Cleavage of a non-conserved target by a specific miR156 isoform in root apexes of Medicago truncatula

Abstract

Micro RNAs (miRNAs) have emerged as an important class of gene expression regulators controlling development, growth and metabolism. These short RNA molecules are 20–24 nucleotides in length and act post-transcriptionally to regulate the cleavage or translation of specific mRNA targets. In the model legume Medicago truncatula, we have recently reported identification of 100 novel and 27 conserved miRNAs in root apexes and nodules. Statistical analysis on sequencing results revealed specific miRNA isoforms for the same family (up to 3 mismatches) showing contrasting expression patterns between these tissues. Here, we report the cleavage of a non-conserved target of miR156 in root apexes complementary to a differentially expressed miR156 isoform. This suggests that changes in the abundance of miRNA isoforms may have functional consequences on the post-transcriptional regulation of new mRNA targets in different organs.

To optimize their growth, plants adapt their root architecture for maximizing the availability of nutrients and water.¹ In legumes, such as Medicago truncatula, roots grown under nitrogen starvation are able to interact with specific Rhizobiaceae bacteria to develop a specialized organ, the nitrogen-fixing root nodule. This organ is morphologically and functionally different to any other plant organ and contains inside the plant cells symbiotic bacteria that have differentiated to fix nitrogen for the plant host.² Small non coding RNAs have emerged as major regulators of gene expression. Short interfering RNAs (siRNAs) and micro-RNAs (miRNAs) are the two major families of non-coding RNAs in plants that regulate gene expression at transcriptional and post-transcriptional level. These small RNAs play critical roles in development or biotic and abiotic stress responses in plants.^3,4

Primary miRNAs (having imperfect stem-loop structures) are generally processed by DICER-LIKE1 (DCL1) to generate a 21 nt miRNA/miRNA* duplex.^5,6 The mature miRNA is then placed into an RNA silencing complex (RISC) that includes the AGO1 protein⁷ and determines the cleavage and/or translational inhibition of their mRNA targets. In plants, the known conserved miRNAs have a high degree of complementarity to their targets and generally direct their slicing between position 10 and 11 of the complementary region. Most conserved miRNAs are encoded by multiple genes and have conserved targets among the plant kingdom. The miRNA families are generally composed of different but highly related (up to 3 mismatches) small RNAs from 20 to 24 nt, referred to as miRNA variants or isoforms. Except few examples processed by DCL4, most canonical 20–21 nt miRNA are produced by DCL1.⁸ In addition, 22-nt forms have frequently been reported.⁹ For instance, the miR168b gene produces both 21-nt and 22-nt variants¹⁰ despite that the 22-nt form was less incorporated into AGO1, suggesting different regulatory roles of the two species. Vazquez et al.¹¹ also described miRNA variants of 23–25 nt, produced by DCL3. Except the rare reports of single miRNA mutants, generally the specificity of expression or function of miRNA isoforms remains difficult to study due to their high level of similarity.

Several Isoforms of miRNAs Accumulate Differentially in Medicago truncatula Roots and Nodules

We have identified 27 conserved miRNA families in nodules and root tips of the model legume Medicago truncatula.¹² Most contained several size variants from 20 to 24 nt in our libraries, although canonical 21-nt forms were generally the most abundant. Forms of 20-nt, retrieved in 16 families, were found generally at low frequency, except for miR156/157 and miR319. Variants of 22-nt (22 families) showed higher read numbers than the 21-nt’s in 2 families (miR167, miR479) and were the only form sequenced for miR393 and miR839. Finally, only a handful of 23–24 nt sequences with very low read numbers (less than 3.5% of the corresponding 21-nt forms) were retrieved, even for miRNA families like miR156 for which Vazquez et al.¹¹ suggested that the 24 nt forms were conserved among plants.

Comparison of total read numbers per family showed differential accumulation of several miRNAs between nodules and root apexes, suggesting a major change in post-transcriptional regulation of the transcriptome between these two tissues. Northern blots and in situ hybridisation confirmed the differential expression of specific miRNAs.¹² Deep sequencing data revealed that, in many conserved families (like miR159, 160, 164, 171 and 390), some 21-nt isoforms showed differential accumulation and sometimes opposite expression profiles in nodules and root apexes. Consistent with this idea, the most abundant 21-nt isoforms found did not always correspond to the Mtr-miRNAs already registered in miRBAse V13. These results thus suggest that differential regulation of genes encoding specific miRNA isoforms exist in diverse organs which in turn may target different mRNAs and play different roles.

A 20-nt miR156 Isoform Cleaves a WD40 Transducin mRNA Isoform in Root Apexes

In plants, miR156 and miR157 have been grouped in one miRNA family because of their high degree of sequence similarity and their conserved target, the Squamosa-promoter Binding like Proteins (SBP). The cleavage of SBP-transcription factors by miR156/157 has been confirmed in Arabidopsis,¹³ maize¹⁴ and moss.¹⁵ MiR156 also inhibits the translation of SBP transcription factors.¹⁶ The miR156-SBP pair was implicated in shoot development14 and flowering.^17,18

In our libraries, five miR156/157 sequences of 21 nt were retrieved (Table 1). Forms 1, 2 and 3 were similar to Ath-miR157 and forms 4 and 5 were miR156-like. In addition, as described in other species (miRBAse, Wu and Poethig¹³), miR156 was mainly represented by a 20-nt variant (called form 6), present in higher read number than any 21-nt isoform. Forms 1, 2 and 5/6 were encoded by at least 2, 1 and 4 genes respectively (Table 1). According to their read numbers, these variants accumulated at higher levels in roots than nodules. As forms 3 and 4 were not registered in miRBAse and were only sequenced once in our libraries, we did not take them into account for further analysis.

Table 1.

Mtr-miR156/157 variants and their SBP and WD40 predicted targets

miR156 form	sRNA (MIRMED)	miR156/157	Precursor (MIRMED)	Precursor (miRBA-seV14)	Na	Ra	length	sequence	Penalty score of predicted targetsb
									SBP	WD40
1	EVSUSMY01CH7KF	mir157	MtrV2chr6-r1702	Mtr-mir156g	13	91	21	TTG ACA GAA GAT AGA GGG CAC	1.5	6.5
2	EVSUSM Y01AZN80	miR157	MtrV2Chr7-r1803	Mtr-miR156f/h Mtr-miR156e	1	14	21	TTG ACA GAA GAT AGA GAG CAC	1	6
3	EVSUSMY01CUEG6	miR157	X	X	1	0	21	TTG ACA GAA GAG. AGA GAG CAC	0	5
4	EVSUSMY01DQI19	miR156	X	X	1	0	21	ATG ACA GAA GAG AGT GAG CAC	2	3
5	E8W526M03HJFS8	miR156	MtrV2Chr1-r3544 MtrV2Chr3-r1440	Mtr-miR156b Mtr-miR156c Mtr-miR156d Mtr-miR156i	0	3	21	TGA CAG AAG AGA GTG AGC ACA	1	4
6	EVSUSMY01CTOEF	miR156	see form 5	see form 5	16	28	20	TGA CAG AAG AGA GTG AGC AC	1	3
total reads					32	136

^aread numbers in the nodule (N) and root tip (R) small RNA libraries.

^bpenalty score of each isoform is shown for the targets TC 121671 (SBP-box transcription factor) and TC 119548 (WD40 transducin like protein). It was calculated as 0.5 points assigned to each G:U wobble, 1 point to each non G:U mismatch and 2 points to each bulged nucleotide in either RNA strand.

All 20 and 21-nt forms of MtrmiR156/157 family were predicted to target a conserved SBP mRNA (TC121671), with penalty scores lower than 3 (Table 1). Score alignments between the targets and the miRNA were calculated following Jones-Rhoades and Bartel¹⁹ rules (gap cost = 2; mismatch cost = 1; GU pair cost = 0.5). An additional target, encoding a WD40 transducin like protein (TC119548), was predicted for the 20-nt miR156 form only (penalty score of 3). WD40-like proteins are involved in chromatin metabolism²⁰ and, recently, Zeng et al.²¹ have demonstrated that one WD40 repeat protein participates in microtubule organization during mitosis in Arabidopsis. Both mRNA targets are expressed in developing stems and roots inoculated by the symbiotic bacteria S. meliloti (gene index, compbio.dfci.harvard.edu/tgi/).

Hence, a 5′ RACE PCR assay was performed to analyze the cleavage of the SBP and WD40 mRNAs by miR156/157 in M. truncatula. 5′-RACE were carried out using the FirstChoice RLM-RACE kit (Ambion) with total RNA from roots tips and mature nodules according to the manufacturer’s instructions. Two genespecific reverse primers for each target were designed and 5′-RACE PCR products were cloned using the pCR2.1 vector (Invitrogen). Cleavage of the SBP mRNA was confirmed in both nodules and root tips (Fig. 1A). All clones sequenced in each sample corresponded to cleavage products after the 10^th nt of miR156 or the 11^th nt of miR157 variants. Such small shifts in the position of cleavage were already reported for conserved miRNAs. For instance, Wu and Poethig¹⁴ reported the preferential cleavage of SPL5 by miR156 between the nt 9 and 10. More recently, Alves et al.²² confirmed the cleavage of several Ath-miR159 targets after nt 9, 10 or 11 and thus accepted these 3 positions in the RNAhybrid algorithm for target prediction. For the WD-40 target, 10/11 clones from root apexes corresponded to a cleavage predicted after the 11^th nt of form 5/6 or the 12^th nt of forms 1 and 2 respectively (Fig. 1B). Due to the high penalty scores and a mismatch at the conventional predicted cleavage site, we think that miR157 could not be involved in the slicing of the WD40 mRNA. As no additional miR156-like variant that could account a conventional cleavage was found in our libraries, we suggest that the 20-nt form of the miR156 is mainly responsible for the WD40 mRNA slicing. Interestingly, no cleavage product was obtained in nodules suggesting that, either the accumulation of the product cleavage is too low to be detected or that miR156-like variants alone are not in sufficient amount to cleave the target in that sample.

Figure 1.

Cleavage sites of the SBP and WD40 mRNA in root tips. The straight lines represent perfect matches, one dot represents a mismatch and two dots represent G-U wobbles. The fraction of cloned products in root tips ending at the indicated cleavage site is mentioned in parentheses.

In this work, we have identified a WD-40-like transcript as a non-conserved target of a miR156/157 isoform in Medicago truncatula roots. Therefore, variable expression patterns of different miRNA isoforms may add alternative mRNA targets into post-transcriptional networks of different organs, adding a level of complexity in the analysis of miRNA action.

Addendum to: Lelandais-Brière C, Naya L, Sallet E, Calenge F, Frugier F, Hartmann C, et al. Genome-wide Medicago truncatula small RNA analysis revealed novel miRNAs and isoforms differentially regulated in roots and nodules. Plant Cell. 2009;21:2780–2796. doi: 10.1105/tpc.109.068130.