scholarly journals Distinct expression, localization and function of two Rab7 proteins encoded by paralogous genes in a free-living model eukaryote.

2011 ◽  
Vol 58 (4) ◽  
Author(s):  
Magdalena Osińska ◽  
Jolanta Wiejak ◽  
Emilia Wypych ◽  
Henryk Bilski ◽  
Rafał Bartosiewicz ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Pcr Analysis ◽  
Transcriptional Gene Silencing ◽  
Post Translational Modification ◽  
Paralogous Genes ◽  
Post Transcriptional Gene Silencing ◽  
2D Gel ◽  
And Function ◽  
Microtubular Structures ◽  
Confocal And Electron Microscopy

Rab7 GTPases are involved in membrane trafficking in the late endosomal/lysosomal pathway. In Paramecium octaurelia Rab7a and Rab7b are encoded by paralogous genes. Antipeptide antibodies generated against divergent C-termini recognize Rab7a of 22.5 kDa and Rab7b of 25 kDa, respectively. In 2D gel electrophoresis two immunoreactive spots were identified for Rab7b at pI about 6.34 and about 6.18 and only one spot for Rab7a of pI about 6.34 suggesting post-translational modification of Rab7b. Mass spectrometry revealed eight identical phosphorylated residues in the both proteins. ProQ Emerald staining and ConA overlay of immunoprecipitated Rab7b indicated its putative glycosylation that was further supported by a faster electrophoretic mobility of this protein upon deglycosylation. Such a post-translational modification and substitution of Ala(140) in Rab7a for Ser(140) in Rab7b may result in distinct targeting to the oral apparatus where Rab7b associates with the microtubular structures as revealed by STED confocal and electron microscopy. Rab7a was mapped to phagosomal compartment. Absolute qReal-Time PCR analysis revealed that expression of Rab7a was 2.6-fold higher than that of Rab7b. Upon latex internalization it was further 2-fold increased for Rab7a and only slightly for Rab7b. Post-transcriptional gene silencing of rab7a suppressed phagosome formation by 70 % and impaired their acidification. Ultrastructural analysis with double immunogold labeling revealed that this effect was due to the lack of V-ATPase recruitment to phagolysosomes. No significant phenotype changes were noticed in cells upon rab7b silencing. In conclusion, Rab7b acquired a new function, whereas Rab7a can be assigned to the phagolysosomal pathway.

scholarly journals FIERY1 promotes microRNA accumulation by suppressing rRNA-derived small interfering RNAs in Arabidopsis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Chenjiang You ◽  
Wenrong He ◽  
Runlai Hang ◽  
Cuiju Zhang ◽  
Xiaofeng Cao ◽  
...  
Keyword(s):  
Rna Degradation ◽  
Transcriptional Gene Silencing ◽  
Rrna Processing ◽  
Small Interfering Rnas ◽  
Post Transcriptional Gene Silencing ◽  
Processing Defects ◽  
And Function ◽  
Mirna Abundance ◽  
Unknown Link

Abstract Plant microRNAs (miRNAs) associate with ARGONAUTE1 (AGO1) to direct post-transcriptional gene silencing and regulate numerous biological processes. Although AGO1 predominantly binds miRNAs in vivo, it also associates with endogenous small interfering RNAs (siRNAs). It is unclear whether the miRNA/siRNA balance affects miRNA activities. Here we report that FIERY1 (FRY1), which is involved in 5′−3′ RNA degradation, regulates miRNA abundance and function by suppressing the biogenesis of ribosomal RNA-derived siRNAs (risiRNAs). In mutants of FRY1 and the nuclear 5′−3′ exonuclease genes XRN2 and XRN3, we find that a large number of 21-nt risiRNAs are generated through an endogenous siRNA biogenesis pathway. The production of risiRNAs correlates with pre-rRNA processing defects in these mutants. We also show that these risiRNAs are loaded into AGO1, causing reduced loading of miRNAs. This study reveals a previously unknown link between rRNA processing and miRNA accumulation.

scholarly journals MicroRNAs in the skin: role in development, homoeostasis and regeneration

2017 ◽  
Vol 131 (15) ◽  
pp. 1923-1940 ◽  
Author(s):  
Steven Horsburgh ◽  
Nicola Fullard ◽  
Mathilde Roger ◽  
Abbie Degnan ◽  
Stephen Todryk ◽  
...  
Keyword(s):  
Therapeutic Interventions ◽  
Transcriptional Gene Silencing ◽  
Biological Functions ◽  
Vast Number ◽  
Regulate Gene Expression ◽  
Post Transcriptional Gene Silencing ◽  
Non Coding Rnas ◽  
And Function ◽  
Molecular Signals

The skin is the largest organ of the integumentary system and possesses a vast number of functions. Due to the distinct layers of the skin and the variety of cells which populate each, a tightly regulated network of molecular signals control development and regeneration, whether due to programmed cell termination or injury. MicroRNAs (miRs) are a relatively recent discovery; they are a class of small non-coding RNAs which possess a multitude of biological functions due to their ability to regulate gene expression via post-transcriptional gene silencing. Of interest, is that a plethora of data demonstrates that a number of miRs are highly expressed within the skin, and are evidently key regulators of numerous vital processes to maintain non-aberrant functioning. Recently, miRs have been targeted as therapeutic interventions due to the ability of synthetic ‘antagomiRs’ to down-regulate abnormal miR expression, thereby potentiating wound healing and attenuating fibrotic processes which can contribute to disease such as systemic sclerosis (SSc). This review will provide an introduction to the structure and function of the skin and miR biogenesis, before summarizing the literature pertaining to the role of miRs. Finally, miR therapies will also be discussed, highlighting important future areas of research.

scholarly journals Post-Translational Modification and Subcellular Distribution of Rac1: An Update

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 263 ◽  
Author(s):  
Abdalla Abdrabou ◽  
Zhixiang Wang
Keyword(s):  
Membrane Trafficking ◽  
Subcellular Distribution ◽  
Bound States ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Guanine Nucleotide ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Rho Family ◽  
And Function

Rac1 is a small GTPase that belongs to the Rho family. The Rho family of small GTPases is a subfamily of the Ras superfamily. The Rho family of GTPases mediate a plethora of cellular effects, including regulation of cytoarchitecture, cell size, cell adhesion, cell polarity, cell motility, proliferation, apoptosis/survival, and membrane trafficking. The cycling of Rac1 between the GTP (guanosine triphosphate)- and GDP (guanosine diphosphate)-bound states is essential for effective signal flow to elicit downstream biological functions. The cycle between inactive and active forms is controlled by three classes of regulatory proteins: Guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine-nucleotide-dissociation inhibitors (GDIs). Other modifications include RNA splicing and microRNAs; various post-translational modifications have also been shown to regulate the activity and function of Rac1. The reported post-translational modifications include lipidation, ubiquitination, phosphorylation, and adenylylation, which have all been shown to play important roles in the regulation of Rac1 and other Rho GTPases. Moreover, the Rac1 activity and function are regulated by its subcellular distribution and translocation. This review focused on the most recent progress in Rac1 research, especially in the area of post-translational modification and subcellular distribution and translocation.

scholarly journals Functional dissection of the ARGONAUTE7 promoter

2018 ◽  
Author(s):  
J. Steen Hoyer ◽  
Jose L. Pruneda-Paz ◽  
Ghislain Breton ◽  
Mariah A. Hassert ◽  
Emily E. Holcomb ◽  
...  
Keyword(s):  
Binding Sites ◽  
Rna Silencing ◽  
Leaf Shape ◽  
Effector Proteins ◽  
Transcriptional Gene Silencing ◽  
Antiviral Defense ◽  
Post Transcriptional Gene Silencing ◽  
And Function ◽  
Central Effector ◽  
Ranked List

AbstractARGONAUTES are the central effector proteins of RNA silencing which bind target transcripts in a small RNA-guided manner. Arabidopsis thaliana has ten ARGONAUTE (AGO) genes, with specialized roles in RNA-directed DNA methylation, post-transcriptional gene silencing, and antiviral defense. To better understand specialization among AGO genes at the level of transcriptional regulation we tested a library of 1497 transcription factors for binding to the promoters of AGO1, AGO10, and AGO7 using yeast 1-hybrid assays. A ranked list of candidate DNA-binding TFs revealed binding of the AGO7 promoter by a number of proteins in two families: the miR156-regulated SPL family and the miR319-regulated TCP family, both of which have roles in developmental timing and leaf morphology. Possible functions for SPL and TCP binding are unclear: we showed that these binding sites are not required for the polar expression pattern of AGO7, nor for the function of AGO7 in leaf shape. Normal AGO7 transcription levels and function appear to depend instead on an adjacent 124-bp region. Progress in understanding the structure of this promoter may aid efforts to understand how the conserved AGO7-triggered TAS3 pathway functions in timing and polarity.

scholarly journals RNA uridylation and decay in plants

2018 ◽  
Vol 373 (1762) ◽  
pp. 20180163 ◽  
Author(s):  
Caroline de Almeida ◽  
Hélène Scheer ◽  
Anthony Gobert ◽  
Veronica Fileccia ◽  
Federico Martinelli ◽  
...  
Keyword(s):  
Rna Degradation ◽  
Flowering Plant ◽  
Future Research ◽  
Transcriptional Gene Silencing ◽  
Messenger Rnas ◽  
Plant Infection ◽  
Post Transcriptional Gene Silencing ◽  
Key Points ◽  
History Of ◽  
And Function

RNA uridylation consists of the untemplated addition of uridines at the 3′ extremity of an RNA molecule. RNA uridylation is catalysed by terminal uridylyltransferases (TUTases), which form a subgroup of the terminal nucleotidyltransferase family, to which poly(A) polymerases also belong. The key role of RNA uridylation is to regulate RNA degradation in a variety of eukaryotes, including fission yeast, plants and animals. In plants, RNA uridylation has been mostly studied in two model species, the green algaeChlamydomonas reinhardtiiand the flowering plantArabidopsis thaliana. Plant TUTases target a variety of RNA substrates, differing in size and function. These RNA substrates include microRNAs (miRNAs), small interfering silencing RNAs (siRNAs), ribosomal RNAs (rRNAs), messenger RNAs (mRNAs) and mRNA fragments generated during post-transcriptional gene silencing. Viral RNAs can also get uridylated during plant infection. We describe here the evolutionary history of plant TUTases and we summarize the diverse molecular functions of uridylation during RNA degradation processes in plants. We also outline key points of future research.This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.

scholarly journals A new method for functional analysis of plastid EMBRYO-DEFECTIVEPPR genes by efficiently constructing cosuppression lines in Arabidopsis

2020 ◽  
Author(s):  
Jingli Chen ◽  
Jirong Huang ◽  
Weihua Huang
Keyword(s):  
Functional Analysis ◽  
Higher Plants ◽  
New Method ◽  
Pcr Analysis ◽  
Transcriptional Gene Silencing ◽  
Transcriptional Level ◽  
Pentatricopeptide Repeat ◽  
Endogenous Gene ◽  
Post Transcriptional Gene Silencing ◽  
In The Wild

Abstract Background: Pentatricopeptide-repeat proteins (PPRs) characterized by tandem arrays of a degenerate 35-amino-acid repeat (PPR motif) can bind a single strand RNA and regulate organell gene expression at the post-transcriptional level, including RNA cleavage, splicing, editing and stability etc. PPRs are conserved in all eukaryotes and extremely expanded in higher plants. Many knockout mutants of PPR genes are embryonic lethal. These genes are named EMB PPRs and their functional analysis is particularly difficult.Results: Here, we report a new method for functional analysis of plastid EMB PPRs by efficiently constructing their cosuppression lines in Arabidopsis. When we overexpressed a mutated full length or fragments lacking one or several PPR repeats of EMB PPRs, such as EMB2279, EMB2654 and EMB976 in the wild-type (WT) background, a large portion of T1 plants displayed chlorosis phenotypes, which is similar to those of their knockdown mutants. RT-PCR analysis showed that the exogenous transgene was overexpressed, while the corresponding endogenous mRNA was significantly downregulated in the transgenic lines. However, when these constructs were overexpressed in the PTGS (Post transcriptional Gene Silencing) deficient mutant, rdr6 (rna dependent rna polymerase 6), none of the T1 plants displayed chlorosis phenotypes. These results indicate that the chlorosis phenotype results from post transcriptional silencing of the corresponding endogenous gene (also known as sense cosuppression). Conclusions: Overexpression of an appropriately truncated PPR CDS in WT leads to a high frequency of cosuppression lines with apparent mutant phenotype, and this method can be employed to study the unknown function of EMB PPR genes. By this method, our preliminary data showed that EMB976 is required for splicing of plastid clpP1 intron 2 and ycf3 intron 1.

scholarly journals RST1 and RIPR connect the cytosolic RNA exosome to the Ski complex in Arabidopsis

2019 ◽  
Author(s):  
Heike Lange ◽  
Simon Y. A. Ndecky ◽  
Carlos Gomez-Diaz ◽  
David Pflieger ◽  
Nicolas Butel ◽  
...  
Keyword(s):  
Domain Architecture ◽  
Transcriptional Gene Silencing ◽  
Functional Link ◽  
Interacting Protein ◽  
Post Transcriptional Gene Silencing ◽  
Evolutionarily Conserved ◽  
Rna Quality Control ◽  
Rna Exosome ◽  
And Function ◽  
Similar Domain

AbstractThe RNA exosome is a key 3’-5’ exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known SKI subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans.

scholarly journals Interactions of Lipid Droplets with the Intracellular Transport Machinery

2021 ◽  
Vol 22 (5) ◽  
pp. 2776
Author(s):  
Selma Yilmaz Dejgaard ◽  
John F. Presley
Keyword(s):  
Membrane Trafficking ◽  
Lipid Droplets ◽  
Intracellular Transport ◽  
Neutral Lipids ◽  
Small Gtpases ◽  
Lipid Phase ◽  
Intracellular Organelles ◽  
And Function ◽  
Lipid Phase Separation ◽  
Endocytic Pathways

Historically, studies of intracellular membrane trafficking have focused on the secretory and endocytic pathways and their major organelles. However, these pathways are also directly implicated in the biogenesis and function of other important intracellular organelles, the best studied of which are peroxisomes and lipid droplets. There is a large recent body of work on these organelles, which have resulted in the introduction of new paradigms regarding the roles of membrane trafficking organelles. In this review, we discuss the roles of membrane trafficking in the life cycle of lipid droplets. This includes the complementary roles of lipid phase separation and proteins in the biogenesis of lipid droplets from endoplasmic reticulum (ER) membranes, and the attachment of mature lipid droplets to membranes by lipidic bridges and by more conventional protein tethers. We also discuss the catabolism of neutral lipids, which in part results from the interaction of lipid droplets with cytosolic molecules, but with important roles for both macroautophagy and microautophagy. Finally, we address their eventual demise, which involves interactions with the autophagocytotic machinery. We pay particular attention to the roles of small GTPases, particularly Rab18, in these processes.

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