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An Rtf2 domain-containing protein influences pre-mRNA splicing and is essential for embryonic development in Arabidopsis thaliana

ABSTRACT: Alternative splicing is prevalent in plants, but little is known about its regulation in the context of developmental and signaling pathways. We describe here a new factor that influences pre-mRNA splicing and is essential for embryonic development in Arabidopsis thaliana. This factor was retrieved in a genetic screen that identified mutants impaired in expression of an alternatively spliced GFP reporter gene. In addition to the known spliceosomal component PRP8, the screen retrieved a previously uncharacterized protein containing a Replication termination factor2 (Rtf2) domain defined by a C2HC2 zinc finger. The Rtf2 protein was discovered in fission yeast, where it stabilizes paused DNA replication forks by an unknown mechanism. When homozygous, a null mutation in Arabidopsis RTF2 (AtRTF2) is embryo-lethal, indicating that it encodes an essential protein. As revealed by quantitative RT-PCR, impaired expression of GFP in atrtf2 and prp8 mutants is attributable to inefficient splicing of the GFP pre-mRNA. A genome-wide analysis using RNA-seq demonstrated that 12% of total introns display a significant degree of retention in atrtf2 mutants. Intron-retaining transcripts are enriched from genes encoding proteins involved in signaling pathways and membrane transport. Affinity purification of AtRTF2 followed by mass spectrometry identified several known and predicted splicing proteins. In a yeast two-hybrid screen, AtRTF2 interacted with Exo70B1, a peripheral subunit of the exocyst, which is involved in vesicle trafficking. Considering these results and previous suggestions that Rtf2 constitutes an ubiquitin-related domain, we discuss possible roles of AtRTF2 in ubiquitin-based regulation of pre-mRNA splicing and membrane signaling to the spliceosome. Rtf2 is SDR1 (= AtRTF2) and was discovered in a genetic suppressor screen using the dms4 mutant. DMS4 was described in Kanno et al (2010) EMBO Rep. 11:65-71.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE63238 | GEO | 2014/11/14

SECONDARY ACCESSION(S): PRJNA267075

REPOSITORIES: GEO

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Project description:Alternative splicing is prevalent in plants, but little is known about its regulation in the context of developmental and signaling pathways. We describe here a new factor that influences pre-mRNA splicing and is essential for embryonic development in Arabidopsis thaliana. This factor was retrieved in a genetic screen that identified mutants impaired in expression of an alternatively spliced GFP reporter gene. In addition to the known spliceosomal component PRP8, the screen retrieved a previously uncharacterized protein containing a Replication termination factor2 (Rtf2) domain defined by a C2HC2 zinc finger. The Rtf2 protein was discovered in fission yeast, where it stabilizes paused DNA replication forks by an unknown mechanism. When homozygous, a null mutation in Arabidopsis RTF2 (AtRTF2) is embryo-lethal, indicating that it encodes an essential protein. As revealed by quantitative RT-PCR, impaired expression of GFP in atrtf2 and prp8 mutants is attributable to inefficient splicing of the GFP pre-mRNA. A genome-wide analysis using RNA-seq demonstrated that 12% of total introns display a significant degree of retention in atrtf2 mutants. Intron-retaining transcripts are enriched from genes encoding proteins involved in signaling pathways and membrane transport. Affinity purification of AtRTF2 followed by mass spectrometry identified several known and predicted splicing proteins. In a yeast two-hybrid screen, AtRTF2 interacted with Exo70B1, a peripheral subunit of the exocyst, which is involved in vesicle trafficking. Considering these results and previous suggestions that Rtf2 constitutes an ubiquitin-related domain, we discuss possible roles of AtRTF2 in ubiquitin-based regulation of pre-mRNA splicing and membrane signaling to the spliceosome. Rtf2 is SDR1 (= AtRTF2) and was discovered in a genetic suppressor screen using the dms4 mutant. DMS4 was described in Kanno et al (2010) EMBO Rep. 11:65-71. Examination of whole-genome DNA methylation status in transgenic Arabidopsis plants

Project description:The essential process of pre-mRNA splicing must occur with high fidelity and efficiency for proper gene expression. The spliceosome employs DExD/H box helicases to promote on-pathway interactions while simultaneously minimizing errors. Prp8 and Snu114, an EF2-like GTPase, regulate the activity of the Brr2 helicase, promoting RNA unwinding by Brr2 at appro-priate points in the splicing cycle and repressing it at others. Mutations linked to Retinitis Pig-mentosa (RP), a disease that causes blindness in humans, map to the Brr2 regulatory region of Prp8. Previous In vitro studies of homologous mutations in Saccharomyces cerevisiae show that Prp8-RP mutants cause defects in spliceosome activation. Here we show a subset of RP muta-tions in Prp8 also cause defects in the transition between the 1st and 2nd catalytic steps of splic-ing. Though Prp8-RP mutants do not cause defects in splicing fidelity, they result in an overall decrease in splicing efficiency. Furthermore, genetic analyses link Snu114 GTP/GDP occupancy to Prp8-dependent regulation of Brr2. Our results implicate the transition between the 1st and 2nd catalytic steps as a critical place in the splicing cycle where Prp8-RP mutants influence splic-ing efficiency. The location of the Prp8-RP mutants, at the â??hingeâ?? that links the Prp8 Jab1-MPN regulatory â??tailâ?? to the globular portion of the domain, suggests that these Prp8-RP mutants inhibit regulated movement of the Prp8 Jab1/MPN domain into the Brr2 RNA binding channel to transiently inhibit Brr2 activity. Therefore, in Prp8-linked RP, disease likely results not only from defects in spliceosome assembly and activation, but also because of defects in splicing ca-talysis. paper to be submitted Two channel microarrays were used. RNA isolated from wt yeast grown simultaneously to the mutant was used as a reference. This reference was used in one of the channels for each hybridization and used in the statistical analysis to obtain an average expression-profile for each mutant relative to the wt. Three independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Strains, both WT and mutant, were grown at 37C until mid-log phase, OD600 of approximately 0.7. The mutated PRP8 gene is present on HIS marked CEN plasmid, and the corresponding genomic copy of PRP8 deleted. Strains labeled as wildtype also have the relevant genomic PRP8 deleted, but complemented with wildtype PRP8 on CEN plasmid.

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An Rtf2 domain-containing protein influences pre-mRNA splicing and is essential for embryonic development in Arabidopsis thaliana

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