Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jun;20(6):1069-1084.
doi: 10.1111/pbi.13786. Epub 2022 Feb 19.

Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine-rich preference for virus- and non-virus-based expression vectors in plants

Yen-Wen Kuo  1 , Bryce W Falk  1
Affiliations

Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine-rich preference for virus- and non-virus-based expression vectors in plants

Yen-Wen Kuo et al. Plant Biotechnol J. 2022 Jun.

Abstract

Artificial microRNA (amiRNA) technology has allowed researchers to direct efficient silencing of specific transcripts using as few as 21 nucleotides (nt). However, not all the artificially designed amiRNA constructs result in selection of the intended ~21-nt guide strand amiRNA. Selection of the miRNA guide strand from the mature miRNA duplex has been studied in detail in human and insect systems, but not so much for plants. Here, we compared a nuclear-replicating DNA viral vector (tomato mottle virus, ToMoV, based), a cytoplasmic-replicating RNA viral vector (tobacco mosaic virus, TMV, based), and a non-viral binary vector to express amiRNAs in plants. We then used deep sequencing and mutational analysis and show that when the structural factors caused by base mismatches in the mature amiRNA duplex were excluded, the nucleotide composition of the mature amiRNA region determined the guide strand selection. We found that the strand with excess purines was preferentially selected as the guide strand and the artificial miRNAs that had no mismatches in the amiRNA duplex were predominantly loaded into AGO2 instead of loading into AGO1 like the majority of the plant endogenous miRNAs. By performing assays for target effects, we also showed that only when the intended strand was selected as the guide strand and showed AGO loading, the amiRNA could provide the expected RNAi effects. Thus, by removing mismatches in the mature amiRNA duplex and designing the intended guide strand to contain excess purines provide better control of the guide strand selection of amiRNAs for functional RNAi effects.

Keywords: artificial microRNA; guide strand selection; small RNA deep sequencing; virus-based expression vector.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The predicted folding structure of pri‐miR319a and partial pri‐amiRNAs. (a) 5p and 3p arms of the miRNA duplex are shown in the box. The mature miR319a duplex region contains 3 mismatches between its 5p and 3p strands. (b) Sequences and the guide strands of amiRA2 and amiRA2c duplex. The mismatches between the 5p and 3p strands were removed from the amiRA2 and amiRA2c constructs. The guide strands of amiRA2 and amiRA2c that were identified from this study was labelled.
Figure 2
Figure 2
Production of amiRNAs in plants. (a) The primary miR319a backbone was used for producing amiRNAs and cloned into three different vectors. The two viral vectors, TRBO and TAV, are in the binary vector pCB301 backbone, and the non‐viral vector is the binary vector pGWB2. Both TRBO and pGWB2 are driven by 35S promoter of cauliflower mosaic virus. (b) The replication of TRBO was confirmed by northern blot hybridization. Genomic RNA, movement protein (MP) subgenomic RNA (sgRNA) and pri‐amiRNA subgenomic RNA were detected from the TRBO‐infiltrated tissues with the probe‐detecting sense (positive strand) RNA. The negative strand of the TRBO genomic RNAs was detected with the probe (positive strand), and the negative strand is only derived during virus replication. The negative controls are the pCB301 vector carrying non‐TMV sequences. (c) PCR was used to confirm the replication of TAV. TAV CR mutant, which contains a 5‐nucleotide mutation in the non‐coding common region of ToMoV to abolish viral replication, was used as a negative control. (d) amiRA2 and amiRA2c accumulation levels produced with the three different vectors were detected by the specific 21‐nt anti‐sense DNA probes by northern blot analysis. P19, the silencing suppressor alone was used as a negative control. (e) Percentages of amiRA2 and amiRA2c guide and star strand reads accumulation level in total mapped 18‐25 nt reads in three different vectors listed in the Table 1 are shown in a bar chart.
Figure 3
Figure 3
Small RNA northern blot analyses for detecting 5p or 3p strand of TAV‐amiRA1, A1c, A3, A3c, G1, G1c, G2, and G2c. Blots were hybridized using probes for a given sequence and exposed to X‐ray film. Then blots were stripped and re‐probed for the other strand. All probes were of the same specific radioactivity, and the same exposure time were applied to the blot that probed for opposite strands. Higher intensity signals indicate more of the specific amiRNA and are interpreted to be the guide strand. The predicted folding structures show that the sequences in the mature amiRNA duplex contain no mismatches in all the amiRNAs. The strands show higher accumulation levels are enclosed in boxes and considered as the guide strand.
Figure 4
Figure 4
Co‐immunoprecipitation results showed that the TAV‐amiRNAs predominantly loaded into AGO2 but not AGO1. Western blot analyses showed that both AGO1 and AGO2 proteins were pulled down by HA antibody conjugated beads. The eluted proteins were detected by anti‐HA antibodies in the Western blot analyses. 3xHA‐AGO1 protein migrated at ~130 kDa and 1xHA‐AGO1 migrated at ~120 kDa in size compared with the protein markers used in Western blot analyses. Small RNA northern blot analyses for the RNA extracted from co‐IP analyses showed the hybridized signals at the 21‐nt position compared with the microRNA ladder. Blots were first probed for 5p strand and, after exposing to X‐ray films, blots were stripped and re‐probed for the 3p strand. In: Input, IP: immunoprecipitated fraction. A1: TAV‐amiRA1, A1c: TAV‐amiRA1c, A2: TAV‐amiRA2, A2c: TAV‐amiRA2c, A3: TAV‐amiRA3, A3c: TAV‐amiRA3c, G1: TAV‐amiRG1, G1c: TAV‐amiRG1c, G2: TAV‐amiRG2, G2c: TAV‐amiRG2c.
Figure 5
Figure 5
EGFP reporter assays indicated only the correct target sequences corresponding to the amiRNA guide strands triggered silencing effects of the EGFP protein accumulation level. (a) The sequence organization of TAV‐amiRNA‐EGFP_target and the TAV‐EGFP_target (empty vector) constructs. The binary vector pCB301 was used for all the TAV viral vector clones. The sequences of TAV‐amiRNA and/or the 35S promotor‐driven EGFP with OCS terminator sequences were cloned in between the T‐DNA right boarder (RB) and left boarder (LB) sequences of the binary vector. The viral common region (CR) was indicated in blue boxes. The viral genes AC1 (Rep; replication associated protein), AC2 (TrAP; transcriptional activator protein), AC3 (REn; replication enhancer protein) and AC4 were indicated in light blue boxes. The up‐stream and down‐stream sequences of the pri‐miR319a backbone were indicated with grey boxes. The amiRNA‐target sequences were cloned at the 3′‐UTR of the EGFP sequence. (b) Results of the Western blot analyses. The samples infiltrated with TAV‐amiRA1‐EGFP_A1_3p (A1/A1_3p) showed silencing effects compared with the accumulation level of the controls: TAV‐amiRA1‐EGFP_A1c_3p (A1/A1c_3p, non‐target) and the TAV‐EGFP_A1_3p (empty vector). The same effects were shown in the TAV‐amiRA2‐EGFP_A2_3p (A2/A2_3p) compared to its controls: TAV‐amiRA2‐EGFP_A2c_3p (A2/A2c_3p, non‐target) and the TAV‐EGFP_A2_3p (empty vector). However, contrary to the amiRA1 and amiRA2, both of which the 3p strand was the purine‐rich guide strand, and the guide strand of the amiRA3 was the 5p. Therefore, the TAV‐amiRA3‐EGFP_A3_3p did not show silencing effects compared to the controls: TAV‐amiRA3‐EGFP_A3c_3p and TAV‐EGFP_A3_3p. The bands from the ponceau S staining (S) were used as loading controls. (c) Quantitative analyses of the Western blot analyses results were presented in bar graphs. Statistical significance was analyzed using T‐test. *P‐value <0.05; **P‐value <0.01; ns: non‐significant.
Figure 6
Figure 6
EGFP reporter assays showed that only the target sequence corresponding to the AGO loading guide strand induced silencing effects at the protein level. (a) The constructs of TAV‐amiRNA‐EGFP_target_5p or _3p were used for these assays. Please see the figure legend of Figure 5 for the details of the sequence/construct information. (b) Representative results of the Western blot analyses. TAV‐amiRA1‐EGFP_A1_3p (A1/A1_3p) showed silencing effects compared to the results of TAV‐amiRA1‐EGFP_A1_5p (A1/A1_5p), while TAV‐amiRA1c‐EGFP‐A1c_5p (A1c/A1c_5p) showed silencing effects compared to the results of TAV‐amiRA1c‐EGFP_A1c_3p (A1c/A1c_3p). Similar results were found in the assays for the TAV‐amiRA2‐EGFP_A2_3p (A2/A2_3p_ or _5p (A2/A2_5p) and the assays for the TAV‐amiRA2c‐EGFP_A2c_5p (A2c/A2c_5p) or _3p (A2c/A2c_3p). However, contrary to the amiRA1, amiRA1c and amiRA2, amiRA2c, the purine‐rich guide strand of amiRA3 was the 5p strand, and the purine‐rich guide strand of the amiRA3c was the 3p strand. Therefore, the TAV‐amiRA3‐EGFP_A3_5p (A3/A3_5p) showed silencing effects compared to the results of TAV‐amiRA3‐EGFP_A3_3p (A3/A3_3p), and the TAV‐amiRA3c‐EGFP_A3c_3p (A3c/A3c_3p) showed silencing effects compared to the results of TAV‐amiRA3c‐EGFP_A3c_5p (A3c/A3c_5p). U: upper leaf; M: middle leaf; L: lower leaf. The bands from the ponceau S staining (S) were used as loading controls. The complete results of these assays were shown in supplemental Figure 4 (Figure S4). (c) Quantitative analyses of the Western blot analyses results were presented in bar graphs. Statistical significance was analyzed using the T‐test. ***P‐value <0.001.
Figure 7
Figure 7
amiRNA strand selection affected silencing effects of the plant endogenous gene, phytoene desaturase (PDS) in Nicotiana benthamiana. (a) The results of the RT‐qPCR analyses showed decreased RNA transcript accumulation level in the amiRPDS1‐, amiRPDS3‐, amiRPDS4‐ and amiRPDS5‐treated plants, while the controls, pCB301 (empty vector), amiRPDS1_TAVmut (non‐replicable viral vector) and amiRPDS6, of which the purine‐rich strand targets the negative strand of the NbPDS gene, showed no (pCB301) or very mild (amiRPDS1_TAVmut and amiRPDS6) silencing effects in the infiltrated tissue. (b) The infiltrated N. benthamiana tissue showed different degrees of mild bleaching that corresponded to the results of NbPDS transcript accumulation level compared to the empty vector control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Ali, I. , Amin, I. , Briddon, R.W. and Mansoor, S . (2013) Artificial microRNA‐mediated resistance against the monopartite begomovirus Cotton leaf curl Burewala virus. Virol J. 10, 231. - PMC - PubMed
    1. Arroyo, J.D. , Gallichotte, E.N. and Tewari, M . (2014) Systematic design and functional analysis of artificial microRNAs. Nucleic Acids Res, 42, 6064–6077. - PMC - PubMed
    1. Bartel, D.P . (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116, 281–297. - PubMed
    1. Basso, M.F. , Ferreira, P.C.G. , Kobayashi, A.K. , Harmon, F.G. , Nepomuceno, A.L. , Molinari, H.B.C. and Grossi‐de‐Sa, M.F . (2019) MicroRNAs and new biotechnological tools for its modulation and improving stress tolerance in plants. Plant Biotechnol J, 17, 1482–1500. - PMC - PubMed
    1. Carbonell, A. , Carrington, J.C. and Daros, J.A . (2016) Fast‐forward generation of effective artificial small RNAs for enhanced antiviral defense in plants. RNA Dis, 3. - PMC - PubMed

Publication types