Further Disruption of the TAS3 Pathway via the Addition of the AGO7 Mutation to the DRB1, DRB2 or DRB4 Mutations Severely Impairs the Reproductive Competence of Arabidopsis thaliana

The previous assignment of functional roles for AGO7, and the DOUBLE-STRANDED RNA BINDING (DRB) proteins, DRB1, DRB2, and DRB4, in either microRNA (miRNA) or trans-acting small-interfering RNA (tasiRNA) production allowed for use of the loss-of-function mutant lines, drb1, drb2, drb4, and ago7, to further functionally characterize the TAS3 pathway in Arabidopsis thaliana (Arabidopsis). Towards achieving this goal, we also describe the developmental and molecular phenotypes expressed by three newly generated Arabidopsis lines, the drb1ago7, drb2ago7, and drb4ago7 double mutants. We show that the previously reported developmental abnormalities displayed by the drb1, drb2, drb4, and ago7 single mutants, are further exacerbated in the drb1ago7, drb2ago7, and drb4ago7 double mutants, with rosette area, silique length, and seed set all impaired to a greater degree in the double mutants. Molecular assessment of the TAS3 pathway in the floral tissues of the seven analyzed mutants revealed that DRB1 is the sole DRB required for miR390 sRNA production. However, DRB2 and DRB4 appear to play secondary roles at this stage of the TAS3 pathway to ensure that miR390 sRNA levels are tightly maintained. We further show that the expression of the TAS3-derived tasiARF target genes, AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4, was altered in drb1ago7, drb2ago7, and drb4ago7 flowers. Altered ARF2, ARF3, and ARF4 expression was in turn demonstrated to lead to changes in the level of expression of KAN1, KAN3, and KAN4, three KANADI transcription factor genes known to be transcriptionally regulated by ARF2, ARF3, and ARF4. Taken together, the demonstrated relationship between altered ARF and KAN gene expression in drb1ago7, drb2ago7 and drb4ago7 flowers, could, in part, explain the more severe developmental defects displayed by the double mutants, compared to milder impact that loss of only a single piece of TAS3 pathway protein machinery was demonstrated to have on drb1, drb2, drb4 and ago7 reproductive development.

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Dr. Jekyll and Mr. Hyde: The Two Faces of the FUS/EWS/TAF15 Protein Family

Sarcoma ◽

10.1155/2011/837474 ◽

2011 ◽

Vol 2011 ◽

pp. 1-13 ◽

Cited By ~ 64

Author(s):

Heinrich Kovar

Keyword(s):

Protein Function ◽

Target Genes ◽

Rna Binding ◽

Rna Binding Proteins ◽

Fusion Gene ◽

Tumor Biology ◽

Central Function ◽

Epithelial Cancers ◽

Transcription Factor Genes

FUS, EWS, and TAF15 form the FET family of RNA-binding proteins whose genes are found rearranged with various transcription factor genes predominantly in sarcomas and in rare hematopoietic and epithelial cancers. The resulting fusion gene products have attracted considerable interest as diagnostic and promising therapeutic targets. So far, oncogenic FET fusion proteins have been regarded as strong transcription factors that aberrantly activate or repress target genes of their DNA-binding fusion partners. However, the role of the transactivating domain in the context of the normal FET proteins is poorly defined, and, therefore, our knowledge on how FET aberrations impact on tumor biology is incomplete. Since we believe that a full understanding of aberrant FET protein function can only arise from looking at both sides of the coin, the good and the evil, this paper summarizes evidence for the central function of FET proteins in bridging RNA transcription, processing, transport, and DNA repair.

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Zebrafish mbnl mutants model physical and molecular phenotypes of myotonic dystrophy

10.1101/665380 ◽

2019 ◽

Author(s):

Melissa N. Hinman ◽

Jared I. Richardson ◽

Rose A. Sockol ◽

Eliza D Aronson ◽

Sarah J. Stednitz ◽

...

Keyword(s):

Alternative Splicing ◽

Myotonic Dystrophy ◽

Protein Function ◽

Rna Binding ◽

Rna Binding Proteins ◽

Genetic Disorder ◽

Loss Of Function ◽

High Fecundity ◽

Molecular Phenotypes ◽

Human Genetic Disorder

AbstractThe muscleblind RNA binding proteins (MBNL1, MBNL2, and MBNL3) are highly conserved across vertebrates and are important regulators of RNA alternative splicing. Loss of MBNL protein function through sequestration by CUG or CCUG RNA repeats is largely responsible for the phenotypes of the human genetic disorder myotonic dystrophy (DM). We generated the first stable zebrafish (Danio rerio) models of DM-associated MBNL loss of function through mutation of the three zebrafish mbnl genes. In contrast to mouse models, zebrafish double and triple homozygous mbnl mutants were viable to adulthood. Zebrafish mbnl mutants displayed disease-relevant physical phenotypes including decreased body size and impaired movement. They also exhibited widespread alternative splicing changes, including the misregulation of many DM-relevant exons. Physical and molecular phenotypes were more severe in compound mbnl mutants than in single mbnl mutants, suggesting partially redundant functions of Mbnl proteins. The high fecundity and larval optical transparency of this complete series of zebrafish mbnl mutants will make them useful for studying DM-related phenotypes and how individual Mbnl proteins contribute to them, and for testing potential therapeutics.

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MIR822 modulates monosporic female gametogenesis through an ARGONAUTE9-dependent pathway in Arabidopsis thaliana

10.1101/2021.10.18.464879 ◽

2021 ◽

Author(s):

ANDREA TOVAR AGUILAR ◽

Daniel GRIMANELLI ◽

Gerardo Acosta Garcia ◽

Jean Philippe Vielle Calzada ◽

Jesus Agustin Badillo-Corona ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Molecular Mechanisms ◽

Target Genes ◽

Flowering Plants ◽

Loss Of Function ◽

Genes Encoding ◽

Functional Megaspore ◽

Dependent Pathway ◽

Female Gametogenesis

In the ovule of flowering plants, the establishment of the haploid generation occurs when a somatic cell differentiates into a Megaspore Mother Cell (MMC) and initiates meiosis. As most flowering plants, Arabidopsis thaliana undergoes a monosporic type of gametogenesis; three meiotically derived cells degenerate without further division, and a single one, the functional megaspore (FM), divides mitotically to form the female gametophyte. In Arabidopsis, the ARGONAUTE4 clade proteins are involved in the control of megasporogenesis. In particular, mutations in ARGONAUTE9 (AGO9) lead to the ectopic differentiation of gametic precursors that can give rise female gametophytes. However, the genetic basis and molecular mechanisms that control monosporic gametogenesis remain largely unknown. Here, we show that Arabidopsis plants carrying loss-of-function mutations in the AGO9-interacting miR822a give rise to extranumerary surviving megaspores that acquire a FM identity and divide without giving rise to differentiated female gametophytes. The overexpression of three miR822a target genes encoding Cysteine/Histidine-Rich C1 domain proteins (DC1) phenocopy mir822a plants. The miR822a targets are overexpressed in ago9 mutant ovules, confirming that miR822a acts through an AGO9-dependent pathway to negatively regulate DC1 domain proteins. Our results identify a new role of miRNAs in the most prevalent form of female gametogenesis in flowering plants

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A Re-examination of the Role of AUXIN RESPONSE FACTOR 8 in Developmental Abnormalities Caused by the P1/HC-Pro Viral Suppressor of RNA Silencing

10.1101/056366 ◽

2016 ◽

Author(s):

Sizolwenkosi Mlotshwa ◽

Gail J. Pruss ◽

John L. Macarthur ◽

Jason W. Reed ◽

Vicki Vance

Keyword(s):

Rna Silencing ◽

Auxin Response Factor ◽

35S Promoter ◽

Insertion Mutant ◽

Response Factor ◽

Loss Of Function ◽

Auxin Response ◽

Developmental Defects ◽

Developmental Abnormalities

AbstractPlant viral suppressors of RNA silencing induce developmental defects similar to those caused by mutations in genes involved in the microRNA (miRNA) pathway. These abnormalities were originally thought to reflect a pleiotropic impact of silencing suppressors on miRNA control of plant development. However, subsequent work with the P1/HC-Pro potyviral suppressor of silencing showed that global impairment of the miRNA pathway was not responsible for the phenotypical anomalies. More recently, developmental defects caused by a P1/HC-Pro transgene under control of the 35S promoter were attributed to moderate upregulation of AUXIN RESPONSE FACTOR 8 (ARF8), a target of miR167. The key piece of evidence in that work was that the developmental defects in the 35S-pro:P1/HC-Pro transgenic Arabidopsis were greatly alleviated in the F1 progeny of a cross with plants carrying the arf8-6 mutation. Arf8-6 is a SALK line T-DNA insertion mutant, a class of mutations prone to inducing transcriptional silencing of transgenes expressed from the 35S promoter. Here we report a re-investigation of the role of ARF8 in P1/HC-Pro-mediated developmental defects. We characterized the progeny of a cross between our 35S-pro:P1/HC-Pro transgenic Arabidopsis line and the same arf8-6 T-DNA insertion mutant used in the earlier study. The T-DNA mutation had little effect in the F1 generation, but almost all arf8-6/P1/HC-Pro progeny had lost the P1/HC-Pro phenotype in the F2 generation. However, this loss of phenotype was not correlated with the number of functional copies of the ARF8 gene. Instead, it reflected transcriptional silencing of the 35S-pro:P1/HC-Pro transgene, as evidenced by a pronounced decrease in P1/HC-Pro mRNA2accompanied by the appearance of 35S promoter siRNAs. Furthermore, arf8-8, an independent loss-of-function point mutation, had no detectable effects on P1/HC-Pro phenotype in either the F1 or F2 generations. Together these data argue against the reported role of increased ARF8 expression in mediating developmental defects in P1/HC-Pro transgenic plants.Author SummaryRNA silencing is an important antiviral defense in plants that uses small RNA molecules to target the invading RNA. Plant viruses, however, have countered with proteins that suppress RNA silencing, and one of the best-studied plant viral suppressors of silencing is P1/HC-Pro. When the genetic model plant Arabidopsis thaliana is bioengineered to express P1/HC-Pro, the resulting plants display distinct developmental abnormalities. These abnormalities are thought to arise because P1/HC-Pro also interferes with the arm of RNA silencing that uses small RNAs called microRNAs (miRNAs) to regulate expression of the plant's own genes. Earlier work, however, showed that interference with all miRNAs in general could not be responsible for these developmental defects. More recently, it was reported that enhanced expression of a single miRNA-controlled gene, AUXIN RESPONSE FACTOR 8 (ARF8), underlies the developmental defects caused by P1/HC-Pro. However, using the same ARF8 mutation as that report, as well as a second, independent ARF8 loss-of function mutation, we now show that mis-regulation of ARF8 is not responsible for those defects. One or a few key miRNA-controlled factors might, in fact, underlie the developmental defects caused by P1/HC-Pro; however, our results show that ARF8 is not one of the key factors.

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Whole-genome association studies of distribution of developmental abnormalities and other breeding-valuable qualitative traits in offspring of the Russian large-white boars

Vavilov Journal of Genetics and Breeding ◽

10.18699/vj20.612 ◽

2020 ◽

Vol 24 (2) ◽

pp. 185-190

Author(s):

A. A. Traspov ◽

O. V. Kostyunina ◽

A. A. Belous ◽

T. V. Karpushkina ◽

N. A. Svejenceva ◽

...

Keyword(s):

Target Genes ◽

Association Studies ◽

Genetic Diseases ◽

Small Sample ◽

The Body ◽

Genome Wide Association Studies ◽

Large White ◽

Developmental Defects ◽

Developmental Abnormalities ◽

Polymorphic Snps

Identifying genome regions that are directly or indirectly associated with developmental defects and malformations in domesticated pigs can help identify genomic traits used as biomarkers of the structural and functional composition of the body, their metabolic status and genetic diseases as well. Such studies are directly related to the improvement of the economic efficiency, as they allow identification and exclusion of defect animals, who may carry target genes not appearing phenotypically, from the breeding process. In the current work, we have searched for these kind of target genes and genome regions with conducting the genome-wide association studies using PorcineSNP60K BeadChips (Illumina, San Diego, USA). A total of 48 boars of a large white breed of the nucleus farm “Znamenskoe” were analyzed for 21 traits of indicated shortcomings of the exterior and defects of development in 39,153 their offspring. Calculations were made using a mixed type linear model in package GEMMA. In this study, we selected only 36,704 polymorphic SNPs from an initial 61,000-strong SNP set. After GWAS, we obtained 24 alleles in 11 corresponding genes (P < 0.1) in the genome of pigs, which are significantly correlated with traits of developmental abnormalities such as anal atresia (ARMC7,FANCC,RND3,ENSSSCG00000017216), limb problems (PAWR,NTM,OPCML,ENSSSCG00000040250, ENSSSCG00000017018) and tremor of piglets (RIC3,ENSSSCG00000032665). Also, co-expression of the NTM,OPCMLand RND3genes was revealed. This study confirms the relevance of using the single SNP detection according to the single trait approach in associative studies, even for small sample numbers.

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Pleiotropic requirements for human TDP-43 in the regulation of cell and organelle homeostasis

10.1101/560144 ◽

2019 ◽

Author(s):

Agnes Roczniak-Ferguson ◽

Shawn M. Ferguson

Keyword(s):

Target Genes ◽

Rna Binding ◽

Complete Loss ◽

Mrna Splicing ◽

Nuclear Pore ◽

Loss Of Function ◽

Nuclear Pore Protein ◽

Morphological Defects ◽

Lateral Sclerosis ◽

Pore Protein

AbstractTDP-43 is an RNA-binding protein that forms cytoplasmic aggregates in multiple neurodegenerative diseases. Although the loss of normal TDP-43 functions likely contributes to disease pathogenesis, the cell biological consequences of human TDP-43 depletion are not well understood. We therefore generated human TDP-43 knockout cells and subjected them to parallel cell biological and transcriptomic analyses. These efforts yielded three important discoveries. First, complete loss of TDP-43 resulted in widespread morphological defects related to multiple organelles including: Golgi, endosomes, lysosomes, mitochondria and the nuclear envelope. Second, we identified a new role for TDP-43 in controlling mRNA splicing of Nup188 (nuclear pore protein). Third, analysis of multiple amyotrophic lateral sclerosis (ALS) causing TDP-43 mutations revealed a broad ability to support splicing of TDP-43 target genes. However, as some TDP-43 disease causing mutants failed to support the regulation of specific target transcripts, our results raise the possibility of mutation-specific loss-of-function contributions to disease pathology.

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Pleiotropic requirements for human TDP-43 in the regulation of cell and organelle homeostasis

Life Science Alliance ◽

10.26508/lsa.201900358 ◽

2019 ◽

Vol 2 (5) ◽

pp. e201900358 ◽

Cited By ~ 5

Author(s):

Agnes Roczniak-Ferguson ◽

Shawn M Ferguson

Keyword(s):

Target Genes ◽

Rna Binding ◽

Complete Loss ◽

Mrna Splicing ◽

Nuclear Pore ◽

Loss Of Function ◽

Nuclear Pore Protein ◽

Morphological Defects ◽

Lateral Sclerosis ◽

Pore Protein

TDP-43 is an RNA-binding protein that forms cytoplasmic aggregates in multiple neurodegenerative diseases. Although the loss of normal TDP-43 functions likely contributes to disease pathogenesis, the cell biological consequences of human TDP-43 depletion are not well understood. We, therefore, generated human TDP-43knockout (KO) cells and subjected them to parallel cell biological and transcriptomic analyses. These efforts yielded three important discoveries. First, complete loss of TDP-43 resulted in widespread morphological defects related to multiple organelles, including Golgi, endosomes, lysosomes, mitochondria, and the nuclear envelope. Second, we identified a new role for TDP-43 in controlling mRNA splicing of Nup188 (nuclear pore protein). Third, analysis of multiple amyotrophic lateral sclerosis causing TDP-43 mutations revealed a broad ability to support splicing of TDP-43 target genes. However, as some TDP-43 disease-causing mutants failed to fully support the regulation of specific target transcripts, our results raise the possibility of mutation-specific loss-of-function contributions to disease pathology.

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Zebrafish mbnl mutants model physical and molecular phenotypes of myotonic dystrophy

Disease Models & Mechanisms ◽

10.1242/dmm.045773 ◽

2021 ◽

Author(s):

Melissa N. Hinman ◽

Jared I. Richardson ◽

Rose A. Sockol ◽

Eliza D Aronson ◽

Sarah J. Stednitz ◽

...

Keyword(s):

Alternative Splicing ◽

Myotonic Dystrophy ◽

Protein Function ◽

Rna Binding ◽

Rna Binding Proteins ◽

Genetic Disorder ◽

Loss Of Function ◽

High Fecundity ◽

Molecular Phenotypes ◽

Human Genetic Disorder

The muscleblind RNA binding proteins (MBNL1, MBNL2, and MBNL3) are highly conserved across vertebrates and are important regulators of RNA alternative splicing. Loss of MBNL protein function through sequestration by CUG or CCUG RNA repeats is largely responsible for the phenotypes of the human genetic disorder myotonic dystrophy (DM). We generated the first stable zebrafish (Danio rerio) models of DM-associated MBNL loss of function through mutation of the three zebrafish mbnl genes. In contrast to mouse models, zebrafish double and triple homozygous mbnl mutants were viable to adulthood. Zebrafish mbnl mutants displayed disease-relevant physical phenotypes including decreased body size and impaired movement. They also exhibited widespread alternative splicing changes, including the misregulation of many DM-relevant exons. Physical and molecular phenotypes were more severe in compound mbnl mutants than in single mbnl mutants, suggesting partially redundant functions of Mbnl proteins. The high fecundity and larval optical transparency of this complete series of zebrafish mbnl mutants will make them useful for studying DM-related phenotypes and how individual Mbnl proteins contribute to them, and for testing potential therapeutics.

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A Comprehensive Genomic Approach Using Gain of Function and Loss of Function Cell Models and ChIP-on-Chip Technology Identifies Novel Promyelocytic Zinc Finger Protein Target Genes.

Blood ◽

10.1182/blood.v108.11.1407.1407 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1407-1407

Author(s):

Kim L. Rice ◽

Ari Melnick ◽

Kenny Ye ◽

Windy Berkofsky-Fessler ◽

Jonathan D. Licht

Keyword(s):

Target Genes ◽

Rna Binding ◽

Zinc Finger Protein ◽

Leukemia Cells ◽

Loss Of Function ◽

Function Model ◽

Gain Of Function ◽

Hematopoietic Stem ◽

Endogenous Expression ◽

On Chip

Abstract The t(11;17)(q23;q21) form of APL involves the production of reciprocal fusion proteins, PLZF-RARα and RARα-PLZF, which mediate malignant transformation by binding to and dysregulating RARα and PLZF target genes. PLZF is expressed in hematopoietic stem cells and is downregulated as cells differentiate. The identification of PLZF target genes including cyclin A2 and MYC is consistent with the hypothesis that PLZF maintains stem cell quiescence by repressing cell cycle driving genes and provides insight into transcriptional pathways disrupted in leukemogenesis. In order to identify additional target genes of PLZF, we constructed a loss of function model in which we suppressed endogenous expression of PLZF using siRNA in KG1a leukemia cells. Our gain of function model consisted of the ectopic expression of PLZF in U937 leukemia cells which do not naturally express PLZF. Expression profiling using GeneChip™ Human Genome U133 Plus 2.0 arrays, which analyze the expression of more than 47,000 transcripts, was performed using both systems. Of the 346 genes identified in the loss of function model, 25% were also regulated by PLZF in the gain of function U937 cell line. Changes in expression of these genes could be direct (through PLZF) or indirect (through secondary effects). In order to determine which genes modulated by changes in PLZF expression are direct transcriptional targets, we immunoprecipitated chromatin using PLZF antibodies in KG1a cells, amplified the products by ligation-mediated PCR and co-hybridized these products with input chromatin to NimbleGen 1.5kB promoter arrays, which represent 24,275 human promoters. Genes bound by PLZF were identified by determining whether consecutively tiled probes were enriched in PLZF-precipitated chromatin as compared to chromatin precipitated with a non-specific antibody. Using a statistical algorithm designed to exclude those probes whose signals of PLZF enrichment might be spuriously identified, we identified 52 genes of the 24,275 on the array as potential PLZF target genes. Strikingly, correlation of these genes with expression analyses revealed that 44% of genes were also significantly regulated by PLZF in the gain of function model and 11% of genes were regulated in the loss of function model. Promoter analyses of a subset of these genes that were identified by ChIP-on-Chip and differentially expressed at least >1.3 fold in PLZF arrays (p<0.05), revealed the presence of a consensus PLZF binding site GTC(C/A)AG in 75% of genes. Analysis of gene ontology for those genes identified by ChIP-on Chip, revealed an enrichment of genes involved in RNA binding and processing as well as genes encoding small G proteins. One gene in particular, RECQL, was directly bound by PLZF in the ChIP-on-Chip assay and transcriptionally regulated by PLZF in both KG1a loss of function and U937 gain of function models. The RECQL protein is a member of the RecQ family of DNA helicases, a class of genes whose mutation is associated with genomic instability tumorigenesis and premature ageing. These data indicate a robust system for the identification of PLZF targets and suggest that PLZF may play a role in genome integrity.

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Loss of function mutations in GEMIN5 cause a neurodevelopmental disorder

Nature Communications ◽

10.1038/s41467-021-22627-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sukhleen Kour ◽

Deepa S. Rajan ◽

Tyler R. Fortuna ◽

Eric N. Anderson ◽

Caroline Ward ◽

...

Keyword(s):

Rna Binding ◽

Neurodevelopmental Disorder ◽

Gene Mutations ◽

Building Blocks ◽

Survival Motor Neuron ◽

Motor Dysfunction ◽

Loss Of Function ◽

Rigor Mortis ◽

Developmental Defects ◽

Pathogenic Variants

AbstractGEMIN5, an RNA-binding protein is essential for assembly of the survival motor neuron (SMN) protein complex and facilitates the formation of small nuclear ribonucleoproteins (snRNPs), the building blocks of spliceosomes. Here, we have identified 30 affected individuals from 22 unrelated families presenting with developmental delay, hypotonia, and cerebellar ataxia harboring biallelic variants in the GEMIN5 gene. Mutations in GEMIN5 perturb the subcellular distribution, stability, and expression of GEMIN5 protein and its interacting partners in patient iPSC-derived neurons, suggesting a potential loss-of-function mechanism. GEMIN5 mutations result in disruption of snRNP complex assembly formation in patient iPSC neurons. Furthermore, knock down of rigor mortis, the fly homolog of human GEMIN5, leads to developmental defects, motor dysfunction, and a reduced lifespan. Interestingly, we observed that GEMIN5 variants disrupt a distinct set of transcripts and pathways as compared to SMA patient neurons, suggesting different molecular pathomechanisms. These findings collectively provide evidence that pathogenic variants in GEMIN5 perturb physiological functions and result in a neurodevelopmental delay and ataxia syndrome.

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