Among alternative splicing events in the human transcriptome, tandem NAGNAG acceptor splice sites represent an appreciable proportion. Both proximal and distal NAG can be used to produce two splicing isoforms differing by three nucleotides. In some cases, the upstream exon can be alternatively spliced as well, which further increases the number of possible transcripts. In this study, we showed that NAG choice in tandem splice site depends considerably not only on the concerned acceptor, but also on the upstream donor splice site sequence. Using an extensive set of experiments with systematically modified two-exonic minigene systems of AFAP1L2 or CSTD gene, we recognized the third and fifth intronic upstream donor splice site position and the tandem acceptor splice site region spanning from −10 to +2, including NAGNAG itself, as the main drivers. In addition, competition between different branch points and their composition were also shown to play a significant role in NAG choice. All these nucleotide effects appeared almost additive, which explained the high variability in proximal versus distal NAG usage.
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We thank Lucie Kopálková for her excellent technical help and Lucie Grodecká for her valuable comments and suggestions, which helped us to improve the quality of the manuscript.
This work was supported by the Ministry of Education, Youth and Sports, Grant No. MUNI/A/1099/2019 and by the Centre for Cardiovascular Surgery and Transplantation, Grant No. 2020001.
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Supplementary file1. Supplementary Fig. S1. Dependence of dNu on 5′ss and 3′ss nucleotide composition in human transcriptome. dNu was counted base on RNA-seq data and sorted according to NAGNAG and particular nucleotide in 3′ss or 5′ss. dNu is presented as boxplot with variable box’s width, with width representing number of analyzed genes (in log scale). Particular group numbers are also indicated in square brackets. Supplementary Fig. S2. Influence of NAGNAG sequence on splicing isoform frequency. a, d Scheme of two analyzed minigenes. b, e Frequency of main splicing isoforms. c, f dNu for FL and Δ2, independently. Error bars represent standard deviations. Significantly distinct dNu (p < 0.05) for NAGNAG variant differing from wild type (AC) is marked with a star (*). Supplementary Fig. S3. dNu is affected by 3′ss nucleotides—CAGCAG variant. A Scheme of AFAP1L2 minigene. B Frequency of main splicing isoforms divided into separated groups differing only in one position. WT sequence is depicted in all groups and is highlighted in red. C dNu for FL and Δe2, independently. Supplementary Fig. S4. dNu is affected by 5′ss nucleotides—CAGCAG variant. A Scheme of AFAP1L2 minigene. B Frequency of main splicing isoforms is divided into separated groups differing in the second exon’s 5′ss only in one position. WT is highlighted in red for each separate group. C dNu for FL and Δe2, independently. Supplementary Fig. S5. dNu negatively correlates with proximal 3′ss strength. Correlations between 3′ss strength for both distal and proximal ss (MaxEnt score) and dNu was evaluated for both FL and Δ2 transcripts independently (PDF 651 kb)
Supplementary file2. Supplementary Table T1. Statical analyses of differences in dNu from RNAseq data. Supplementary Table T2. Dependence of dNu on used upstream 5′ss in human transcriptome. Supplementary Table T3. Summary of used minigene variants. All studied combinations of 5′ss, 3′ss and BP used in minigene AFAP1L2 and CTSD gene variants. Supplementary Table T4. Statistical analyses of differences in dNu for all studied combinations in AFAP1L2 and CTSD minigenes. Supplementary Table T5. Primers used in the study (XLS 251 kb)
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Hujová, P., Souček, P., Radová, L. et al. Nucleotides in both donor and acceptor splice sites are responsible for choice in NAGNAG tandem splice sites. Cell. Mol. Life Sci. 78, 6979–6993 (2021). https://doi.org/10.1007/s00018-021-03943-2