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RNA-Seq mapping enabled quantitative analysis of gene expression differences between WT and rbm48 mutants in Zea mays (Maize).

ABSTRACT: 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.

ORGANISM(S): Zea mays

PROVIDER: GSE118505 | GEO | 2019/01/01

REPOSITORIES: GEO

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Project description:U12-type introns were originally recognized based on their highly conserved non-consensus AT-AC termini1,2, which are spliced by a separate minor spliceosome3,4. Padgett and Krainer groups later showed that terminal dinucleotides do not differentiate U12-type from U2-type introns, as there are U12-type introns with GT-AG termini and U2-type introns with AT-AC termini5,6. Rather, U12-type introns are recognized by their divergent and highly conserved 5’ splice site (5’ss) and branch point sequences, which both differ from the consensus sequences found in U2-type introns. To date, no functional differences have been ascribed to AT-AC or GT-AG subtypes of U12-type introns, nor have RNAseq analyses of minor spliceosome diseases reported any subtype specificity. Here, we describe a novel protein component of the minor spliceosome, encoded by the CENATAC locus, that is required for accurate splicing of AT-AC but not GT-AG type minor introns. CENATAC was initially identified in a subset of Mosaic Variegated Aneuploidy (MVA) patients with mutations in CENATAC, which lead to chromosome congression defects during mitosis. Earlier large-scale proteomic analyses tentatively classified CENATAC as a spliceosome component and phylogenetic analyses showed co-segregation of CENATAC with minor spliceosome components. Targeted depletion of CENATAC in HeLa cells, followed by RNAseq revealed global retention of AT-AC minor subtype introns with more than 60% showing statistically significant (up to 90%) intron retention (IR). Additionally, U12-type introns with 5’-AT, but divergent 3’-terminal dinucleotides also showed significant IR. We also detected cryptic U2-type splice site activation near affected AT-AC introns. In contrast, about 10% of GT-AG subtype introns responded to CENATAC depletion. Co-IP experiments revealed that CENATAC is not a U11/U12 di-snRNP component as expected for a specificity factor, but rather associates with the U4atac/U6atac.U5 tri-snRNP via interaction with PRPF3/4, suggesting a role for minor tri-snRNP in initial 5’ss recognition. CENATAC also interacts with TXNL4B, a paralog of TXNL4A in the major tri-snRNP. Finally, several genes encoding chromosome congression factors harbor U12 AT-AC-type introns that were highly retained in CENATAC depleted cells, potentially explaining the aneuploidy phenotype observed in MVA patients.

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RNA-Seq mapping enabled quantitative analysis of gene expression differences between WT and rbm48 mutants in Zea mays (Maize).

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