1. Progress report Yiming Zhang 02/10/2012

2. All AS events in ASIP Intron retention Exon skipping Alternative Acceptor site NAGNAG AltA Alternative Donor site GYNGYN AltD Alternative both sites (AltP)

3. NAGNAG alternative splicing Figure 1. NAGNAG alternative splicing with E and I sites and isoforms. NAGNAG alternaive splicing can result in one of three possibilities (Figure 1) - constitutive use of the first acceptor (the so-called exonic, or “E” variant), constitutive use of the second acceptor (the so-called intronic, or “I” variant), or use of both acceptors, that is,alternative splicing (the “EI” variant). Sinha et al. 2010

4. GYNGYN alternative splicing Figure 2. GYNGYN alternative splicing with e and i sites and isoforms. Hilller et al. 2006

5. All introns ConstitutiveNAGNAG-ENAGNAG-IGYNGYN-eGYNGYN-I……AlternativeIntronRExonSAltANAGNAG-ei……AltDGYNGYN-ei……Multiple AS……Unclear

6. Intron statistics from ASIP ATBDGMLJMTOSPPPTSBSLVVTotal Cons.EST>=4all36040760162031056635132393153063347901416819000131151 NAG-E149430583391951299533102317693495010 NAG-I522521213744003313696261391854 GYN-e128340621342461406748135403583825356 GYN-i48914296138970524171168272082321 EST>=10all133662205697406250914409660611293366689409152488 NAG-E5741018978556822040117241481982 NAG-I19917433216010711311255685 GYN-e46752321710063733036163281772192 GYN-i1527947393038825461488863 Alt.EST>=2IntronR4197501669763278233200425616719180619380 AltA_all648101891149926404389224972488 AltA_NAG1284313617844327430458 AltD_all305299547575453156416711652 ALtD_GYN90011103010227 AltP510321018827239016356 ExonS47653711172210042979304592384144 Table 1. Intron statistics from ASIP. 4 species which have small amount of data are not listed here. All statistics are intron-based instead of event-based which means redundancy has been removed. The most common type of alternative intron type is IntronR, second common type is ExonS. NAGNAG AS occurs much more frequently in AltA than GYNGYN AS occurs in AltD.

7. Background NAGNAG alternative splicing which can insert or delete a single amino acid in the protein, is very common and well studied in animals. The NAGNAG motif is present in 30% of human genes and is functional in at least 5% of the genes. Hiller et al. 2004 NAGNAG AS is frame-preserving, the vast majority of cases should lead to different proteins. Studies so far have found evidence of both cases where such proteins have variations in function, as well as those in which there is no noticeable difference. Akerman et al. 2006 Iida et al. 2008 The GO analyses in some studies shows genes with specific GO term DNA binding to be statistically significant and more than half of all AS-NAGNAG events affected polar amino acid residues. Iida et al. 2008 Sinha et al. 2010

8. Background The studies of NAGNAG AS in plant is few right now (Only 3 species: Arabidopsis, Rice and Physcomitrala). One study found 321 and 372 AS-NAGNAG events in Arabidopsis and rice, respectively. Another study found 6% of all introns and 21% of all annotated genes in Arabidopsis harbor a genomic NAGNAG acceptor motif. Iida et al. 2008 Schindler et al. 2008 In addition, the GO analysis is agreed with previous study in human that the specific GO term DNA binding is statistically significant. Some study indicates that NAGNAG acceptors frequently occur in the Arabidopsis genome and are particularly prevalent in SR and SR-related protein-coding genes. Sinha et al 2010 Schindler et al. 2008

9. Background The state-of-the-art in silico studies for prediction of NAGNAG splice site are done by Sinha's group for both human and plant species. They achieved high balanced specificity and sensitivity for both human and plant species. The most informative features they found are the nucleotides in the NAGNAG and in its immediate vicinity, along with the splice sites scores. The model they trained on human data also can achieve high AUC on plant data shows that NAGNAG splicing in plants is similar to that in animals. Sinha et al. 2009, 2010

10. NAGNAG dataset I tried to predict NAGNAG events (thus to predict EI, I or E isoforms) based on the dataset I generated from ASIP using Random Forest. Strict criteria has been used to identify NAGNAG events from ASIP database: For E and I events, at least 10 ESTs or cDNAs support them, and for EI events at lease 2 EST or cDNA support each isoform. After removing redundancy, I got 458 EI form alternative NAGNAG introns, 1988 E form constitutive introns and 685 I form constitutive introns in 15 plant species.

11. Features Figure 3. A total of 28 features which each represented a nucleotide, and thus had four possible values (A, C, G, T). U1, U2, U3 are the first three nucleotides in the upstream exon. D1, D2, D3 are the first three nucleotides in the downstream exon. A weak polypyrimidine tract (PPT) can contribute to AS. So P1-P20 are PPT upstream of NAGNAG. Finally, I also use intron length as an additional feature.

12. Classifier evaluation Random Forest with 200 trees has been used and 5 fold cross validation has been applied. TP rateFP ratePrecisionRecallF-measureROC areaClass 0.9920.0890.9510.9920.9710.995E 0.9530.0230.920.9530.9360.995I 0.6570.0170.870.6570.7490.967EI The evaluation results strongly agree with Sinha’s paper (For Physcomitrella) in which AUC = 0.96, 0.99 and 0.98 for the EI, E and I forms, respectively.

13. Figure 4. The EI class, or AS, harder to predict (AUC = 0.967) than the two constitutive variants, E and I (AUC = 0.995 for both).

14. Most informative features Figure 5. Most informative features according to information gain.

15. Sequence Logos Figure 6a. Figure 6b.

16. Figure 6c. Figure 6d. Figure 6a-6d. Sequence logos of NAGNAG splice sites. 6a: E sites; 6b: I sites; 6c: EI sites; 6d: all splice sites. Position 1-3 is U1-U3. Position 4-24 are P20-P1. Position 30-32 are D1-D3.

17. Conclusion NAGNAG-AS can be predicted with high accuracy. Using carefully constructed training and test datasets, an in silico performance of AUC = 0.967, 0.995 and 0.995 was achieved for the EI, E and I forms, respectively. The most informative features are the nucleotides in the NAGNAG and in its immediate vicinity. NAGNAG AS in plants is similar to that in animals and is largely dependent on the splice site and its immediate neighborhood.