Intron retention and 3′-UTR analysis of Arabidopsis Dicer-like 2 transcripts
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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.
KeywordsAlternative splicing Dicer-like protein Intron retention 3′ Untranslated region
Green fluorescence protein
RNA-induced silencing complex
Small interfering RNA
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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