scholarly journals Transcript Lifetime Is Balanced between Stabilizing Stem-Loop Structures and Degradation-Promoting Polyadenylation in Plant Mitochondria

2001 ◽  
Vol 21 (3) ◽  
pp. 731-742 ◽  
Author(s):  
Josef Kuhn ◽  
Ulrike Tengler ◽  
Stefan Binder
Keyword(s):  
Plant Mitochondria ◽  
Rna Stability ◽  
Processing System ◽  
Stem Loop ◽  
Functional Studies ◽  
In Vitro Processing ◽  
Rapid Amplification ◽  
And Function

ABSTRACT To determine the influence of posttranscriptional modifications on 3′ end processing and RNA stability in plant mitochondria, peaatp9 and Oenothera atp1 transcripts were investigated for the presence and function of 3′ nonencoded nucleotides. A 3′ rapid amplification of cDNA ends approach initiated at oligo(dT)-adapter primers finds the expected poly(A) tails predominantly attached within the second stem or downstream of the double stem-loop structures at sites of previously mapped 3′ ends. Functional studies in a pea mitochondrial in vitro processing system reveal a rapid removal of the poly(A) tails up to termini at the stem-loop structure but little if any influence on further degradation of the RNA. In contrast 3′ poly(A) tracts at RNAs without such stem-loop structures significantly promote total degradation in vitro. To determine the in vivo identity of 3′ nonencoded nucleotides more accurately, pea atp9 transcripts were analyzed by a direct anchor primer ligation-reverse transcriptase PCR approach. This analysis identified maximally 3-nucleotide-long nonencoded extensions most frequently of adenosines combined with cytidines. Processing assays with substrates containing homopolymer stretches of different lengths showed that 10 or more adenosines accelerate RNA processivity, while 3 adenosines have no impact on RNA life span. Thus polyadenylation can generally stimulate the decay of RNAs, but processivity of degradation is almost annihilated by the stabilizing effect of the stem-loop structures. These antagonistic actions thus result in the efficient formation of 3′ processed and stable transcripts.

scholarly journals Host poly(A) polymerases PAPD5 and PAPD7 provide two layers of protection that ensure the integrity and stability of hepatitis B virus RNA

2021 ◽  
Author(s):  
Fei Liu ◽  
Amy C.H Lee ◽  
Fang Guo ◽  
Andrew S. Kondratowicz ◽  
Holly M Micolochick Steuer ◽  
...  
Keyword(s):  
Dominant Role ◽  
Regulatory Element ◽  
Rna Stability ◽  
Antiviral Effect ◽  
Inhibitory Effect ◽  
Stem Loop ◽  
Knock Out ◽  
The Individual

Noncanonical poly(A) polymerases PAPD5 and PAPD7 (PAPD5/7) stabilize HBV RNA via the interaction with the viral post-transcriptional regulatory element (PRE), representing new antiviral targets to control HBV RNA metabolism, HBsAg production and viral replication. Inhibitors targeting these proteins are being developed as antiviral therapies, therefore it is important to understand how PAPD5/7 coordinate to stabilize HBV RNA. Here, we utilized a potent small-molecule AB-452 as a chemical probe, along with genetic analyses to dissect the individual roles of PAPD5/7 in HBV RNA stability. AB-452 inhibits PAPD5/7 enzymatic activities and reduces HBsAg both in vitro (EC50 ranged from 1.4 to 6.8 nM) and in vivo by 0.93 log10. Our genetic studies demonstrate that the stem-loop alpha sequence within PRE is essential for both maintaining HBV poly(A) tail integrity and determining sensitivity towards the inhibitory effect of AB-452. Although neither single knock-out (KO) of PAPD5 nor PAPD7 reduces HBsAg RNA and protein production, PAPD5 KO does impair poly(A) tail integrity and confers partial resistance to AB-452. In contrast, PAPD7 KO could not result in any measurable phenotypic changes, but displays a similar antiviral effect as AB-452 treatment when PAPD5 is depleted simultaneously. PAPD5/7 double KO confers complete resistance to AB-452 treatment. Our results thus indicate that PAPD5 plays a dominant role in stabilizing viral RNA by protecting the integrity of its poly(A) tail, while PAPD7 serves as a second line of protection. These findings inform PAPD5 targeted therapeutic strategies and open avenues for further investigating PAPD5/7 in HBV replication.

scholarly journals Small cis-Acting Sequences That Specify Secondary Structures in a Chloroplast mRNA Are Essential for RNA Stability and Translation

1999 ◽  
Vol 19 (12) ◽  
pp. 8479-8491 ◽  
Author(s):  
David C. Higgs ◽  
Risa S. Shapiro ◽  
Karen L. Kindle ◽  
David B. Stern
Keyword(s):  
Untranslated Region ◽  
Rna Stability ◽  
Dimethyl Sulfate ◽  
Secondary Structures ◽  
Rna Modification ◽  
Range Interaction ◽  
Stem Loop ◽  
Chloroplast Mrna

ABSTRACT Nucleus-encoded proteins interact with cis-acting elements in chloroplast transcripts to promote RNA stability and translation. We have analyzed the structure and function of three such elements within the Chlamydomonas petD 5′ untranslated region; petD encodes subunit IV of the cytochromeb 6/f complex. These elements were delineated by linker-scanning mutagenesis, and RNA secondary structures were investigated by mapping nuclease-sensitive sites in vitro and by in vivo dimethyl sulfate RNA modification. Element I spans a maximum of 8 nucleotides (nt) at the 5′ end of the mRNA; it is essential for RNA stability and plays a role in translation. This element appears to form a small stem-loop that may interact with a previously described nucleus-encoded factor to block 5′→3′ exoribonucleolytic degradation. Elements II and III, located in the center and near the 3′ end of the 5′ untranslated region, respectively, are essential for translation, but mutations in these elements do not affect mRNA stability. Element II is a maximum of 16 nt in length, does not form an obvious secondary structure, and appears to bind proteins that protect it from dimethyl sulfate modification. Element III spans a maximum of 14 nt and appears to form a stem-loop in vivo, based on dimethyl sulfate modification and the sequences of intragenic suppressors of element III mutations. Furthermore, mutations in element II result in changes in the RNA structure near element III, consistent with a long-range interaction that may promote translation.

scholarly journals Collagenase Impacts the Quantity and Quality of Native Mesenchymal Stem/Stromal Cells Derived during Processing of Umbilical Cord Tissue

2018 ◽  
Vol 27 (1) ◽  
pp. 181-193 ◽  
Author(s):  
Rouzbeh R. Taghizadeh ◽  
Kyle J. Cetrulo ◽  
Curtis L. Cetrulo
Keyword(s):  
Umbilical Cord ◽  
Processing System ◽  
Enzymatic Digestion ◽  
Cell Recovery ◽  
Surface Marker Expression ◽  
Fold Reduction ◽  
Umbilical Cord Tissue ◽  
And Function

Enzymes are commonly used as a biochemical means to liberate cells from a host of tissues for use in in vitro studies and/or in vivo transplantations. However, very little understanding exists of the biological and functional effects that enzymes have on cells during the process of releasing the native cells from a given tissue. One specific reason for this is that no technology has existed as a nonenzymatic control to compare baseline biology and function for a given processed tissue. We have developed a sterile, onetime use, disposable system (referred to as the AuxoCell Processing System or AC:Px®) that allows for processing of solid tissue in a closed, standardized system using mechanical means to liberate cells without the need and/or use of any biochemical, enzymatic digestion. In this report, for the first time, we directly compare the cellular outputs derived from processing the same umbilical cord tissue (UCT) in the presence and absence of collagenase. In the presence of collagenase, we observed on average, approximately a 2.7-fold reduction in native mesenchymal stem/stromal cell (MSC) yields and a reduction in MSC-specific markers CD90, CD29, CD105, CD73, CD44, CD36, CD49b, CD49a, CD146, CD295, and CD166 and in endothelial marker CD31. These data directly exhibit that the use of collagenase to process UCT to release cells impacts cell recovery with respect to number and cell surface marker expression and, hence, could affect the in vivo function of the recovered native cellular population.

scholarly journals The role of Mfn2 in the structure and function of ER-mitochondrial tethering in vivo

2021 ◽  
Author(s):  
Song Han ◽  
Fanpeng Zhao ◽  
Jeffrey Hsia ◽  
Xiaopin Ma ◽  
Yi Liu ◽  
...  
Keyword(s):  
Pyramidal Neurons ◽  
Critical Role ◽  
Biochemical Changes ◽  
Conditional Knockout ◽  
Functional Studies ◽  
And Function ◽  
Close Contacts

The mitochondria-ER contacts (MERCs) plays an essential role in multiple cell physiological process. While Mfn2 was the first protein implicated in the formation of MERCs, it is debated whether it acts as a tether or antagonizer, largely based on in vitro studies. To understand the role of Mfn2 in MERCs in vivo, we characterized ultrastructural and biochemical changes of MERCs in pyramidal neurons of hippocampus in Mfn2 conditional knockout (KO) mice and in Mfn2 overexpression (OE) mice and found Mfn2 ablation caused reduced close contacts while Mfn2 OE caused increased close contacts between ER and mitochondria in vivo. Functional studies on SH-SY5Y cells with Mfn2 KO or overexpression demonstrating similar biochemical changes found that mitochondrial calcium uptake along with IP3R3-Grp75 interaction was decreased in Mfn2 KO cells but increased in the Mfn2 OE cells. Lastly, we found Mfn2 KO decreased and Mfn2 OE increased the interaction between the ER-mitochondria tethering pair of VAPB-PTPIP51. In conclusion, our study supports the notion that Mfn2 plays a critical role in ER-mitochondrial tethering and the formation of close contacts in neuronal cells in vivo.

scholarly journals Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

2020 ◽  
pp. 1-14
Author(s):  
Shelby Shrigley ◽  
Fredrik Nilsson ◽  
Bengt Mattsson ◽  
Alessandro Fiorenzano ◽  
Janitha Mudannayake ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Functional Recovery ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Alternative Source ◽  
And Function

Background: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. Objective: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. Methods: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. Results: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. Conclusion: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

scholarly journals POLRMT mutations impair mitochondrial transcription causing neurological disease

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Monika Oláhová ◽  
Bradley Peter ◽  
Zsolt Szilagyi ◽  
Hector Diaz-Maldonado ◽  
Meenakshi Singh ◽  
...  
Keyword(s):  
Progressive External Ophthalmoplegia ◽  
Global Developmental Delay ◽  
Disease Mechanism ◽  
Mitochondrial Transcription ◽  
Functional Studies ◽  
Mtdna Deletions ◽  
Molecular Nature ◽  
Important Disease

AbstractWhile >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.

scholarly journals Extracellular vesicles as mediators and markers of acute organ injury: current concepts

2021 ◽  
Author(s):  
Birte Weber ◽  
Niklas Franz ◽  
Ingo Marzi ◽  
Dirk Henrich ◽  
Liudmila Leppik
Keyword(s):  
Extracellular Vesicles ◽  
Inflammatory Diseases ◽  
Organ Injury ◽  
Isolation And Characterization ◽  
Communication Processes ◽  
Incidence And Mortality ◽  
New Biomarkers ◽  
And Function

AbstractDue to the continued high incidence and mortality rate worldwide, there is a need to develop new strategies for the quick, precise, and valuable recognition of presenting injury pattern in traumatized and poly-traumatized patients. Extracellular vesicles (EVs) have been shown to facilitate intercellular communication processes between cells in close proximity as well as distant cells in healthy and disease organisms. miRNAs and proteins transferred by EVs play biological roles in maintaining normal organ structure and function under physiological conditions. In pathological conditions, EVs change the miRNAs and protein cargo composition, mediating or suppressing the injury consequences. Therefore, incorporating EVs with their unique protein and miRNAs signature into the list of promising new biomarkers is a logical next step. In this review, we discuss the general characteristics and technical aspects of EVs isolation and characterization. We discuss results of recent in vitro, in vivo, and patients study describing the role of EVs in different inflammatory diseases and traumatic organ injuries. miRNAs and protein signature of EVs found in patients with acute organ injury are also debated.

scholarly journals Modelling the Human Placental Interface In Vitro—A Review

Micromachines ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 884
Author(s):  
Marta Cherubini ◽  
Scott Erickson ◽  
Kristina Haase
Keyword(s):  
Drug Testing ◽  
Cell Types ◽  
In Vitro Models ◽  
Placental Development ◽  
Scientific Challenge ◽  
Induced Pluripotent ◽  
And Function ◽  
And Control

Acting as the primary link between mother and fetus, the placenta is involved in regulating nutrient, oxygen, and waste exchange; thus, healthy placental development is crucial for a successful pregnancy. In line with the increasing demands of the fetus, the placenta evolves throughout pregnancy, making it a particularly difficult organ to study. Research into placental development and dysfunction poses a unique scientific challenge due to ethical constraints and the differences in morphology and function that exist between species. Recently, there have been increased efforts towards generating in vitro models of the human placenta. Advancements in the differentiation of human induced pluripotent stem cells (hiPSCs), microfluidics, and bioprinting have each contributed to the development of new models, which can be designed to closely match physiological in vivo conditions. By including relevant placental cell types and control over the microenvironment, these new in vitro models promise to reveal clues to the pathogenesis of placental dysfunction and facilitate drug testing across the maternal–fetal interface. In this minireview, we aim to highlight current in vitro placental models and their applications in the study of disease and discuss future avenues for these in vitro models.

scholarly journals Therapeutic Application of Brain-Specific Angiogenesis Inhibitor 1 for Cancer Therapy

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3562
Author(s):  
Mitra Nair ◽  
Chelsea Bolyard ◽  
Tae Jin Lee ◽  
Balveen Kaur ◽  
Ji Young Yoo
Keyword(s):  
Angiogenesis Inhibitor ◽  
Suppressor Gene ◽  
Chemotherapeutic Agents ◽  
Tumor Models ◽  
In Vivo Models ◽  
Herpes Simplex Viruses ◽  
Multiple Tumor ◽  
And Function

Brain-specific angiogenesis inhibitor 1 (BAI1/ADGRB1) is an adhesion G protein-coupled receptor that has been found to play key roles in phagocytosis, inflammation, synaptogenesis, the inhibition of angiogenesis, and myoblast fusion. As the name suggests, it is primarily expressed in the brain, with a high expression in the normal adult and developing brain. Additionally, its expression is reduced in brain cancers, such as glioblastoma (GBM) and peripheral cancers, suggesting that BAI1 is a tumor suppressor gene. Several investigators have demonstrated that the restoration of BAI1 expression in cancer cells results in reduced tumor growth and angiogenesis. Its expression has also been shown to be inversely correlated with tumor progression, neovascularization, and peri-tumoral brain edema. One method of restoring BAI1 expression is by using oncolytic virus (OV) therapy, a strategy which has been tested in various tumor models. Oncolytic herpes simplex viruses engineered to express the secreted fragment of BAI1, called Vasculostatin (Vstat120), have shown potent anti-tumor and anti-angiogenic effects in multiple tumor models. Combining Vstat120-expressing oHSVs with other chemotherapeutic agents has also shown to increase the overall anti-tumor efficacy in both in vitro and in vivo models. In the current review, we describe the structure and function of BAI1 and summarize its application in the context of cancer treatment.

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