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Maize seedling mRNA-seq of splicing factor mutants and their wild-type siblings

ABSTRACT: The maize Rough endosperm3 (Rgh3) gene encodes an ortholog of the human essential splicing factor, ZRSR2. To test whether a mutation in Rgh3 affects mRNA splicing, we compared rgh3 mutants and wild-type sibling transcriptomes in an RNA-seq experiment. Twelve libraries were constructed with mRNA extracted from the roots and shoots of three seedlings of each genotype. The libraries were multiplexed and sequenced on one lane of the HiSeq 2000 platform. The run produced 149 million paired-end 100 bp reads that mapped to 35,028 genes. Two approaches were used to analyze the dataset. In the first approach, Mosaik2, FreeBayes, GSNAP, and Cufflinks were used to identify differences in transcript isoform abundance in a SNP-tolerant fashion. During reverse-transcription PCR validation, six examples of intron retention were found to occur more frequently in rgh3 seedlings, and all six introns were members of a rare class of introns called U12-type introns. The second approach utilized a t-test to determine whether more reads were mapped to U12-type introns in rgh3 libraries relative to wild-type libraries. Out of all U12-type introns within genes that are expressed at a early seedling stage, 43% exhibit splicing defects in rgh3 mutants. These U12-type intron splicing defects include intron retention and cryptic splice site activation. We report that the rgh3 mutation specifically impairs the U12-type intron splicing.

ORGANISM(S): Zea mays

PROVIDER: GSE57466 | GEO | 2017/02/13

SECONDARY ACCESSION(S): PRJNA246502

REPOSITORIES: GEO

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Project description:Purpose: The goals of this study are to compare NGS-derived transcriptomes (RNA-Seq) derived from rbm48 mutant and WT maize endosperm tissue. Homozygous rbm48 mutants exhibit a seed defect and are seedling lethal. Maize Rbm48 is homologouse to a human protein (rbm48) that is thought o be invoilved in the splicing of minor (U12) introns. This study characterizes DEGs between rbm48 alleles umu1 and umu2 and their WT counterparts and serves to identify U12 introns exhibiting retention as evidenced by RNA-seq read density within U12 introns in rbm49 mutants relative to WT controls. Methods: Endosperm mRNA profiles from 16-18 days after pollination (DAP) normal and rbm48 kernels in the Zea mays W22 background were generated by deep RNA-Seq. Total RNA was prepared for 4 biological replicates of paired rbm48 and normal sibling pools. 4 total RNA samples of rbm48 umu1 and their WT controls, and 4 samples of rbm48 umu2 and their WT controls were obtained (16 total). Non strand specific Tru-Seq illumine libraries were constructed and sequenced on the Illumina HiSeq. The sequence reads that passed quality filters were aligned with GSNAP, read counts/gene were determined with the HTSeq-Count utility in the HTSeq package and DEG analysis performed with DESeq2. Counts to U2 and U12 introns were also assessed and intron retention was assessed by measuring the Percent Splice Out (PSO) proportions for individual introns between mutant and WT samples. (PSO) calculations are based on the established methods for examining exon skip proportions (PSI). Conclusions: Differential gene expression analysis of rbm48 identifies large-scale changes. Considering counts in introns, the major U2-type introns are largely unaffected with only 3-5% of introns in expressed genes have a ΔPSO >20% By contrast, 65% and 53% of U12-type introns in rbm48-umu1 and rbm48-umu2, respectively, have a ΔPSO >20% suggesting more than half of U12-type introns are retained in rbm48 mutants. These RNA-Seq data help demonstrate that rbm48 plays a role in splicing U12 introns.

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Maize seedling mRNA-seq of splicing factor mutants and their wild-type siblings

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