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Intron retention and 3′-UTR analysis of Arabidopsis Dicer-like 2 transcripts

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Abstract

Arabidopsis thaliana Dicer-like protein 2 (AtDCL2) plays an essential role in the RNA interference pathway. The function of AtDCL2 and other DCLs has been much studied but little has been done to characterize the DCLs transcripts before they are translated into proteins. Here, we investigated AtDCL2 transcripts and showed that all 21 introns of AtDCL2 except intron 9, 18, 20 and 21 could be retained although spliced sequences usually predominated. Intron 10 was more frequently retained and transient expression assays in Nicotiana benthamiana leaves showed that when AG/C at the 3′ splicing site of the intron was changed to AG/G, the intron was more frequently spliced out. Conversely, a high retention of intron 18 was obtained if the AG/G at the 3′ splicing site was changed to AG/C. These results suggest that the sequence at the 3′ splicing site affects the efficiency of intron splicing. The 3′-UTRs of AtDCL2 had lengths between 54 and 154 nts, and the different 3′-UTRs differentially affected the transcriptional levels of fused GFP expressed transiently in N. benthamiana. Further comparisons and mutation experiments suggested that a putative SBF-1 binding site and an AU-rich element in the 3′-UTR both down-regulated expression of the upstream GFP fused to the 3′-UTR. Conversely, a second poly(A) consensus signal sequence in one 3′-UTR up-regulated gene expression. Our results provide insight into the character of AtDCL2 transcripts and demonstrate the potential complexity of factors that affect the frequency and patterns of alternative splicing.

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Abbreviations

AGO:

Argonaute proteins

ARE:

AU-rich element

AS:

Alternative splicing

DCL:

Dicer-like protein

GFP:

Green fluorescence protein

M4T:

Oligo(dT)M4

miRNA:

microRNA

nt:

Nucleotides

Poly(A):

Polyadenylation

RISC:

RNA-induced silencing complex

siRNA:

Small interfering RNA

ta-siRNAs:

Trans-acting siRNAs

UTR:

Untranslated region

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Acknowledgments

This work was financially supported by National Basic Research Program of China (973 program, No. 2010CB126203), International Science and Technology Cooperation Project of MOST of China (2007DFB30350), Major Project of New Varieties of Genetically Modified Organism of China (2009ZX08001-006B; 2008ZX08001-002), and Natural Science Foundation of Zhejiang Province (Y3080417). We thank Professor M J Adams, Rothamsted Research, Harpenden, UK for help in correcting the English of the manuscript.

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Authors and Affiliations

  1. College of Chemistry and Life Science, Zhejiang Normal University, Jinhua, 321004, People’s Republic of China

    Qiongji He

  2. Stake Key Laboratory Breeding Base for Sustainable Control of Pest and Disease, MOA and Zhejiang Provincial Key Laboratory of Plant Virology, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China

    Qiongji He, Jiejun Peng, Fei Yan, Lin Lin, Yuwen Lu, Hongying Zheng & Jianping Chen

  3. Plant Protection College, Yunnan Agricultural University, Kunming, 650205, People’s Republic of China

    Jiejun Peng & Hairu Chen

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  1. Qiongji He
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Correspondence to Jianping Chen.

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Qiongji He and Jiejun Peng contributed equally in this work.

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11033_2011_1095_MOESM1_ESM.tif

Supplementary Fig. 1: Demonstration of hybridization between the intron-spliced and intron-retained sequences following PCR for retention analysis of intron 10. a the electrophoretic map of PCR products showing the large (L), middle (M) and small (S) fragments. b the electrophoretic map of PCR products using the separately purified fragments and a mixture of the middle and small fragments (M + S) (TIFF 812 kb)

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He, Q., Peng, J., Yan, F. et al. Intron retention and 3′-UTR analysis of Arabidopsis Dicer-like 2 transcripts. Mol Biol Rep 39, 3271–3280 (2012). https://doi.org/10.1007/s11033-011-1095-5

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