Genome-Wide Characterization and Expression Analysis of KH Family Genes Response to ABA and SA in Arabidopsis thaliana

K-homologous (KH) family is a type of nucleic acid-binding protein containing the KH domain and has been found to affect splicing and transcriptional regulation. However, KH family genes haven’t been investigated in plant species systematically. In this study, we identified 30 genes that belonged to the KH family based on HMM of the KH domain in Arabidopsis thaliana. Phylogenetic tree analysis showed that the KH family is grouped into three subgroups. Synteny analysis showed that AtKH9 and AtKH29 have the conserved synteny relationship between A. thaliana and the other five species. The AtKH9 and AtKH29 were located in the cytoplasm and nucleus. The seed germination rates of the mutants atkh9 and atkh29 were higher than wild-type after abscisic acid (ABA) and salicylic acid (SA) treatments. In addition, the expression of ABA-related genes, such as ABRE-binding factor 2 (ABF2), ABRE-binding factor 4 (ABF4), and delta 1-pyrroline-5-carboxylate synthase (P5CS), and an SA-related gene pathogenesis-related proteins b (PR1b) were downregulated after ABA and SA treatments, respectively. These results suggested that atkh9 and atkh29 mutants inhibit the effect of ABA and SA on seed germination. In conclusion, our results provide valuable information for further exploration of the function of KH family genes and propose directions and ideas for the identification and characterization of KH family genes in other plants.

Regulation of KHNYN antiviral activity by the extended di-KH domain and nucleo-cytoplasmic trafficking

The zinc finger antiviral protein (ZAP) restricts a broad range of viruses by binding CpG dinucleotides in viral RNA to target it for degradation and inhibit its translation. KHNYN was recently identified as an antiviral protein required for ZAP to inhibit retroviral replication, though little is known about its functional determinants. KHNYN contains an N-terminal extended di-KH-like domain, a PIN endoribonuclease domain and a C-terminal CUBAN domain that binds NEDD8 and ubiquitin. We show that deletion of the extended di-KH domain reduces its antiviral activity. However, despite its similarity to RNA binding KH domains, the extended di-KH domain in KHNYN does not appear to bind RNA. Mutation of residues in the CUBAN domain that bind NEDD8 increase KHNYN abundance but do not alter its antiviral activity, suggesting that this interaction regulates KHNYN homeostatic turnover. In contrast, a CRM1-dependent nuclear export signal (NES) at the C-terminus of the CUBAN domain is required for antiviral activity. Deletion of this signal retains KHNYN in the nucleus and inhibits its interaction with ZAP. Interestingly, this NES appeared in the KHNYN lineage at a similar time as when ZAP evolved in tetrapods, indicating that these proteins may have co-evolved to restrict viral replication.

Differential expression of QKI, KH domain-containing RNA-binding in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding QKI, KH domain-containing RNA-binding, QKI, when comparing primary tumors of the breast to the tissue of origin, the normal breast. QKI mRNA was present at significantly lower quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of QKI in primary tumors of the breast was correlated with overall survival in patients with luminal B subtype cancer, demonstrating a relationship between primary tumor expression of a differentially expressed gene and patient survival outcomes influenced by PAM50 molecular subtype. QKI may be of relevance to initiation, maintenance or progression of cancers of the female breast.

Mask, the Drosophila Ankyrin Repeat and KH domain-containing protein, affects microtubule stability

Proper regulation of microtubule (MT) stability and dynamics is vital for essential cellular processes, including axonal transportation and synaptic growth and remodeling in neurons. Here, we demonstrate that Mask negatively affects MT stability in both fly larval muscles and motor neurons. In larval muscles, loss-of-function of mask increases MT polymer length, and in motor neurons, loss of mask function results in overexpansion of the presynaptic terminal at the larval neuromuscular junctions (NMJs). mask genetically interacts with stathmin (stai), a neuronal modulator of MT stability, in the regulation of axon transportation and synaptic terminal stability. Our structure/function analysis on Mask revealed that its Ankyrin Repeats domain-containing N-terminal portion is sufficient to mediate Mask's impact to MT stability. Furthermore, we discovered that Mask negatively regulates the abundance of the microtubule-associated protein Jupiter in motor neuron axons, and that neuronal knocking down of Jupiter partially suppresses mask loss-of-function phenotypes at the larval NMJs. Together, our studies demonstrated that Mask is a novel regulator for microtubule stability, and such a role of Mask requires normal function of Jupiter.

Silencing of StRIK in potato suggests a role in periderm related to RNA processing and stress

Background The periderm is a protective barrier crucial for land plant survival, but little is known about genetic factors involved in its development and regulation. Using a transcriptomic approach in the cork oak (Q. suber) periderm, we previously identified an RS2-INTERACTING KH PROTEIN (RIK) homologue of unknown function containing a K homology (KH)-domain RNA-binding protein, as a regulatory candidate gene in the periderm. Results To gain insight into the function of RIK in the periderm, potato (S. tuberosum) tuber periderm was used as a model: the full-length coding sequence of RIK, hereafter referred to as StRIK, was isolated, the transcript profile analyzed and gene silencing in potato performed to analyze the silencing effects on periderm anatomy and transcriptome. The StRIK transcript accumulated in all vegetative tissues studied, including periderm and other suberized tissues such as root and also in wounded tissues. Downregulation of StRIK in potato by RNA interference (StRIK-RNAi) did not show any obvious effects on tuber periderm anatomy but, unlike Wild type, transgenic plants flowered. Global transcript profiling of the StRIK-RNAi periderm did show altered expression of genes associated with RNA metabolism, stress and signaling, mirroring the biological processes found enriched within the in silico co-expression network of the Arabidopsis orthologue. Conclusions The ubiquitous expression of StRIK transcript, the flower associated phenotype and the differential expression of StRIK-RNAi periderm point out to a general regulatory role of StRIK in diverse plant developmental processes. The transcriptome analysis suggests that StRIK might play roles in RNA maturation and stress response in the periderm.

KH domain containing RNA-binding proteins coordinate with microRNAs to regulate Caenorhabditis elegans development

MicroRNAs (miRNAs) and RNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level, but the extent to which these key regulators of gene expression coordinate their activities and the precise mechanisms of this coordination are not well understood. RBPs often have recognizable RNA binding domains that correlate with specific protein function. Recently, several RBPs containing K homology (KH) RNA binding domains were shown to work with miRNAs to regulate gene expression, raising the possibility that KH domains may be important for coordinating with miRNA pathways in gene expression regulation. To ascertain whether additional KH domain proteins functionally interact with miRNAs during Caenorhabditis elegans development, we knocked down twenty-four genes encoding KH-domain proteins in several miRNA sensitized genetic backgrounds. Here, we report that a majority of the KH domain-containing genes genetically interact with multiple miRNAs and Argonaute alg-1. Interestingly, two KH domain genes, predicted splicing factors sfa-1 and asd-2, genetically interacted with all of the miRNA mutants tested, whereas other KH domain genes showed genetic interactions only with specific miRNAs. Our domain architecture and phylogenetic relationship analyses of the C. elegans KH domain-containing proteins revealed potential groups that may share both structure and function. Collectively, we show that many C. elegans KH domain RBPs functionally interact with miRNAs, suggesting direct or indirect coordination between these two classes of post-transcriptional gene expression regulators.

The Flowering Time Regulator FLK Controls Pathogen Defense in Arabidopsis thaliana

Plant disease resistance is a complex process that is maintained in an intricate balance with development. Increasing evidence indicates the importance of post-transcriptional regulation of plant defense by RNA binding proteins. The K homology (KH) repeat is an ancient RNA binding motif found in proteins from diverse organisms. The role of KH domain proteins in pathogen resistance is not well known. From a genetic screen aimed to uncover novel defense genes in Arabidopsis, we identified a new allele of the canonical flowering regulatory gene, FLOWERING LOCUS KH Domain (FLK), encoding a putative triple KH-repeat protein. 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 found that the flk mutations compromised basal defense and defense signaling mediated by salicylic acid and led to increased reactive oxygen species (ROS) scavenging, likely through FLK’s regulation of the ROS scavenging enzyme catalases. RNA-seq data revealed that major defense signaling genes are regulated by FLK, providing a molecular basis for FLK’s contribution to pathogen defense. Together our data support that FLK is a multifunctional protein regulating pathogen defense and development of plants.