Skip to main content

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

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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

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

References

  • Barbazuk WB, Fu Y, McGinnis KM (2008) Genome-wide analyses of alternative splicing in plants: opportunities and challenges. Genome Res 18:1381–1392

    PubMed  Article  CAS  Google Scholar 

  • Blevins T, Rajeswaran R, Shivaprasad PV, Beknazariants D, Si-Ammour A, Park HS, Vazquez F, Robertson D, Meins F Jr, Hohn T, Pooggin MM (2006) Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing. Nucleic Acids Res 34:6233–6246

    PubMed  Article  CAS  Google Scholar 

  • Brett D, Pospisil H, Valcarcel J, Reich J, Bork P (2002) Alternative splicing and genome complexity. Nat Genet 30:29–30

    PubMed  Article  CAS  Google Scholar 

  • Brodersen P, Voinnet O (2006) The diversity of RNA silencing pathways in plants. Trends Genet 22:268–280

    PubMed  Article  CAS  Google Scholar 

  • del Prete MJ, Vernal R, Dolznig H, Mullner EW, Garcia-Sanz JA (2007) Isolation of polysome-bound mRNA from solid tissues amenable for RT-PCR and profiling experiments. RNA 13:414–421

    PubMed  Article  Google Scholar 

  • Deleris A, Gallego-Bartolome J, Bao J, Kasschau KD, Carrington JC, Voinnet O (2006) Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science 313:68–71

    PubMed  Article  CAS  Google Scholar 

  • Donaire L, Barajas D, Martinez-Garcia B, Martinez-Priego L, Pagan I, Llave C (2008) Structural and genetic requirements for the biogenesis of tobacco rattle virus-derived small interfering RNAs. J Virol 82:5167–5177

    PubMed  Article  CAS  Google Scholar 

  • Dong Z, Han MH, Fedoroff N (2008) The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1. Proc Natl Acad Sci USA 105:9970–9975

    PubMed  Article  CAS  Google Scholar 

  • Fang Y, Spector DL (2007) Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants. Curr Biol 17:818–823

    PubMed  Article  CAS  Google Scholar 

  • Fusaro AF, Matthew L, Smith NA, Curtin SJ, Dedic-Hagan J, Ellacott GA, Watson JM, Wang MB, Brosnan C, Carroll BJ, Waterhouse PM (2006) RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway. EMBO Rep 7:1168–1175

    PubMed  Article  CAS  Google Scholar 

  • Gasciolli V, Mallory AC, Bartel DP, Vaucheret H (2005) Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Curr Biol 15:1494–1500

    PubMed  Article  CAS  Google Scholar 

  • Gingerich TJ, Feige JJ, LaMarre J (2004) AU-rich elements and the control of gene expression through regulated mRNA stability. Anim Health Res Rev 5:49–63

    PubMed  Article  CAS  Google Scholar 

  • Grzybowska EA, Wilczynska A, Siedlecki JA (2001) Regulatory functions of 3′UTRs. Biochem Biophys Res Commun 288:291–295

    PubMed  Article  CAS  Google Scholar 

  • Harrison MJ, Lawton MA, Lamb CJ, Dixon RA (1991) Characterization of a nuclear protein that binds to three elements within the silencer region of a bean chalcone synthase gene promoter. Proc Natl Acad Sci USA 88:2515–2519

    PubMed  Article  CAS  Google Scholar 

  • Henderson IR, Zhang X, Lu C, Johnson L, Meyers BC, Green PJ, Jacobsen SE (2006) Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat Genet 38:721–725

    PubMed  Article  CAS  Google Scholar 

  • Hiraguri A, Itoh R, Kondo N, Nomura Y, Aizawa D, Murai Y, Koiwa H, Seki M, Shinozaki K, Fukuhara T (2005) Specific interactions between Dicer-like proteins and HYL1/DRB-family dsRNA-binding proteins in Arabidopsis thaliana. Plant Mol Biol 57:173–188

    PubMed  Article  CAS  Google Scholar 

  • Howell MD, Fahlgren N, Chapman EJ, Cumbie JS, Sullivan CM, Givan SA, Kasschau KD, Carrington JC (2007) Genome-wide analysis of the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 pathway in Arabidopsis reveals dependency on miRNA- and tasiRNA-directed targeting. Plant Cell 19:926–942

    PubMed  Article  CAS  Google Scholar 

  • Imaishi H, Matsumoto Y, Ishitobi U, Ohkawa H (1999) Encoding of a cytochrome P450-dependent lauric acid monooxygenase by CYP703A1 specifically expressed in the floral buds of petunia hybrida. Biosci Biotechnol Biochem 63:2082–2090

    PubMed  Article  CAS  Google Scholar 

  • Kasschau KD, Fahlgren N, Chapman EJ, Sullivan CM, Cumbie JS, Givan SA, Carrington JC (2007) Genome-wide profiling and analysis of Arabidopsis siRNAs. PLoS Biol 5:e57

    PubMed  Article  Google Scholar 

  • Kim SJ, Martinson HG (2003) Poly(A)-dependent transcription termination: continued communication of the poly(A) signal with the polymerase is required long after extrusion in vivo. J Biol Chem 278:41691–41701

    PubMed  Article  CAS  Google Scholar 

  • Kurihara Y, Takashi Y, Watanabe Y (2006) The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis. RNA 12:206–212

    PubMed  Article  CAS  Google Scholar 

  • Lareau LF, Green RE, Bhatnagar RS, Brenner SE (2004) The evolving roles of alternative splicing. Curr Opin Struct Biol 14:273–282

    PubMed  Article  CAS  Google Scholar 

  • Lawton MA, Dean SM, Dron M, Kooter JM, Kragh KM, Harrison MJ, Yu L, Tanguay L, Dixon RA, Lamb CJ (1991) Silencer region of a chalcone synthase promoter contains multiple binding sites for a factor, SBF-1, closely related to GT-1. Plant Mol Biol 16:235–249

    PubMed  Article  CAS  Google Scholar 

  • Lee Y, Yoo SK, Lee JS, Kwon YM (2000) Genomic structure of ornithine carbamoyltransferase gene from Canavalia lineata. Mol Cells 10:480–485

    PubMed  CAS  Google Scholar 

  • Liu B, Li P, Li X, Liu C, Cao S, Chu C, Cao X (2005) Loss of function of OsDCL1 affects microRNA accumulation and causes developmental defects in rice. Plant Physiol 139:296–305

    PubMed  Article  CAS  Google Scholar 

  • Liu B, Chen Z, Song X, Liu C, Cui X, Zhao X, Fang J, Xu W, Zhang H, Wang X, Chu C, Deng X, Xue Y, Cao X (2007) Oryza sativa dicer-like4 reveals a key role for small interfering RNA silencing in plant development. Plant Cell 19:2705–2718

    PubMed  Article  CAS  Google Scholar 

  • Loke JC, Stahlberg EA, Strenski DG, Haas BJ, Wood PC, Li QQ (2005) Compilation of mRNA polyadenylation signals in Arabidopsis revealed a new signal element and potential secondary structures. Plant Physiol 138:1457–1468

    PubMed  Article  CAS  Google Scholar 

  • Mlotshwa S, Pruss GJ, Peragine A, Endres MW, Li J, Chen X, Poethig RS, Bowman LH, Vance V (2008) DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis. PLoS ONE 3:e1755

    PubMed  Article  Google Scholar 

  • Modrek B, Lee C (2002) A genomic view of alternative splicing. Nat Genet 30:13–19

    PubMed  Article  CAS  Google Scholar 

  • Moissiard G, Voinnet O (2006) RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins. Proc Natl Acad Sci USA 103:19593–19598

    PubMed  Article  CAS  Google Scholar 

  • Moissiard G, Parizotto EA, Himber C, Voinnet O (2007) Transitivity in Arabidopsis can be primed, requires the redundant action of the antiviral Dicer-like 4 and Dicer-like 2, and is compromised by viral-encoded suppressor proteins. RNA 13:1268–1278

    PubMed  Article  CAS  Google Scholar 

  • Ner-Gaon H, Leviatan N, Rubin E, Fluhr R (2007) Comparative cross-species alternative splicing in plants. Plant Physiol 144:1632–1641

    PubMed  Article  CAS  Google Scholar 

  • Papp I, Mette MF, Aufsatz W, Daxinger L, Schauer SE, Ray A, van der Winden J, Matzke M, Matzke AJ (2003) Evidence for nuclear processing of plant micro RNA and short interfering RNA precursors. Plant Physiol 132:1382–1390

    PubMed  Article  CAS  Google Scholar 

  • Pesole G, Mignone F, Gissi C, Grillo G, Licciulli F, Liuni S (2001) Structural and functional features of eukaryotic mRNA untranslated regions. Gene 276:73–81

    PubMed  Article  CAS  Google Scholar 

  • Pikaard CS (2006) Cell biology of the Arabidopsis nuclear siRNA pathway for RNA-directed chromatin modification. Cold Spring Harb Symp Quant Biol 71:473–480

    PubMed  Article  CAS  Google Scholar 

  • Pontes O, Li CF, Nunes PC, Haag J, Ream T, Vitins A, Jacobsen SE, Pikaard CS (2006) The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell 126:79–92

    PubMed  Article  CAS  Google Scholar 

  • Reddy AS (2007) Alternative splicing of pre-messenger RNAs in plants in the genomic era. Annu Rev Plant Biol 58:267–294

    PubMed  Article  CAS  Google Scholar 

  • Simpson CG, Lewandowska D, Fuller J, Maronova M, Kalyna M, Davidson D, McNicol J, Raczynska D, Jarmolowski A, Barta A, Brown JW (2008) Alternative splicing in plants. Biochem Soc Trans 36:508–510

    PubMed  Article  CAS  Google Scholar 

  • Song L, Han MH, Lesicka J, Fedoroff N (2007) Arabidopsis primary microRNA processing proteins HYL1 and DCL1 define a nuclear body distinct from the Cajal body. Proc Natl Acad Sci USA 104:5437–5442

    PubMed  Article  CAS  Google Scholar 

  • Stamm S, Ben-Ari S, Rafalska I, Tang Y, Zhang Z, Toiber D, Thanaraj TA, Soreq H (2005) Function of alternative splicing. Gene 344:1–20

    PubMed  Article  CAS  Google Scholar 

  • Tagami Y, Motose H, Watanabe Y (2009) A dominant mutation in DCL1 suppresses the hyl1 mutant phenotype by promoting the processing of miRNA. Rna 15:450–458

    PubMed  Article  CAS  Google Scholar 

  • Tebo J, Der S, Frevel M, Khabar KS, Williams BR, Hamilton TA (2003) Heterogeneity in control of mRNA stability by AU-rich elements. J Biol Chem 278:12085–12093

    PubMed  Article  CAS  Google Scholar 

  • Tian B, Hu J, Zhang H, Lutz CS (2005) A large-scale analysis of mRNA polyadenylation of human and mouse genes. Nucleic Acids Res 33:201–212

    PubMed  Article  CAS  Google Scholar 

  • Vaucheret H (2006) Post-transcriptional small RNA pathways in plants: mechanisms and regulations. Genes Dev 20:759–771

    PubMed  Article  CAS  Google Scholar 

  • Vazquez F, Vaucheret H, Rajagopalan R, Lepers C, Gasciolli V, Mallory AC, Hilbert JL, Bartel DP, Crete P (2004) Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol Cell 16:69–79

    PubMed  Article  CAS  Google Scholar 

  • Voinnet O, Vain P, Angell S, Baulcombe DC (1998) Systemic spread of sequence-specific transgene RNA degradation in plants is initiated by localized introduction of ectopic promoterless DNA. Cell 95:177–187

    PubMed  Article  CAS  Google Scholar 

  • Wang BB, Brendel V (2006) Genomewide comparative analysis of alternative splicing in plants. Proc Natl Acad Sci USA 103:7175–7180

    PubMed  Article  CAS  Google Scholar 

  • Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman D, Jacobsen SE, Carrington JC (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2:E104

    PubMed  Article  Google Scholar 

  • Xing Y, Lee C (2006) Alternative splicing and RNA selection pressure—evolutionary consequences for eukaryotic genomes. Nat Rev Genet 7:499–509

    PubMed  Article  CAS  Google Scholar 

  • Yan F, Peng J, Lu Y, Lin L, Zheng H, Chen H, Chen J, Adams MJ (2009a) Identification of novel splice variants of the Arabidopsis DCL2 gene. Plant Cell Rep 28:241–246

    PubMed  Article  CAS  Google Scholar 

  • Yan F, Peng J, Lu Y, Lin L, Zheng H, Chen H, Chen J, Adams MJ (2009b) Molecular cloning and characterization of the Dicer-like 2 gene from Brassica rapa. Mol Biol Rep 36:1283–1289

    PubMed  Article  CAS  Google Scholar 

  • Yoshikawa M, Peragine A, Park MY, Poethig RS (2005) A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev 19:2164–2175

    PubMed  Article  CAS  Google Scholar 

  • Zhang H, Hu J, Recce M, Tian B (2005) PolyA_DB: a database for mammalian mRNA polyadenylation. Nucleic Acids Res 33:D116–D120

    PubMed  Article  CAS  Google Scholar 

  • Zhou Y, Zhou C, Ye L, Dong J, Xu H, Cai L, Zhang L, Wei L (2003) Database and analyses of known alternatively spliced genes in plants. Genomics 82:584–595

    Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jianping Chen.

Additional information

Qiongji He and Jiejun Peng contributed equally in this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-011-1095-5

Keywords

  • Alternative splicing
  • Dicer-like protein
  • Intron retention
  • 3′ Untranslated region