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Rtf2 is Important for Replication Fork Barrier Activity of RTS1 via Splicing of Rtf1 [Pu-Seq]

ABSTRACT: This study investigated the role Rtf2 plays on the replication fork barrier (RFB) activity of RTS1 in S. pombe. We confirm Rtf2 is important for the full barrier activity of the RTS1-Rtf1 RFB. We show that Rtf2 does not enact its enhancing effect on RTS1 RFB activity via binding to Region A of RTS1, but instead we find it to be closely associated with several splicing factors. Analysis of cells deleted for rtf2 by cDNA-Seq reveal splicing defects specifically effecting intron retention, including for the Rtf1 transcript. We show that intron retention within rtf1 is responsible for the reduced barrier activity of RTS1 when rtf2 is deleted. This study reveals Rtf2 to be an important factor for maintaining efficient splicing within the cell.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE192342 | GEO | 2021/12/24

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

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Rtf2 is Important for Replication Fork Barrier Activity of RTS1 via Splicing of Rtf1 [Pu-Seq]

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