A 5′-uridine amplifies miRNA/miRNA* asymmetry in Drosophilaby promoting RNA-induced silencing complex formation
© Seitz et al; licensee BioMed Central Ltd. 2011
Received: 26 January 2011
Accepted: 7 June 2011
Published: 7 June 2011
MicroRNA (miRNA) are diverse in sequence and have a single known sequence bias: they tend to start with uridine (U).
Our analyses of fly, worm and mouse miRNA sequence data reveal that the 5′-U is recognized after miRNA production. Only one of the two strands can be assembled into Argonaute protein from a single miRNA/miRNA* molecule: in fly embryo lysate, a 5′-U promotes miRNA loading while decreasing the loading of the miRNA*.
We suggest that recognition of the 5′-U enhances Argonaute loading by a mechanism distinct from its contribution to weakening base pairing at the 5′-end of the prospective miRNA and, as recently proposed in Arabidopsis and in humans, that it improves miRNA precision by excluding incorrectly processed molecules bearing other 5′-nt.
MicroRNA (miRNA) are approximately 22-nt regulatory RNA that direct members of the Argonaute protein family to their mRNA targets . Together, miRNA guide and the Argonaute protein form the core of the RNA-induced silencing complex (RISC), which recognizes its mRNA targets primarily through its seed sequence, nt 2 through nt 7 .
The RNase III enzymes Drosha and Dicer excise most animal miRNA from long primary transcripts (pri-miRNA). Drosha cleaves pri-miRNA to release an approximately 65-nt pre-miRNA; Dicer cleaves the pre-miRNA to liberate a miRNA/miRNA* duplex. The duplex is then loaded into an Argonaute protein. The geometry of the miRNA/miRNA* duplex during the loading reaction determines the fate of each small RNA: the miRNA binds tightly to Argonaute, with its 5′-nt anchored in a positively charged pocket in the Mid domain of the protein [3, 4]. The miRNA* assumes the same position as subsequent mRNA targets and is held to the complex predominantly by seed sequence base pairing. A seed sequence mismatch between the miRNA and its miRNA* is believed to promote miRNA* dissociation [5, 6]. A subset of Argonaute proteins can cleave the miRNA* if it is extensively paired to the miRNA, triggering its destruction [7–10]. The orientation of the duplex during Argonaute loading is not random: the miRNA is usually the strand with the less stably paired 5′-end in the duplex [11, 12]. Consequently, the duplex liberated by Dicer determines the identity of the miRNA.
miRNA sequences are diverse, and only one common sequence motif has been identified. Most miRNA begin with a 5′-uridine (5′-U). In plants, a 5′-U directs miRNA to AGO1, small RNA that begin with adenosine (A) load AGO2 and those that start with cytidine (C) load AGO5 [13–15]. Likewise, the 5′-nt of fly small RNA participates in sorting, with a 5′-U directing small RNA toward Ago1 and a 5′-C favoring Ago2 [16–19]. In mammals, the Mid domain of Ago2, the homolog of Drosophila Ago1, specifically recognizes a 5′-U or 5′-A , explaining why miRNA tend to start with those nucleotides, but fly and worm miRNA typically begin with 5′-U but not 5′-A. Moreover, small RNA sorting in flies and worms also reflects the secondary structure of the miRNA/miRNA* duplex, with centrally paired duplexes preferentially loaded into one Argonaute, - Ago2 in flies and RDE-1 in worms, - and duplexes bearing a central mismatch directed toward the major miRNA-binding Argonautes, - Ago1 in flies and the paralogous ALG-1/ALG-2 proteins in worms [5, 6, 17–19, 21–23].
We investigated the function of 5′-U in animal miRNA. Our statistical analyses of sequencing data from flies, worms and mice reveal that 5′-U is recognized after miRNA/miRNA* production by Dicer cleavage of the pre-miRNA. Our experimental results show that 5′-U facilitates loading of miRNA while decreasing loading of miRNA*, consistent with the view that only one of the two strands can be assembled from a single miRNA/miRNA* molecule. Our data support the view that 5′-U enhances RISC assembly by a mechanism distinct from its contribution to destabilizing base pairing at the 5′-end of miRNA. Similarly to what has been proposed in Arabidopsis thaliana and in Homo sapiens [13, 20], our data also suggest that recognition of the first miRNA nucleotide during loading may select against incorrectly processed molecules bearing 5′-nt other than 5′-U.
Results and discussion
5′-U acts after miRNA processing
In theory, a 5′-U might facilitate Drosha cleavage of the pri-miRNA or pre-miRNA export from the nucleus. Such a role for a 5′-U would be reflected in a greater likelihood of both miRNA and miRNA* derived from the 5′-arm of the pre-miRNA stem to begin with U compared to those residing in the 3′ arm. We compared the approximately 40% of fly, 35% of worm and 50% of mouse miRNA that reside in the 5′-arm of their pre-miRNA to their 3′ counterparts. Our analysis argues against a role for a 5′-U in Drosha processing or nuclear export. miRNA tend to start with a U, regardless of their position in the pre-miRNA (Figure 1, Additional file 1, Figure S1, and Additional file 2, Figure S2). Moreover, miRNA* sequences tend not to begin with U, even when they derive from the pre-miRNA 5′-arm. Our data similarly exclude a role for a 5′-U in cleavage of the pre-miRNA by Dicer, which would favor a 5′-U for miRNA and miRNA* derived from the 3′-arm.
miRNA asymmetry correlates with first nucleotide identity
Strikingly, the most asymmetric miRNA also exhibit a lower than expected frequency of 5′-A (Figure 2, top left), whereas the thermodynamic stability rule would have predicted a high frequency of both U and A. This observation suggests that 5′-nt identity, not just thermodynamic asymmetry, contributes to the differential loading of miRNA and miRNA* in vivo.
Initial nucleotide identity influences miRNA loading in vitro
Both authentic miR-2a and miR-2a-1* begin with U; the 5′-U of miR-2a is paired to A19 of miR-2a-1*. Inverting this U:A base pair so that miR-2a began with A nearly halved the amount of miRNA assembled into RISC and more than doubled the amount of miR-2a-1* (Figure 3A). Thus, a change in the identity of the first nucleotide of the miRNA decreased the efficiency of assembly of the miRNA into RISC and increased assembly of the miRNA* while preserving the relative thermodynamic asymmetry of the duplex.
When the initial U:A base pair of miR-2a/miR-2a-1* was altered, UU assembled more miRNA into RISC than did AA (Figure 3B). Notably, an AA mismatch at the 5′-end of the miRNA more than doubled the amount of miRNA* incorporated into RISC. Next, we examined a series of miR-2a/miR-2a* derivatives in which the 19th base of miR-2a* was always C, ensuring that duplex stability was the same when the miRNA began with U or A. Again, a 5′-U favored miRNA loading and disfavored miRNA* loading (Figure 3C). When the 5′-U was replaced with inosine, which can pair to the miRNA* C at position 19, only slightly less miRNA was assembled into RISC than that observed for an A/C mismatch. We conclude that the identity of the first miRNA nucleotide contributes more to the loading of miR-2a than do differences in the stability of the duplex termini. Reciprocally, when the first miRNA nucleotide was C, the identity of miRNA* nt 19 did not have any significant effect on miRNA or miRNA* loading (Figure 3D), demonstrating that the effect shown in Figure 3A reflects a mutation of the first miRNA nucleotide, not the change in miRNA* nt 19. Experiments using miR-14 and miR-184 gave similar results (Additional file 5, Figure S5).
Strikingly, the order of preference for nt 1 was not the same across the three tested miRNA: miR-2a preferred U > A > C (Figure 3), miR-14 preferred U ~ C > A and miR-184 preferred U ~ A > C (Additional file 6, Figure S6). Hence additional features in the miRNA/miRNA* duplex must influence the order of preference for miRNA nt 1. Mutating the overhanging nucleotide in miR-184* did not alter the efficiency of loading miR-184 (Additional file 7, Figure S7), excluding a role for base pairing between nt 1 and the 3′ overhang of the miRNA*.
Covarying features in miRNA/miRNA* duplexes suggest that the identity of nt 2 affects the order of preference for miRNA nt 1
Strikingly, the influence of nt 2 on nt 1 seems to be specific for flies. Neither worm nor mouse miRNA/miRNA* show such covariation (Additional file 8, Figure S8). Caenorhabditis elegans miRNA nt 1 covaries mostly with the base-pairing status of miRNA nt 18 and the identity of the miRNA* nucleotide facing miRNA nt 3. In mouse, nt 1 covaries with the identity of miRNA nt 12 as well as several positions at the 3′ end of the miRNA strand. The sequence composition of miRNA differs greatly between flies and humans , suggesting that the nucleotide preference of the miRNA loading machinery has evolved since the divergence of protostomes and deuterostomes, with only the overall tendency for miRNA to start with U remaining conserved.
Our data support the view that a U at the 5′-end of a miRNA favors RISC loading in flies and, given both our informatics data and the broad phylogenetic conservation of the 5′-U bias among miRNA in worms and mice, likely in animals generally.
The Drosophila Ago1 loading machinery remains to be identified, although chaperones have been implicated in assembling miRNA into RISC [6, 27, 28]. It is tempting to speculate that the requirement for the miRNA 5′-end to be the less thermodynamically stable in a miRNA/miRNA* duplex reflects the need for the first nucleotide to be single-stranded to present it to components of the RISC loading machinery or to Ago1 itself.
Why has the miRNA pathway evolved to prefer a 5′-U? The likely answer is that preferential loading of miRNA starting with U improves the precision of the miRNA 5′-end . Drosha and Dicer generate pools of miRNA/miRNA* duplexes with alternative 5′- and 3′-ends; loading of these duplexes into Drosophila Ago2, -which prefers 5′-C, - has been shown to purify this population of miRNA , loading preferentially the miRNA isoforms bearing a 5′-C [19, 25]. The preference of the Ago1 loading machinery or of Ago1 itself for 5′-U could similarly restrict entry into the Ago1 pathway by loading only miRNA isoforms that begin with U. Consistent with this idea, the pre-miRNA nucleotides flanking miRNA nt 1 tend to be depleted in U (Additional file 9, Figure S9). Such a purifying selection could ensure that most mature miRNA have the correct 5′-end and therefore the correct seed sequence, ensuring that they regulate the appropriate mRNA targets.
Synthetic oligonucleotides used in this studya
Sequence (5′ to 3′)
miR-2a with P1 U
UAU CAC AGC CAG CUU UGA UGA GC
miR-2a with P1 A
AAU CAC AGC CAG CUU UGA UGA GC
miR-2a with P1 I
IAU CAC AGC CAG CUU UGA UGA GC
miR-2a with P1 C
CAU CAC AGC CAG CUU UGA UGA GC
miR-2a with P1 G
GAU CAC AGC CAG CUU UGA UGA GC
miR-2a-1* with P19 A
UCU CAA AGU GGU UGU GAA AUG
miR-2a-1* with P19 U
UCU CAA AGU GGU UGU GAA UUG
miR-2a-1* with P19 C
UCU CAA AGU GGU UGU GAA CUG
miR-2a-1* with P19 I
UCU CAA AGU GGU UGU GAA IUG
miR-184 with P1 U
UGG ACG GAG AAC UGA UAA GGG C
miR-184 with P1 A
AGG ACG GAG AAC UGA UAA GGG C
miR-184 with P1 C
CGG ACG GAG AAC UGA UAA GGG C
miR-184 with P1 T
TGG ACG GAG AAC UGA UAA GGG C
miR-184 with P1 G
GGG ACG GAG AAC UGA UAA GGG C
miR-184* with P19
CCU UAU CAU UCU CUC GCC CCG
miR-184* with P19
CCU UAU CAU UCU CUC GCC ACG
miR-184* with P21
CCU UAU CAU UCU CUC GCC CCC
miR-184* with P21 U
CCU UAU CAU UCU CUC GCC CCU
miR-184* with P21 A
CCU UAU CAU UCU CUC GCC CCA
miR-14 with P1 U
UCA GUC UUU UUC UCU CUC CUA
miR-14* with P1 A
GGA GCG AGA CGG GGA CUC ACU
miR-14 with P1 A
ACA GUC UUU UUC UCU CUC CUA
miR-14* with P19 U
GGA GCG AGA CGG GGA CUC UCU
miR-2c with P1 U
UAU CAC AGC CAG CUU UGA UGG GC
miR-2c* with P20 A
CAU CAA AAA GGG CUG AAG AAA G
Oligo to capture miR-2a and miR-2c
Bio-mAmUmGmU mUmGmG mCmUmC mAmUmC mAmAmA mGmCmU mGmGmC mUmGmU mGmAmU mCmUmG mCmUmG mA
Oligo to capture miR-2a-1*
Bio-mAmUmG mUmUmG mCmAmC mUmUmC mAmCmA mAmCmC mAmCmU mUmUmG mAmGmA mUmGmC mUmGmA
Oligo to capture miR-184
Bio-mAmUmG mUmUmG mGmCmC mCmUmU mAmUmC mAmGmU mUmCmU mCmCmG mUmCmC mCmUmG mCmUmG mA
Oligo to capture miR-184*
Bio-mAmUmG mUmUmG mCmGmG mGmGmC mGmAmG mAmGmA mAmUmG mAmUmA mAmGmG mUmGmC mUmGmA
Oligo to capture miR-14
Bio-mAmUmG mUmUmG mUmAmG mGmAmG mAmGmA mGmAmA mAmAmA mGmAmC mUmGmC mUmGmC mUmGmA
Oligo to capture miR-14*
Bio-mAmUmG mUmUmG mAmGmC mGmAmG mUmCmC mCmCmG mUmCmU mCmGmC mUmCmC mUmGmC mUmGmA
Oligo to capture miR-2c*
Bio-mAmUmG mUmUmG mCmUmU mUmCmU mUmCmA mGmCmC mCmUmU mUmUmU mGmAmU mGmUmG mCmUmG mA
pre-miR-2a-1 loop (extended by 4 nt)
CAU UUC CGC UUU GCG CGG CAU AUC
miR-2a (shortened by 4 nt)
ACA GCC AGC UUU GAU GAG C
DNA splint for pre-miR-2a-1 ligation
GCT AAG CTC ATC AAA GCT GGC TGT GAT ATG CCG CGC AAA GCG GAA ATG CAT TTC ACA ACC ACT TTG AGA GCT TA
UCU CAA AGU GGU UGU GAA AUG CAU UUC CGC UUU GCG CGG CAU AUC ACA GCC AGC UUU GAU GAG C
miR-2a with U at position 1 and U at position 2
UUU CAC AGC CAG CU UUG AUG AGC
miR-2a with A at position 1 and U at position 2
AUU CAC AGC CAG CUU UGA UGA GC
miR-2a with C at position 1 and U at position 2
CUU CAC AGC CAG CUU UGA UGA GC
miR-184 with U at position 1 and C at position 2
UCG ACG GAG AAC UGA UAA GGG C
miR-184 with A at position 1 and C at position 2
ACG ACG GAG AAC UGA UAA GGG C
miR-184 with C at position 1 and C at position 2
CCG ACG GAG AAC UGA UAA GGG C
High throughput sequencing data used in this studya
GSM139137, GSM297742, GSM297743, GSM297744, GSM297745, GSM297746, GSM297747, GSM297748, GSM297750, GSM297751
GSM180328, GSM180329, GSM180330, GSM180331, GSM180332, GSM180333, GSM180334, GSM180335, GSM180336, GSM180337, GSM239041, GSM239052, GSM239054, GSM239056, GSM240749, GSM246084, GSM272651, GSM272652, GSM272653, GSM275691, GSM280082, GSM280085, GSM286602, GSM286603, GSM286604, GSM286605, GSM286606, GSM286607, GSM286611, GSM286613, GSM322208, GSM322219, GSM322245, GSM322338, GSM322533, GSM322543, GSM343832, GSM343833, GSM360256, GSM360257, GSM360260, GSM360262, GSM361908, GSM364902, GSM371638, GSM385744, GSM385748, GSM385821, GSM385822, GSM399100, GSM399101, GSM399105, GSM399106, GSM399107, GSM399110, GSM609217, GSM609218, GSM609219, GSM609220, GSM609221, GSM609222, GSM609223, GSM609224, GSM609225, GSM609226, GSM609227, GSM609228, GSM609229, GSM609234, GSM609235, GSM609238, GSM609239, GSM609240, GSM609241, GSM609242, GSM609243, GSM609244, GSM609246, GSM609247, GSM609248, GSM609249, GSM609250, GSM609251
GSM237107, GSM237110, GSM261957, GSM261959, GSM304914, GSM314552, GSM314558
We thank Yukihide Tomari, Tomoko Kawamata and Miyuki Mitomi for dcr-2 embryo lysate and sharing unpublished data, as well as members of the Zamore laboratory for critical comments on the manuscript. This work was supported in part by National Institutes of Health grants GM62862 and GM65236 (to PDZ) and European Molecular Biology Organization long-term (ALTF 9102004) and Human Frontier Science Program (LT00575/2005-L) fellowships (to HS).
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