Kinase Motifs Research Articles

Uncovering the profile of ubiquitination motif in catalytic proteins using sequence contextual features

Ubiquitin molecules modify their target protein by conjugating the amino group of lysine side chain, thereby regulating the activity and function of most intracellular proteins. However, within multiple lysine residues on a target protein, only particular residues can be modified by ubiquitin molecules. The occurrence of ubiquitination varies on different lysine residues. The ubiquitination of a lysine residue is ubiquitinated depends on the environment of amino acid sequences near the lysine residue; this sequence is called a motif. Although the interaction of motifs with ubiquitin molecules have been extensively studied, there have been no reports on the ubiquitination motifs. In the current study, 133 878 ubiquitinated peptide data from 27 762 proteins in 30 articles and the mUbiSiDa database in the past decade were collected and analyzed. A total of 208 motifs were found with a score above 6, covering most of the ubiquitination peptides. Of these, six motifs with higher scores and significant regularity were found to belong to zinc finger proteins and serine/threonine kinases, termed zinc finger and serine/threonine kinase motifs, respectively. Analysis on four zinc finger motifs revealed that the ubiquitination sites in zinc finger motifs, xxHxxxxxxE_K_xxxCxxCxxx and xxxKxxHxxx_K_xxxxxExxKx, were located between two zinc finger domains, while the ubiquitination sites in zinc finger motifs, xxxxxxxxCG_K_xFxxxxxxxx and CxxxxxxFxQ_K_xxxxxxxxxx were located within zinc chelation structure near one Cys residue. Different proteins contain different types and quantities of zinc finger motifs. The results of the 3D structure showed that the ubiquitination sites in motif CxxxxxxFxQ_K_xxxxxxxxxx were located in the ring structure of the zinc finger domain. This may affect the interaction between zinc finger domains and DNA. Meanwhile, the ubiquitination sites of the other three zinc finger motifs located outside the interaction structure between zinc finger domains and DNA may not serve to block interaction but degradation. The analysis of two serine/threonine kinase motifs revealed that some residues near the ubiquitination site in two serine/threonine kinase motifs were completely identical in different proteins; however, these conserved residues were completely different in different motifs, suggesting the varied function of the two motifs. Moreover, it was found that no protein simultaneously contains both serine/threonine kinase motifs. Although there were significant differences in the amino acid sequences of these motifs, their spatial structures were highly conserved. These results suggest that the regulatory functions of the two motifs may differ. This is because two motifs may be recognized by different ubiquitinases or deubiquitinases. Overall, these results suggest that the motifs of ubiquitination sites exhibit high regularity and special structural characteristics in different types of proteins. This study sheds light on the profile and regularity of ubiquitination motifs, promoting the prediction of the function, structure, and modified sites of ubiquitinated proteins.

Inducible auto-phosphorylation regulates a widespread family of nucleotidyltransferase toxins

Nucleotidyltransferases (NTases) control diverse physiological processes, including RNA modification, DNA replication and repair, and antibiotic resistance. The Mycobacterium tuberculosis NTase toxin family, MenT, modifies tRNAs to block translation. MenT toxin activity can be stringently regulated by diverse MenA antitoxins. There has been no unifying mechanism linking antitoxicity across MenT homologues. Here we demonstrate through structural, biochemical, biophysical and computational studies that despite lacking kinase motifs, antitoxin MenA1 induces auto-phosphorylation of MenT1 by repositioning the MenT1 phosphoacceptor T39 active site residue towards bound nucleotide. Finally, we expand this predictive model to explain how unrelated antitoxin MenA3 is similarly able to induce auto-phosphorylation of cognate toxin MenT3. Our study reveals a conserved mechanism for the control of tuberculosis toxins, and demonstrates how active site auto-phosphorylation can regulate the activity of widespread NTases.

PTMoreR-enabled cross-species PTM mapping and comparative phosphoproteomics across mammals

To support PTM proteomic analysis and annotation in different species, we developed PTMoreR, a user-friendly tool that considers the surrounding amino acid sequences of PTM sites during BLAST, enabling a motif-centric analysis across species. By controlling sequence window similarity, PTMoreR can map phosphoproteomic results between any two species, perform site-level functional enrichment analysis, and generate kinase-substrate networks. We demonstrate that the majority of real P-sites in mice can be inferred from experimentally derived human P-sites with PTMoreR mapping. Furthermore, the compositions of 129 mammalian phosphoproteomes can also be predicted using PTMoreR. The method also identifies cross-species phosphorylation events that occur on proteins with an increased tendency to respond to the environmental factors. Moreover, the classic kinase motifs can be extracted across mammalian species, offering an evolutionary angle for refining current motifs. PTMoreR supports PTM proteomics in non-human species and facilitates quantitative phosphoproteomic analysis.

Expression and clinical significance of U2AF homology motif kinase 1 in oral squamous cell carcinoma

ObjectiveU2AF homology motif kinase 1 (UHMK1) is a newly discovered molecule that may have multiple functions. Recent studies have revealed that UHMK1 had aberrant expression in many tumors and was associated with tumor progression. However, UHMK1 was rarely reported in oral squamous cell carcinoma (OSCC). Study DesignIn this study, Western blot, quantitative real-time polymerase chain reaction (PCR), and immunohistochemistry were used to detect the expression of UHMK1 in OSCC and peritumoral non-neoplastic tissues. Then, its relationship with clinicopathologic parameters was analyzed. The Kaplan–Meier method and Cox regression model were used to analyze the effects of UHMK1 expression on the prognosis and survival of OSCC patients. ResultsOur results showed that UHMK1 had higher expression in OSCC tissues compared with in peritumoral non-neoplastic tissues, and its high expression was associated with high TNM stage and lymph node metastasis. High UHMK1 expression was related to short overall and disease-free survival times. Moreover, UHMK1 expression was identified as an independent prognostic factor that influences overall and disease-free survival of OSCC patients. ConclusionHigh expression of UHMK1 is associated with the poor prognosis of patients, and it can be used as a potential prognostic molecule for OSCC.

Identification and Characterization of Entamoeba histolytica Choline Kinase.

Entamoeba histolytica is one of the death-causing parasites in the world. Study on its lipid composition revealed that it is predominated by phosphatidylcholine and phosphatidylethanolamine. Further study revealed that its phosphorylated metabolites might be produced by the Kennedy pathway. Here, we would like to report on the characterizations of enzymes from this pathway that would provide information for the design of novel inhibitors against these enzymes in future. E. histolytica HM-1:IMSS genomic DNA was isolated and two putative choline/ethanolamine kinase genes (EhCK1 and EhCK2) were cloned and expressed from Escherichia coli BL21 strain. Enzymatic characterizations were further carried out on the purified enzymes. EhCK1 and EhCK2 were identified from E. histolytica genome. The deduced amino acid sequences were more identical to its homologues in human (35-48%) than other organisms. The proteins were clustered as ethanolamine kinase in the constructed phylogeny tree. Sequence analysis showed that they possessed all the conserved motifs in choline kinase family: ATP-binding loop, Brenner's phosphotransferase motif, and choline kinase motif. Here, the open reading frames were cloned, expressed, and purified to apparent homogeneity. EhCK1 showed activity with choline but not ethanolamine. The biochemical characterization showed that it had a Vmax of 1.9 ± 0.1µmol/min/mg. Its Km for choline and ATP was 203 ± 26µM and 3.1 ± 0.4mM, respectively. In contrast, EhCK2 enzymatic activity was only detected when Mn2+ was used as the co-factor instead of Mg2+ like other choline/ethanolamine kinases. Highly sensitive and specific antibody against EhCK1 was developed and used to confirm the endogenous EhCK1 expression using immunoblotting. With the understanding of EhC/EK importance in phospholipid metabolism and their unique characteristic, EhC/EK could be a potential target for future anti-amoebiasis study.

Transcriptome analysis of Haemaphysalis flava female using Illumina HiSeq 4000 sequencing: de novo assembly, functional annotation and discovery of SSR markers

BackgroundTicks are ectoparasites capable of directly damaging their hosts and transmitting vector-borne diseases. The ixodid tick Haemaphysalis flava has a broad distribution that extends from East to South Asia. This tick is a reservoir of severe fever with thrombocytopenia syndrome virus (SFTSV) that causes severe hemorrhagic disease, with cases reported from China, Japan and South Korea. Recently, the distribution of H. flava in South Korea was found to overlap with the occurrence of SFTSV.MethodsThis study was undertaken to discover the molecular resources of H. flava female ticks using the Illumina HiSeq 4000 system, the Trinity de novo sequence assembler and annotation against public databases. The locally curated Protostome database (PANM-DB) was used to screen the putative adaptation-related transcripts classified to gene families, such as angiotensin-converting enzyme, aquaporin, adenylate cyclase, AMP-activated protein kinase, glutamate receptors, heat shock proteins, molecular chaperones, insulin receptor, mitogen-activated protein kinase and solute carrier family proteins. Also, the repeats and simple sequence repeats (SSRs) were screened from the unigenes using RepeatMasker (v4.0.6) and MISA (v1.0) software tools, followed by the designing of SSRs flanking primers using BatchPrimer 3 (v1.0) software.ResultsThe transcriptome produced a total of 69,822 unigenes, of which 46,175 annotated to the homologous proteins in the PANM-DB. The unigenes were also mapped to the EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) specializations. Promiscuous presence of protein kinase, zinc finger (C2H2-type), reverse transcriptase, and RNA recognition motif domains was observed in the unigenes. A total of 3480 SSRs were screened, of which 1907 and 1274 were found as tri- and dinucleotide repeats, respectively. A list of primer sequences flanking the SSR motifs was detailed for validation of polymorphism in H. flava and the related tick species.ConclusionsThe reference transcriptome information on H. flava female ticks will be useful for an enriched understanding of tick biology, its competency to act as a vector and the study of species diversity related to disease transmission.Graphical abstract

Functional annotation of proteins for signaling network inference in non-model species

Molecular biology aims to understand cellular responses and regulatory dynamics in complex biological systems. However, these studies remain challenging in non-model species due to poor functional annotation of regulatory proteins. To overcome this limitation, we develop a multi-layer neural network that determines protein functionality directly from the protein sequence. We annotate kinases and phosphatases in Glycine max. We use the functional annotations from our neural network, Bayesian inference principles, and high resolution phosphoproteomics to infer phosphorylation signaling cascades in soybean exposed to cold, and identify Glyma.10G173000 (TOI5) and Glyma.19G007300 (TOT3) as key temperature regulators. Importantly, the signaling cascade inference does not rely upon known kinase motifs or interaction data, enabling de novo identification of kinase-substrate interactions. Conclusively, our neural network shows generalization and scalability, as such we extend our predictions to Oryza sativa, Zea mays, Sorghum bicolor, and Triticum aestivum. Taken together, we develop a signaling inference approach for non-model species leveraging our predicted kinases and phosphatases.

Rational Design of Covalent Kinase Inhibitors by an Integrated Computational Workflow (Kin-Cov).

Covalent kinase inhibitors (CKIs) hold great promise for drug development. However, examples of computationally guided design of CKIs are still scarce. Here, we present an integrated computational workflow (Kin-Cov) for rational design of CKIs. The design of the first covalent leucine-zipper and sterile-α motif kinase (ZAK) inhibitor was presented as an example to showcase the power of computational workflow for CKI design. The two representative compounds, 7 and 8, inhibited ZAK kinase with half-maximal inhibitory concentration (IC50) values of 9.1 and 11.5 nM, respectively. Compound 8 displayed an excellent ZAK target specificity in Kinome profiling against 378 wild-type kinases. Structural biology and cell-based Western blot washout assays validated the irreversible binding characteristics of the compounds. Our study presents a rational approach for the design of CKIs based on the reactivity and accessibility of nucleophilic amino acid residues in a kinase. The workflow is generalizable and can be applied to facilitate CKI-based drug design.

Resistance screening and in silico characterization of cloned novel RGA from multi-race resistant lentil germplasm against Fusarium wilt (Fusarium oxysporum f. sp. lentis).

Fusarium wilt caused by Fusarium oxysporum f. sp. lentis (Fol) is the most devastating disease of lentil present worldwide. Identification of multi-race fusarium wilt resistance genes and their incorporation into existing cultivars will help to reduce yield losses. In the present study, 100 lentil germplasms belonging to seven lentil species were screened against seven prevalent races of Fol, and accessions IC201561 (Lens culinaris subsp. culinaris), EC714243 (L. c. subsp. odemensis), and EC718238 (L. nigricans) were identified as resistant. The typical R gene codes for the nucleotide-binding site and leucine-rich repeats (NBS-LRR) at the C terminal are linked to either the Toll/interleukin 1-like receptor (TIR) or coiled coil (CC) at the N terminal. In the present study, degenerate primers, designed from the NBS region amplifying the P-loop to the GLPLA motif, isolated forty-five resistance gene analogues (RGAs) from identified resistant accessions. The sequence alignment identified both classes of RGAs, TIR and non-TIR, based on the presence of aspartate (D) and tryptophan (W) at the end of the kinase motif, respectively. The phylogenetic analysis grouped the RGAs into six classes, from LRGA1 to LRGA6, which determined the diversity of the RGAs present in the host. Grouping of the RGAs identified from Lens nigricans, LnRGA 2, 9, 13 with I2 revealed the structural similarity with the fusarium resistance gene. The similarity index ranged from 27.85% to 86.98% among the RGAs and from 26.83% to 49.41% among the known R genes, I2, Gpa2, M, and L6. The active binding sites present along the conserved motifs grouped the RGAs into 13 groups. ADP/ATP, being the potential ligand, determines the ATP binding and ATP hydrolysis activity of the RGAs. The isolated RGAs can be used to develop markers linked to the functional R gene. Furthermore, expression analysis and full-length gene isolation pave the path to identifying the molecular mechanism involved in resistance.

UHMK1 promotes lung adenocarcinoma oncogenesis by regulating the PI3K/AKT/mTOR signaling pathway.

Effective targeted therapy for lung adenocarcinoma (LUAD), the number one cancer killer worldwide, continues to be a difficult problem because of the limitation of number of applicable patients and acquired resistance. Identifying more promising drug targets for LUAD treatment holds immense clinical significance. Recent studies have revealed that the U2 auxiliary factor (U2AF) homology motif kinase 1 (UHMK1) is a robust pro-oncogenic factor in many cancers. However, its biological functions and the underlying molecular mechanisms in LUAD have not been investigated. The UHMK1 expression in LUAD cells and tissues was evaluated by bioinformatics analysis, immunohistochemistry (IHC), western blotting (WB), and real time quantitative polymerase chain reaction (RT-qPCR) assays. A series of gain- and loss-of-function experiments for UHMK1 were carried out to investigate its biological functions in LUAD in vitro and in vivo. The mechanisms underlying UHMK1's effects in LUAD were analyzed by transcriptome sequencing and WB assays. UHMK1 expression was aberrantly elevated in LUAD tumors and cell lines and positively correlated with tumor size and unfavorable patient prognosis. Functionally, UHMK1 displayed robust pro-oncogenic capacity in LUAD and mechanistically exerted its biological effects via the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway. UHMK1 is a potent oncogene in LUAD. Targeting UHMK1 may significantly improve the effect of LUAD treatment via inhibiting multiple biological ways of LUAD progression.

MTORC2 is a physiological hydrophobic motif kinase of S6 Kinase 1

Ribosomal protein S6 kinase 1 (S6K1), a major downstream effector molecule of mTORC1, regulates cell growth and proliferation by modulating protein translation and ribosome biogenesis. We have recently identified eIF4E as an intermediate in transducing signals from mTORC1 to S6K1 and further demonstrated that the role of mTORC1 is restricted to inducing eIF4E phosphorylation and interaction with S6K1. This interaction relieves S6K1 auto-inhibition and facilitates its hydrophobic motif (HM) phosphorylation and activation as a consequence. These observations underscore a possible involvement of mTORC1 independent kinase in mediating HM phosphorylation. Here, we report mTORC2 as an in-vivo/physiological HM kinase of S6K1. We show that rapamycin-resistant S6K1 truncation mutant ∆NH∆CT continues to display HM phosphorylation with selective sensitivity toward Torin-1. We also show that HM phosphorylation of wildtype S6K1and ∆NH∆CT depends on the presence of mTORC2 regulatory subunit-rictor. Furthermore, truncation mutagenesis and molecular docking analysis reveal the involvement of a conserved 19 amino acid stretch of S6K1 in mediating interaction with rictor. We finally show that deletion of the 19 amino acid region from wildtype S6K1 results in loss of interaction with rictor, with a resultant loss of HM phosphorylation regardless of the presence of functional TOS motif. Our data demonstrate that mTORC2 acts as a physiological HM kinase that can activate S6K1 after its auto-inhibition is overcome by mTORC1. We, therefore, propose a novel mechanism for S6K1 regulation where mTOR complexes 1 and 2 act in tandem to activate the enzyme.

UHMK1 is a novel splicing regulatory kinase

The U2AF Homology Motif Kinase 1 (UHMK1) is the only kinase that contains the U2AF homology motif, a common protein interaction domain among splicing factors. Through this motif, UHMK1 interacts with the splicing factors SF1 and SF3B1, known to participate in the 3' splice site recognition during the early steps of spliceosome assembly. Although UHMK1 phosphorylates these splicing factors invitro, the involvement of UHMK1 in RNA processing has not previously been demonstrated. Here, we identify novel putative substrates of this kinase and evaluate UHMK1 contribution to overall gene expression and splicing, by integrating global phosphoproteomics, RNA-seq, and bioinformatics approaches. Upon UHMK1 modulation, 163 unique phosphosites were differentially phosphorylated in 117 proteins, of which 106 are novel potential substrates of this kinase. Gene Ontology analysis showed enrichment of terms previously associated with UHMK1 function, such as mRNA splicing, cell cycle, cell division, and microtubule organization. The majority of the annotated RNA-related proteins are components of the spliceosome but are also involved in several steps of gene expression. Comprehensive analysis of splicing showed that UHMK1 affected over 270 alternative splicing events. Moreover, splicing reporter assay further supported UHMK1 function on splicing. Overall, RNA-seq data demonstrated that UHMK1 knockdown had a minor impact on transcript expression and pointed to UHMK1 function in epithelial-mesenchymal transition. Functional assays demonstrated that UHMK1 modulation affects proliferation, colony formation, and migration. Taken together, our data implicate UHMK1 as a splicing regulatory kinase, connecting protein regulation through phosphorylation and gene expression in key cellular processes.

Genome-wide identification of calcineurin B-like protein-interacting protein kinase gene family reveals members participating in abiotic stress in the ornamental woody plant Lagerstroemia indica.

Calcineurin B-like protein-interacting protein kinases (CIPKs) play important roles in plant responses to stress. However, their function in the ornamental woody plant Lagerstroemia indica is remains unclear. In this study, the LiCIPK gene family was analyzed at the whole genome level. A total of 37 LiCIPKs, distributed across 17 chromosomes, were identified. Conserved motif analysis indicated that all LiCIPKs possess a protein kinase motif (S_TKc) and C-terminal regulatory motif (NAF), while seven LiCIPKs lack a protein phosphatase interaction (PPI) motif. 3D structure analysis further revealed that the N-terminal and C-terminal 3D-structure of 27 members are situated near to each other, while 4 members have a looser structure, and 6 members lack intact structures. The intra- and interspecies collinearity analysis, synonymous substitution rate (Ks) peaks of duplicated LiCIPKs, revealed that ∼80% of LiCIPKs were retained by the two whole genome duplication (WGD) events that occurred approximately 56.12–61.16 million year ago (MYA) and 16.24–26.34 MYA ago. The promoter of each LiCIPK contains a number of auxin, abscisic acid, gibberellic acid, salicylic acid, and drought, anaerobic, defense, stress, and wound responsive cis-elements. Of the 21 members that were successfully amplified by qPCR, 18 LiCIPKs exhibited different expression patterns under NaCl, mannitol, PEG8000, and ABA treatments. Given that LiCIPK30, the AtSOS2 ortholog, responded to all four types of stress it was selected for functional verification. LiCIPK30 complements the atsos2 phenotype in vivo. 35S:LiCIPK-overexpressing lines exhibit increased leaf area increment, chlorophyll a and b content, reactive oxygen species scavenging enzyme activity, and expression of ABF3 and RD22, while the degree of membrane lipid oxidation decreases under NaCl treatment compared to WT. The evolutionary history, and potential mechanism by which LiCIPK30 may regulate plant tolerance to salt stress were also discussed. In summary, we identified LiCIPK members involved in abiotic stress and found that LiCIPK30 transgenic Arabidopsis exhibits more salt and osmotic stress tolerance than WT. This research provides a theoretical foundation for further investigation into the function of LiCIPKs, and for mining gene resources to facilitate the cultivation and breeding of new L. indica varieties in coastal saline-alkali soil.

ZAKβ Alleviates Oxidized Low-density Lipoprotein (ox-LDL)-Induced Apoptosis and B-type Natriuretic Peptide (BNP) Upregulation in Cardiomyoblast.

Oxidized low-density lipoprotein (ox-LDL) is a type of modified cholesterol that promotes apoptosis and inflammation and advances the progression of heart failure. Leucine-zipper and sterile-α motif kinase (ZAK) is a kinase of the MAP3K family which is highly expressed in the heart and encodes two variants, ZAKα and ZAKβ. Our previous study serendipitously found opposite effects of ZAKα and ZAKβ in which ZAKβ antagonizes ZAKα-induced apoptosis and hypertrophy of the heart. This study aims to test the hypothesis of whether ZAKα and ZAKβ are involved in the damaging effects of ox-LDL in the cardiomyoblast. Cardiomyoblast cells H9c2 were treated with different concentrations of ox-LDL. Cell viability and apoptosis were measured by MTT and TUNEL assay, respectively. Western blot was used to detect apoptosis, hypertrophy, and pro-survival signaling proteins. Plasmid transfection, pharmacological inhibition with D2825, and siRNA transfection were utilized to upregulate or downregulate ZAKβ, respectively. Ox-LDL concentration-dependently reduces the viability and expression of several pro-survival proteins, such as phospho-PI3K, phospho-Akt, and Bcl-xL. Furthermore, ox-LDL increases cleaved caspase-3, cleaved caspase-9 as indicators of apoptosis and increases B-type natriuretic peptide (BNP) as an indicator of hypertrophy. Overexpression of ZAKβ by plasmid transfection attenuates apoptosis and prevents upregulation of BNP. Importantly, these effects were abolished by inhibiting ZAKβ either by D2825 or siZAKβ application. Our results suggest that ZAKβ upregulation in response to ox-LDL treatment confers protective effects on cardiomyoblast.

Reversible RNA phosphorylation stabilizes tRNA for cellular thermotolerance

Post-transcriptional modifications have critical roles in tRNA stability and function1–4. In thermophiles, tRNAs are heavily modified to maintain their thermal stability under extreme growth temperatures5,6. Here we identified 2′-phosphouridine (Up) at position 47 of tRNAs from thermophilic archaea. Up47 confers thermal stability and nuclease resistance to tRNAs. Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by Up47. The 2′-phosphate of Up47 protrudes from the tRNA core and prevents backbone rotation during thermal denaturation. In addition, we identified the arkI gene, which encodes an archaeal RNA kinase responsible for Up47 formation. Structural studies showed that ArkI has a non-canonical kinase motif surrounded by a positively charged patch for tRNA binding. A knockout strain of arkI grew slowly at high temperatures and exhibited a synthetic growth defect when a second tRNA-modifying enzyme was depleted. We also identified an archaeal homologue of KptA as an eraser that efficiently dephosphorylates Up47 in vitro and in vivo. Taken together, our findings show that Up47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions.

Adaptive translational reprogramming of metabolism limits the response to targeted therapy in BRAFV600 melanoma

Despite the success of therapies targeting oncogenes in cancer, clinical outcomes are limited by residual disease that ultimately results in relapse. This residual disease is often characterized by non-genetic adaptive resistance, that in melanoma is characterised by altered metabolism. Here, we examine how targeted therapy reprograms metabolism in BRAF-mutant melanoma cells using a genome-wide RNA interference (RNAi) screen and global gene expression profiling. Using this systematic approach we demonstrate post-transcriptional regulation of metabolism following BRAF inhibition, involving selective mRNA transport and translation. As proof of concept we demonstrate the RNA processing kinase U2AF homology motif kinase 1 (UHMK1) associates with mRNAs encoding metabolism proteins and selectively controls their transport and translation during adaptation to BRAF-targeted therapy. UHMK1 inactivation induces cell death by disrupting therapy induced metabolic reprogramming, and importantly, delays resistance to BRAF and MEK combination therapy in multiple in vivo models. We propose selective mRNA processing and translation by UHMK1 constitutes a mechanism of non-genetic resistance to targeted therapy in melanoma by controlling metabolic plasticity induced by therapy.

UHMK1-dependent phosphorylation of Cajal body protein coilin alters 5-FU sensitivity in colon cancer cells

Resistance to 5-fluorouracil (5-FU) in chemotherapy and recurrence of colorectal tumors is a serious concern that impedes improvements to clinical outcomes. In the present study, we found that conditioned medium (CM) derived from 5-FU-resistant HCT-8/FU cells reduced 5-FU chemosensitivity in HCT-8 colon cancer cells, with corresponding changes to number and morphology of Cajal bodies (CBs) as observable nuclear structures. We found that U2AF homology motif kinase 1 (UHMK1) altered CB disassembly and reassembly and regulated the phosphorylation of coilin, a major component of CBs. This subsequently resulted in a large number of variations in RNA alternative splicing that affected cell survival following 5-FU treatment, induced changes in intracellular phenotype, and transmitted preadaptive signals to adjacent cells in the tumor microenvironment (TME). Our findings suggest that CBs may be useful for indicating drug sensitivity or resistance in tumor cells in response to stress signals. The results also suggest that UHMK1 may be an important factor for maintaining CB structure and morphology by regulating splicing events, especially following cellular exposure to cytotoxic drugs.Graphical EzAUFb-Sx6xbWL9vLuNL_4Video

An atypical class of non-coding small RNAs is produced in rice leaves upon bacterial infection

Non-coding small RNAs (sRNA) act as mediators of gene silencing and regulate plant growth, development and stress responses. Early insights into plant sRNAs established a role in antiviral defense and they are now extensively studied across plant–microbe interactions. Here, sRNA sequencing discovered a class of sRNA in rice (Oryza sativa) specifically associated with foliar diseases caused by Xanthomonas oryzae bacteria. Xanthomonas-induced small RNAs (xisRNAs) loci were distinctively upregulated in response to diverse virulent strains at an early stage of infection producing a single duplex of 20–22 nt sRNAs. xisRNAs production was dependent on the Type III secretion system, a major bacterial virulence factor for host colonization. xisRNA loci overlap with annotated transcripts sequences, with about half of them encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and xisRNA loci predominantly coincide with the coding sequence of a conserved kinase motif. xisRNAs exhibit features of small interfering RNAs and their biosynthesis depend on canonical components OsDCL1 and OsHEN1. xisRNA induction possibly mediates post-transcriptional gene silencing but they do not broadly suppress cis-genes expression on the basis of mRNA-seq data. Overall, our results identify a group of unusual sRNAs with a potential role in plant–microbe interactions.

Bladder cancer-associated transcript 1 promotes melanoma cell proliferation and invasion via the miR-374b-5p/U2-associated factor homology motif kinase 1 axis.

Melanoma is a malignancy derived from melanocytes and is associated with high mortality rates worldwide. Long noncoding RNAs (lncRNAs) have been confirmed to be pivotal regulators in multiple types of cancer. Many lncRNAs are aberrantly expressed in tumors and perform vital functions in cancer progression. Nevertheless, the biological role of lncRNA bladder cancer-associated transcript 1 (BLACAT1) in melanoma progression remains unexplored. In this study, the collected data showed that BLACAT1 was highly expressed in melanoma. Mechanistically, miR-374b-5p bound to BLACAT1, and U2-associated factor homology motif kinase 1 (UHMK1) was a downstream target of miR-374b-5p. BLACAT1 upregulated UHMK1 expression by acting as a competing endogenous RNA for miR-374-5b. BLACAT1 deficiency resulted in the upregulation of miR-374b-5p expression and the downregulation of UHMK1 expression in melanoma cells. Moreover, BLACAT1 activated PI3K and AKT signaling by upregulating UHMK1 expression, as shown by western blotting analyses. Functionally, UHMK1 overexpression or miR-374b-5p knockdown reversed the suppressive effect of BLACAT1 depletion on melanoma cell proliferation and invasion. In conclusion, BLACAT1 promotes melanoma cell proliferation and invasion by upregulating UHMK1 expression via miR-374b-5p to activate the PI3K/AKT pathway. These results might provide promising insight into the investigation of prognostic biomarkers of melanoma.

Hsa-miR-374b-5p regulates expression of the gene U2AF homology motif (UHM) kinase 1.

We aimed to identify a microRNA (miRNA) that is significantly upregulated in blood and in cells of the oral mucosa upon exposure to the periodontitis main risk factors oral inflammation and tobacco smoke, to subsequently identify its target gene and to describe the molecular mechanism of gene regulation. miRNAs are associated with many disorders. Array-based miRNA expression studies indicated a number of differentially expressed miRNAs in the pathology of oral diseases. However, these miRNAs mostly lacked replication, and their target genes have remained unknown. 863 miRNAs were analyzed in blood from 18 PD cases and 70 controls (Geniom Biochip). Selected miRNAs were analyzed for upregulation in the inflamed oral mucosa of PD patients using published miRNA expression profiling studies from gingival cells. hsa-miR-374b-5p mimic was overexpressed in primary gingival fibroblasts (pGFs) from 3 donors, and genome-wide mRNA expression was quantified (Clarion Array). Gene-specific regulation was validated by qRT-PCR and Luciferase activity in HeLa cells. hsa-miR-374b-5p showed >twofold change (FC) in 3 independent studies performed in blood, gingival tissues, and cells. After hsa-miR-374b-5p overexpression, genome-wide expression analysis showed UHMK1 as top 1 downregulated gene in pGFs (p=2.5×10-04 , fold change=-1.8). Reporter genes demonstrated that hsa-miR-374b-5p downregulates mRNA levels (p=.02; FC=-1.5), leading to reduction in protein activity (p=.013, FC=-1.3). hsa-miR-374b-5p is upregulated in blood and ginvial cells exposed to oral inflammation and tobacco smoke and regulates UHMK1, which has a role in osteoclast differentiation.