TY - JOUR

T1 - Many rice genes are differentially spliced between roots and shoots but cytokinin has minimal effect on splicing

AU - Freese, Nowlan H.

AU - Estrada, April R.

AU - Blakley, Ivory C.

AU - Duan, Jinjie

AU - Loraine, Ann E.

PY - 2019/5/1

Y1 - 2019/5/1

N2 - Alternatively spliced genes produce multiple spliced isoforms, called transcript variants. In differential alternative splicing, transcript variant abundance differs across sample types. Differential alternative splicing is common in animal systems and influences cellular development in many processes, but its extent and significance is not as well known in plants. To investigate differential alternative splicing in plants, we examined RNA-Seq data from rice seedlings. The data included three biological replicates per sample type, approximately 30 million sequence alignments per replicate, and four sample types: roots and shoots treated with exogenous cytokinin delivered hydroponically or a mock treatment. Cytokinin treatment triggered expression changes in thousands of genes but had negligible effect on splicing patterns. However, many genes were differentially spliced between mock-treated roots and shoots, indicating that our methods were sufficiently sensitive to detect differential splicing between data sets. Quantitative fragment analysis of reverse transcriptase-PCR products made from newly prepared rice samples confirmed 9 of 10 differential splicing events between rice roots and shoots. Differential alternative splicing typically changed the relative abundance of splice variants that co-occurred in a data set. Analysis of a similar (but less deeply sequenced) RNA-Seq data set from Arabidopsis showed the same pattern. In both the Arabidopsis and rice RNA-Seq data sets, most genes annotated as alternatively spliced had small minor variant frequencies. Of splicing choices with abundant support for minor forms, most alternative splicing events were located within the protein-coding sequence and maintained the annotated reading frame. A tool for visualizing protein annotations in the context of genomic sequence (ProtAnnot) together with a genome browser (Integrated Genome Browser) were used to visualize and assess effects of differential splicing on gene function. In general, differentially spliced regions coincided with conserved protein domains, indicating that differential alternative splicing is likely to affect protein function between root and shoot tissue in rice.

AB - Alternatively spliced genes produce multiple spliced isoforms, called transcript variants. In differential alternative splicing, transcript variant abundance differs across sample types. Differential alternative splicing is common in animal systems and influences cellular development in many processes, but its extent and significance is not as well known in plants. To investigate differential alternative splicing in plants, we examined RNA-Seq data from rice seedlings. The data included three biological replicates per sample type, approximately 30 million sequence alignments per replicate, and four sample types: roots and shoots treated with exogenous cytokinin delivered hydroponically or a mock treatment. Cytokinin treatment triggered expression changes in thousands of genes but had negligible effect on splicing patterns. However, many genes were differentially spliced between mock-treated roots and shoots, indicating that our methods were sufficiently sensitive to detect differential splicing between data sets. Quantitative fragment analysis of reverse transcriptase-PCR products made from newly prepared rice samples confirmed 9 of 10 differential splicing events between rice roots and shoots. Differential alternative splicing typically changed the relative abundance of splice variants that co-occurred in a data set. Analysis of a similar (but less deeply sequenced) RNA-Seq data set from Arabidopsis showed the same pattern. In both the Arabidopsis and rice RNA-Seq data sets, most genes annotated as alternatively spliced had small minor variant frequencies. Of splicing choices with abundant support for minor forms, most alternative splicing events were located within the protein-coding sequence and maintained the annotated reading frame. A tool for visualizing protein annotations in the context of genomic sequence (ProtAnnot) together with a genome browser (Integrated Genome Browser) were used to visualize and assess effects of differential splicing on gene function. In general, differentially spliced regions coincided with conserved protein domains, indicating that differential alternative splicing is likely to affect protein function between root and shoot tissue in rice.

KW - alternative splicing

KW - cytokinin

KW - rice

KW - RNA-Seq

KW - root

KW - shoot

UR - http://www.scopus.com/inward/record.url?scp=85066472481&partnerID=8YFLogxK

U2 - 10.1002/pld3.136

DO - 10.1002/pld3.136

M3 - Journal article

C2 - 31245776

AN - SCOPUS:85066472481

VL - 3

JO - Plant Direct

JF - Plant Direct

SN - 2475-4455

IS - 5

M1 - e00136

ER -