- Award ID(s):
- NSF-PAR ID:
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Page Range / eLocation ID:
- 3490 to 3506
- Medium: X
- Sponsoring Org:
- National Science Foundation
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OZ1, an RNA editing factor that controls the editing of 14 cytidine targets in Arabidopsis chloroplasts, contains two RanBP2-type zinc finger (Znf) domains. The RanBP2 Znf is a C4-type member of the broader zinc finger family with unique functions and an unusually diverse distribution in plants. The domain can mediate interactions with proteins or RNA and appears in protein types such as proteases, RNA editing factors, and chromatin modifiers; however, few characterized Arabidopsis proteins containing RanBP2 Znfs have been studied specifically with the domain in mind. In humans, RanBP2 Znf-containing proteins are involved in RNA splicing, transport, or transcription initiation. We present a phylogenetic overview of Arabidopsis RanBP2 Znf proteins and the functional niches that these proteins occupy in plants. OZ1 and its four-member family represent a branch of this family with major impact on the RNA biology of chloroplasts and mitochondria in Arabidopsis. We discuss what is known about other plant proteins carrying the RanBP2 Znf domain and point out how phylogenetic information can provide clues to functions of uncharacterized Znf proteins.more » « less
Abstract Plant disease resistance is a complex process that is maintained in an intricate balance with development. Increasing evidence indicates the importance of posttranscriptional regulation of plant defense by RNA binding proteins. In a genetic screen for suppressors of Arabidopsis (Arabidopsis thaliana) accelerated cell death 6-1 (acd6-1), a small constitutive defense mutant whose defense level is grossly in a reverse proportion to plant size, we identified an allele of the canonical flowering regulatory gene FLOWERING LOCUS K HOMOLOGY DOMAIN (FLK) encoding a putative protein with triple K homology (KH) repeats. The KH repeat is an ancient RNA binding motif found in proteins from diverse organisms. The relevance of KH-domain proteins in pathogen resistance is largely unexplored. In addition to late flowering, the flk mutants exhibited decreased resistance to the bacterial pathogen Pseudomonas syringae and increased resistance to the necrotrophic fungal pathogen Botrytis cinerea. We further found that the flk mutations compromised basal defense and defense signaling mediated by salicylic acid (SA). Mutant analysis revealed complex genetic interactions between FLK and several major SA pathway genes. RNA-seq data showed that FLK regulates expression abundance of some major defense- and development-related genes as well as alternative splicing of a number of genes. Among the genes affected by FLK is ACD6, whose transcripts had increased intron retentions influenced by the flk mutations. Thus, this study provides mechanistic support for flk suppression of acd6-1 and establishes that FLK is a multifunctional gene involved in regulating pathogen defense and development of plants.more » « less
Alternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.
We recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.
Mass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRASG12Vand KRASQ61Hcells, respectively, 997 of which were shared (
p< 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its variant RBM45M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS. Conclusion
Proteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.
Abstract Brassinosteroids (BRs) are a group of steroid hormones regulating plant growth and development. Since BRs do not undergo transport among plant tissues, their metabolism is tightly regulated by transcription factors (TFs) and feedback loops. BAS1 (CYP734A1, formerly CYP72B1) and SOB7 (CYP72C1) are two BR-inactivating cytochrome P450s identified in Arabidopsis thaliana. We previously found that a TF ATAF2 (ANAC081) suppresses BAS1 and SOB7 expression by binding to the Evening Element (EE) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)-binding site (CBS) on their promoters. Both the EE and CBS are known binding targets of the circadian regulatory protein CCA1. Here, we confirm that CCA1 binds the EE and CBS motifs on BAS1 and SOB7 promoters, respectively. Elevated accumulations of BAS1 and SOB7 transcripts in the CCA1 null mutant cca1-1 indicate that CCA1 is a repressor of their expression. When compared with either cca1-1 or the ATAF2 null mutant ataf2-2, the cca1-1 ataf2-2 double mutant shows higher SOB7 transcript accumulations and a stronger BR-insensitive phenotype of hypocotyl elongation in white light. CCA1 interacts with ATAF2 at both DNA–protein and protein–protein levels. ATAF2, BAS1, and SOB7 are all circadian regulated with distinct expression patterns. These results demonstrate that CCA1 and ATAF2 differentially suppress BAS1- and SOB7-mediated BR inactivation.more » « less
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