequencing of alRT-PCR product of MYB33 mRNA, showing uridine addition (=) at the 3′ end of the cleavage site (▽). (B) Nonencoded oligouridine signature from Arabidopsis (At), mouse (Mm), and Epstein-Barr virus (EBV), with number (†) and frequency (§) of signature-bearing clones among clones (‡) derived from miRNA-directed cleavage. NA, not available. (C) Summary of cRT-PCR result of MYB33 from tobacco acid pyrophosphatase (TAP)- and calf intestine alkaline phosphatase (CIP)/TAP-treated RNA (1), showing 3′ uridine addition (=) and 5′ shortening (number). Each line represents one or multiple (in parentheses) clones. Also shown are PCR primers (P1, P2, and P3), distance (nt) from P1 to transcript start and P3 to cleavage site (▽), and presence (+) versus absence (–) and correlations (5c/3s) of 5′ cap (5c) and 3′ signature (3s).

On the basis of this finding in Arabidopsis, we revised our approach for identifying miRNA-directed cleavage by using alRT-PCR to select amplified 3′ oligouridinated RNA (1). We used this approach to clone the decaying intermediate of mouse HoxB8 mRNA and, using a human cell culture system, validated that the cleavage is directed by miR196 (2, 3). Note that nonencoded oligouridines were also found downstream (3′) of the cleavage site of the mouse HoxB8 mRNA (Fig. 1B).

Nonencoded oligonucleotides (5 to 24 nt), mostly adenine and uridine, have been reported at the 3′ termini of the shorter transcript of the Epstein-Barr (EB) virus DNA polymerase mRNA (Fig. 1B) (4). A recent report suggests that this shorter transcript is a result of cleavage directed by the virus-encoded miRNA (5). The added nucleotides in the cloned EB virus intermediates, however, are often longer (Fig. 1B), possibly because of the protection from 3′-5′ exonucleases by the polyadenylate tails that follow (4). On the basis of our results from Arabidopsis and mouse and of the EB virus reports (4, 5), we conclude that the presence of 3′ added oligouridine is a signature of miRNA-directed mRNA cleavage.

To investigate possible roles of the oligouridine signature in (de)stabilizing the cleaved transcript, we analyzed two Arabidopsis genes, ARF10 and MYB33, by an RT-PCR approach that relies on mRNA circularization (cRT-PCR) and that allows for the simultaneous analysis of the 5′ and 3′ ends of the same mRNA molecule. For both mRNAs analyzed, this strategy confirmed the presence of the 3′ oligouridine signature (Fig. 1C) (2). Although the 3′ uridine additions are variable, the degree of 5′ shortening is more marked among 5′ cleavage products (Fig. 1C) (2), which suggests that, besides 3′ decay, the 5′ cleavage products are primarily degraded from the 5′ end. In both mRNAs, it also revealed a correlation between 3′ oligouridine addition and 5′ shortening (Fig. 1C) (2), suggesting a role for the 3′ oligouridine in enhancing mRNA 5′ decay.

miRNA functions by recruiting the RNA-induced silencing complex and guiding mRNA cleavage near the center of the target site complementary to the miRNA (Fig. 1A). It is believed that the two mRNA fragments thus generated, 5′ and 3′ cleavage products, are subsequently degraded at the newly exposed ends by the 3′-5′ exonuclease–containing exosome and by 5′-3′ exonucleases such as AtXRN4 (6), respectively. Our result further shows that after miRNA-directed cleavage, an oligouridine track is added to the 3′ end of the 5′ cleavage product, and as a result, presumably enhances 5′ end decay of the later. This is consistent with the often unusually rapid decay of the 5′ cleavage products (6, 7), some of which might otherwise be translated into potentially toxic proteins.

The finding of an oligouridine signature posttranscriptionally added to miRNA-directed cleavage products in species as diverse as Arabidopsis, mouse, and EB virus implies that it has general importance. One of its roles, as suggested by our study in Arabidopsis, is likely to enhance decay of the cleaved transcripts. Future studies should reveal the mechanisms of uridine addition (sometimes adenine as well), its biological functions, and the extent to which currently known RNA processing machinery is involved.

Supporting Online Material

www.sciencemag.org/cgi/content/full/306/5698/997/DC1

Materials and Methods

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