Open Access Research article

Identification and characterization of NAGNAG alternative splicing in the moss Physcomitrella patens

Rileen Sinha15, Andreas D Zimmer23, Kathrin Bolte247, Daniel Lang23, Ralf Reski345, Matthias Platzer6, Stefan A Rensing245* and Rolf Backofen145*

Author affiliations

1 Bioinformatics group, University of Freiburg, Georges-Koehler-Allee 106, 79110 Freiburg, Germany

2 Faculty of Biology, University of Freiburg, Hauptstrasse 1, 79104 Freiburg, Germany

3 Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestrasse 1, 79104 Freiburg, Germany

4 Freiburg Initiative for Systems Biology (FRISYS), University of Freiburg, Schaenzlestrasse 1, 79104 Freiburg, Germany

5 Centre for Biological Signalling Studies (bioss), University of Freiburg, Albertstr. 19, 79104 Freiburg, Germany

6 Genome Analysis, Leibniz Institute for Age Research - Fritz Lipmann Institute, Beutenbergstr. 11, 07745 Jena, Germany

7 Philipps-Universität Marburg, Laboratorium für Zellbiologie, Karl-von-Frisch Str., 35032 Marburg, Germany

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Citation and License

BMC Plant Biology 2010, 10:76  doi:10.1186/1471-2229-10-76

Published: 28 April 2010



Alternative splicing (AS) involving tandem acceptors that are separated by three nucleotides (NAGNAG) is an evolutionarily widespread class of AS, which is well studied in Homo sapiens (human) and Mus musculus (mouse). It has also been shown to be common in the model seed plants Arabidopsis thaliana and Oryza sativa (rice). In one of the first studies involving sequence-based prediction of AS in plants, we performed a genome-wide identification and characterization of NAGNAG AS in the model plant Physcomitrella patens, a moss.


Using Sanger data, we found 295 alternatively used NAGNAG acceptors in P. patens. Using 31 features and training and test datasets of constitutive and alternative NAGNAGs, we trained a classifier to predict the splicing outcome at NAGNAG tandem splice sites (alternative splicing, constitutive at the first acceptor, or constitutive at the second acceptor). Our classifier achieved a balanced specificity and sensitivity of ≥ 89%. Subsequently, a classifier trained exclusively on data well supported by transcript evidence was used to make genome-wide predictions of NAGNAG splicing outcomes. By generation of more transcript evidence from a next-generation sequencing platform (Roche 454), we found additional evidence for NAGNAG AS, with altogether 664 alternative NAGNAGs being detected in P. patens using all currently available transcript evidence. The 454 data also enabled us to validate the predictions of the classifier, with 64% (80/125) of the well-supported cases of AS being predicted correctly.


NAGNAG AS is just as common in the moss P. patens as it is in the seed plants A. thaliana and O. sativa (but not conserved on the level of orthologous introns), and can be predicted with high accuracy. The most informative features are the nucleotides in the NAGNAG and in its immediate vicinity, along with the splice sites scores, as found earlier for NAGNAG AS in animals. Our results suggest that the mechanism behind NAGNAG AS in plants is similar to that in animals and is largely dependent on the splice site and its immediate neighborhood.