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.KEYWORDS alternative splicing; C2HC2 zinc finger; intron retention; Rtf2 domain; ubiquitin ligase I T is increasingly recognized that cotranscriptional and posttranscriptional gene regulation is comparable to transcriptional regulation in intricacy and importance. Pre-mRNA splicing is a cotranscriptional process and a major determinant of transcript abundance and complexity (Reddy et al. 2013). Constitutive splicing refers to the use of only one set of splice sites to generate a single mature mRNA. By contrast, alternative splicing occurs when variable splice sites are selected, leading to the generation of more than one processed RNA product from a single pre-messenger RNA (mRNA). An individual gene can thus potentially encode multiple proteins, leading to a substantial increase in proteomic diversity (Chen and Manley 2009;Syed et al. 2012;Reddy et al. 2013).Recent work has established that alternative splicing is common in plants, affecting 60% of intron-containing genes . Alternative splicing has important roles in plant growth, development, abiotic stress tolerance, circadian rhythms, and pathogen defense (Staiger and Brown 2013). The most common outcome of alternative splicing in plants is intron retention Lan et al. 2013), which occurs when an intron fails to be spliced out of the premRNA. Retained introns frequently contain premature termination codons (PTCs) that can channel the transcript into the nonsense-mediated decay (NMD) pathway. Intron retention provides a means for "transcriptome tuning" (Braunschweig et al. 2014) and contributes to the post-transcriptional regulation of gene expression by reducing levels of inappropriately expressed transcripts Ge and Porse 2013).Alternative splicing is subject to elaborate regulation that relies on general and specific trans-acting factors as ...

A genetic screen implicates a CWC16/Yju2/CCDC130 protein and SMU1 in alternative splicing in Arabidopsis thaliana

“…In contrast to smu1-4 and cwc16a-1, the two GFP-weak mutants reported here, prp8-10 and rtf2-4 showed generally reduced splicing efficiency of GFP premRNA, leading to increased accumulation of the unspliced primary transcript. The overall reduced splicing efficiency in these mutants is consistent with core spliceosomal roles of PRP8 (Pleiss et al 2007) and possibly RTF2 (Larson et al 2016), although a regulatory role through ubiquitination has also been suggested for RTF2 (Sasaki et al 2015).…”

“…Whether SMU1 has this activity in Arabidopsis remains to be investigated. Several Arabidopsis splicing factors acquire ubiquitination, including the catalytic site core protein PRP8, which was identified among the GFPweak mutants in our screens (Sasaki et al 2015; this study). Homozygous smu1-1 and smu1-2 T-DNA insertion mutants of Arabidopsis were reported to be nonviable or show multiple, nonlethal developmental defects, respectively (Chung et al 2009).…”

“…The downstream gray stippled area and ATG represent an unused promoter and initiation codon, respectively. The 3 ′ splice sites for the GT-AG and AT-AC introns are separated by only 3 nt with the noncanonical AC on the outside (Sasaki et al 2015;Kanno et al 2016).…”

“…To identify proteins that impact splicing efficiency and alternative splicing in plants, we are performing a forward genetic screen based on an alternatively spliced GFP reporter gene in Arabidopsis. The usefulness of this genetic system has been validated by the identification of a novel factor, RTF2 (Replication Termination Factor2), which may participate in ubiquitin-based regulation of the spliceosome (Sasaki et al 2015), and the finding of an unexpected role for the Cajal body marker protein coilin in attenuating splicing efficiency of a small subset of stress-related genes (Kanno et al 2016). Here we report three new mutants identified in the screen that are defective, respectively, in splicing factors SMU1, SmF, and CWC16, which are related to budding yeast first step factor Yju2 and human CCDC130.…”

Recruitment of the NineTeen Complex to the activated spliceosome requires AtPRMT5

“…S2B). A recent study showed that Arabidopsis Prp8 is involved in modulating the splicing of the GFP reporter gene in a genetic screen (28) and long noncoding transcript COOLAIR to regulate FLC transcription (25). Protein sequence alignment of Prp8 homologs showed that P1141 of Prp8 is in the linker region between the RNA recognition motif (RRM) and the U5-snRNA binding domain and is highly conserved from yeast to humans, and P347 is in the linker region between the N-terminal domain and the PROCN domain and is moderately conserved (Fig.…”