Dataset Information

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-messengerRNA (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 recovered Arabidopsis RTF2 (AtRTF2), a previously uncharacterized, evolutionarily conserved protein containing a replication termination factor 2 (Rtf2) domain. A homozygous null mutation in AtRTF2 is embryo lethal, indicating that AtRTF2 is an essential protein. Quantitative RT-PCR demonstrated that impaired expression of GFP in atrtf2 and prp8 mutants is due to inefficient splicing of the GFP pre-mRNA. A genome-wide analysis using RNA sequencing indicated that 13-16% of total introns are retained to a significant degree in atrtf2 mutants. Considering these results and previous suggestions that Rtf2 represents an ubiquitin-related domain, we discuss the possible role of AtRTF2 in ubiquitin-based regulation of pre-mRNA splicing.

SUBMITTER: Sasaki T

PROVIDER: S-EPMC4492377 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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An Rtf2 Domain-Containing Protein Influences Pre-mRNA Splicing and Is Essential for Embryonic Development in Arabidopsis thaliana.

Sasaki Taku T Kanno Tatsuo T Liang Shih-Chieh SC Chen Pao-Yang PY Liao Wen-Wei WW Lin Wen-Dar WD Matzke Antonius J M AJ Matzke Marjori M

Genetics 20150327 2

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-messengerRNA (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 recove ...[more]

PMID: 25819795

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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:Ubiquitin-like proteins play important roles in the regulation of many biological processes. UBL5 (Ubiquitin-like protein 5)/Hub1 (Homologous to ubiquitin 1), a member of the ubiquitin family, acts as a ubiquitin-like modifier on a specific target, the spliceosomal protein Snu66, in yeast and human cells. The 22nd aspartic acid (Asp22) is involved in the attachment of Hub1 to the Hub1 interaction domain (HIND) of Snu66 in yeast to modulate spliceosomal activity. Hub1 differs from other modifiers which interact covalently with their targets. It modulates pre-mRNA splicing by binding to Snu66 non-covalently in both yeast and human cells. However, the molecular mechanisms of Hub1-mediated pre-mRNA splicing in plant systems remains unclear. To better understand the function of Hub1 in plants, we examined the role of this ubiquitin-like modifier in Arabidopsis thaliana, which has two Hub1 homologues. Arabidopsis UBL5/Hub1(UBL5) is highly conserved at the amino acid level, compared to eukaryotic homologues in both plants and animals. In this study, phenotypic analysis of A. thaliana with reduced UBL5 gene expression, generated by RNA interference of AtUBL5a and AtUBL5b were performed. Interestingly, knock down plants of AtUBL5 showed abnormalities in root elongation, plant development, and auxin response. AtUBL5b is highly expressed in the vascular tissue of the leaf, stem, and root tissue. Yeast two-hybrid analysis revealed that AtUBL5a and AtUBL5b interact with the putative splicing factor AtPRP38 through its C-terminal domain (AtPRP38C). Knock down of AtUBL5b resulted in a pattern of insufficient pre-mRNA splicing in several introns of AtCDC2, and in introns of IAA1, IAA4, and IAA5. Defects of pre-mRNA splicing in an AtPRP38 mutant resulted in an insufficient pre-mRNA splicing pattern in the intron of IAA1. Based on these results, we showed that AtUBL5b positively regulates plant root elongation and development through pre-mRNA splicing with AtPRP38C in A. thaliana.

Dataset Information

An Rtf2 Domain-Containing Protein Influences Pre-mRNA Splicing and Is Essential for Embryonic Development in Arabidopsis thaliana.

Publications

An Rtf2 Domain-Containing Protein Influences Pre-mRNA Splicing and Is Essential for Embryonic Development in Arabidopsis thaliana.

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