Abstract
Alternatively spliced introns are the ones that are usually spliced but can be occasionally retained in a transcript isoform. They are the most frequently used alternative splice form in plants (~50% of alternative splicing events). Chlamydomonas reinhardtii, a unicellular alga, is a good model to understand alternative splicing (AS) in plants from an evolutionary perspective as it diverged from land plants a billion years ago. Using over 7 million cDNA sequences from both pyrosequencing and Sanger sequencing, we found that a much higher percentage of genes (~20% of multi-exon genes) undergo AS than previously reported (3–5%). We found a full component of SR and SR-like proteins possibly involved in AS. The most prevalent type of AS event (40%) was retention of introns, most of which were supported by multiple cDNA evidence (72%) while only 20% of them have coding capacity. By comparing retained and constitutive introns, we identified sequence features potentially responsible for the retention of introns, in the framework of an “intron definition” model for splicing. We find that retained introns tend to have a weaker 5′ splice site, more Gs in their poly-pyrimidine tract and a lesser conservation of nucleotide ‘C’ at position −3 of the 3′ splice site. In addition, the sequence motifs found in the potential branch-point region differed between retained and constitutive introns. Furthermore, the enrichment of G-triplets and C-triplets among the first and last 50 nt of the introns significantly differ between constitutive and retained introns. These could serve as intronic splicing enhancers. All the alternative splice forms can be accessed at http://bioinfolab.miamioh.edu/cgi-bin/PASA_r20140417/cgi-bin/status_report.cgi?db=Chre_AS.
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Abbreviations
- 5′ss:
-
5′ Splice site
- 3′ss:
-
3′ Splice site
- CDS:
-
Coding sequence
- ESE:
-
Exonic splicing enhancer
- ESS:
-
Exonic splicing silencer
- GFF3:
-
General feature format
- ISE:
-
Intronic splicing enhancer
- ISS:
-
Intronic splicing silencer
- nt:
-
Nucleotide
- PSSM:
-
Position specific scoring matrix
- NMD:
-
Non-mediated decay
- AS:
-
Alternative splicing
- PASA:
-
Program to assemble spliced alignment
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Acknowledgements
Funding for this project is provided by a grant award from the Ohio Plant Biotechnology Consortium to CL. This work was partially supported by the National Institutes of Health (1R15GM94732-1 A1 to CL) and Centre National de la Recherche Scientifique (ANR-11-LABX-0011-DYNAMO to OV). The authors thank Mario Stanke, Lin Liu and Trey Moler for their participation in this project and Marina Cavaioulo for giving access to her mapping of Illumina reads.
Authors’ contributions
CL managed and coordinated the project. PKRK and CL conceived the study. PKRK carried out implementation and drafted the manuscript. OV conducted SR protein analysis and individual intron inspection and helped devise validation tests. All authors participated in manuscript writing.
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11103_2017_605_MOESM1_ESM.tif
Supplemental Fig. 1 Example of alternative splicing event supported only by Augustus annotation. a. Splicing graph of the gene structure as viewed in PASA website [http://bioinfolab.miamioh.edu/cgi-bin/PASA_r20140417/cgi-bin/assembly_alt_splice_info.cgi?db=Chre_AS&cdna_acc=asmbl_94&SHOW_ALL=1&SHOW_ALIGNMENTS]. b and c show alternative splicing forms “alternative acceptor” and “retained intron” as inferred from comparing two isoforms called asmbl_94 and asmbl_95. Splice sites are shown in light blue, exons in black, coding regions as predicted by PASA in red, and spliced out introns by the thin connecting lines. Highlighted purple box denote the change between isoforms. Both isoforms asmbl_94 and asmbl_95 are supported by only Augustus annotations “Cre01.g003100.t1.3” and “Cre01.g003100.t2.1” respectively. An intron retained between the coding exons is denoted as CDS retained intron (TIF 458 KB)
11103_2017_605_MOESM2_ESM.xlsx
Supplemental Table 1 Chlamydomonas SR proteins. SR protein family assignments were based on (Barbosa-Morais et al. 2006; Barta et al. 2008). RRM: RNA recognition motif; RS: arginine/serine-rich region; ZnK: Zinc knuckle domain; SWAP/Surp: suppressor-of-white-apricot domain; PWI: Pro-Trp-Ile domain. Parentheses within a “Known Domains” field entry indicate a weak domain feature. Au9 gene models, annotations, and corresponding browser links showing PASA transcript models are provided in the right column. (XLSX 13 KB)
11103_2017_605_MOESM3_ESM.xlsx
Supplemental Table 2 Retained introns and their validation scores. Retained intron found in the PASA model with their genomic coordinates, validation scores and results of individual inspection where available. (XLSX 112 KB)
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Raj-Kumar, PK., Vallon, O. & Liang, C. In silico analysis of the sequence features responsible for alternatively spliced introns in the model green alga Chlamydomonas reinhardtii . Plant Mol Biol 94, 253–265 (2017). https://doi.org/10.1007/s11103-017-0605-9
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DOI: https://doi.org/10.1007/s11103-017-0605-9