scholarly journals Broad role for YBX1 in defining the small noncoding RNA composition of exosomes

2017 ◽  
Vol 114 (43) ◽  
pp. E8987-E8995 ◽  
Author(s):  
Matthew J. Shurtleff ◽  
Jun Yao ◽  
Yidan Qin ◽  
Ryan M. Nottingham ◽  
Morayma M. Temoche-Diaz ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Rna Binding ◽  
Hek293t Cell ◽  
Full Length ◽  
Protein Coding ◽  
Transfer Rnas ◽  
Small Noncoding Rna ◽  
Small Noncoding Rnas ◽  
Small Ncrnas ◽  
Terminal Oligopyrimidine

RNA is secreted from cells enclosed within extracellular vesicles (EVs). Defining the RNA composition of EVs is challenging due to their coisolation with contaminants, lack of knowledge of the mechanisms of RNA sorting into EVs, and limitations of conventional RNA-sequencing methods. Here we present our observations using thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) to characterize the RNA extracted from HEK293T cell EVs isolated by flotation gradient ultracentrifugation and from exosomes containing the tetraspanin CD63 further purified from the gradient fractions by immunoisolation. We found that EV-associated transcripts are dominated by full-length, mature transfer RNAs (tRNAs) and other small noncoding RNAs (ncRNAs) encapsulated within vesicles. A substantial proportion of the reads mapping to protein-coding genes, long ncRNAs, and antisense RNAs were due to DNA contamination on the surface of vesicles. Nevertheless, sequences mapping to spliced mRNAs were identified within HEK293T cell EVs and exosomes, among the most abundant being transcripts containing a 5′ terminal oligopyrimidine (5′ TOP) motif. Our results indicate that the RNA-binding protein YBX1, which is required for the sorting of selected miRNAs into exosomes, plays a role in the sorting of highly abundant small ncRNA species, including tRNAs, Y RNAs, and Vault RNAs. Finally, we obtained evidence for an EV-specific tRNA modification, perhaps indicating a role for posttranscriptional modification in the sorting of some RNA species into EVs. Our results suggest that EVs and exosomes could play a role in the purging and intercellular transfer of excess free RNAs, including full-length tRNAs and other small ncRNAs.

scholarly journals A broad role for YBX1 in defining the small non-coding RNA composition of exosomes

2017 ◽  
Author(s):  
Matthew J. Shurtleff ◽  
Jun Yao ◽  
Yidan Qin ◽  
Ryan M. Nottingham ◽  
Morayma Temoche-Diaz ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Hek293t Cell ◽  
Full Length ◽  
Protein Coding ◽  
Non Coding Rna ◽  
Terminal Oligopyrimidine ◽  
Non Coding Rnas

AbstractRNA is secreted from cells enclosed within extracellular vesicles (EVs). Defining the RNA composition of EVs is challenging due to their co-isolation with contaminants, a lack of knowledge of the mechanisms of RNA sorting into EVs and limitations of conventional RNA-seq methods. Here we present our observations using thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) to characterize the RNA extracted from HEK293T cell EVs isolated by flotation gradient ultracentrifugation and from exosomes containing the tetraspannin CD63 further purified from the gradient fractions by immunoisolation. We found that EV-associated transcripts are dominated by full-length, mature tRNAs and other small non-coding RNAs encapsulated within vesicles. A substantial proportion of the reads mapping to protein-coding genes, long non-coding, and antisense RNAs were due to DNA contamination on the surface of vesicles. Nevertheless, sequences mapping to spliced mRNAs were identified within HEK293T cell EVs and exosomes, among the most abundant being transcripts containing a 5’ terminal oligopyrimidine (5’ TOP) motif. Our results indicate that the RNA-binding protein YBX1, which we showed previously is required for the sorting of selected miRNAs into exosomes, plays a role in the sorting of highly abundant small non-coding RNA species, including tRNAs, Y RNAs, and Vault RNAs. Finally, we obtained evidence for an EV-specific tRNA modification, perhaps indicating a role for post-transcriptional modification in the sorting of some RNA species into EVs. The identification of full-length small non-coding RNAs within EVs suggests a role for EVs in the export and possible intercellular functional transfer of abundant cellular transcripts.Statement of SignificanceCells release vesicles containing selectively packaged cargo, including RNA, into the extracellular environment. Prior studies have identified RNA inside extracellular vesicles (EVs) but, due to limitations of conventional sequencing methods, highly structured and post-transcriptionally modified RNA species were not effectively captured. Using an alternative sequencing approach (TGIRT-seq), we found that EVs contain abundant small non-coding RNA species, including full-length tRNAs and Y RNAs. Using a knockout cell line, we obtained evidence that the RNA-binding protein YBX1 plays a role in sorting small non-coding RNAs into a subpopulation of extracellular vesicles termed exosomes. These experiments expand our understanding of EV-RNA composition and provide insights into how RNA is sorted into EVs for export from the cell.

Long and small noncoding RNAs during oocyte-to-embryo transition in mammals

2017 ◽  
Vol 45 (5) ◽  
pp. 1117-1124 ◽  
Author(s):  
Petr Svoboda
Keyword(s):  
Noncoding Rna ◽  
Transcriptional Control ◽  
Noncoding Rnas ◽  
Rna Degradation ◽  
Early Embryo ◽  
Protein Coding ◽  
Small Noncoding Rnas ◽  
Major Genome ◽  
Silencing Pathways ◽  
And Function

Oocyte-to-embryo transition is a process during which an oocyte ovulates, is fertilized, and becomes a developing embryo. It involves the first major genome reprogramming event in life of an organism where gene expression, which gave rise to a differentiated oocyte, is remodeled in order to establish totipotency in blastomeres of an early embryo. This remodeling involves replacement of maternal RNAs with zygotic RNAs through maternal RNA degradation and zygotic genome activation. This review is focused on expression and function of long noncoding RNAs (lncRNAs) and small RNAs during oocyte-to-embryo transition in mammals. LncRNAs are an assorted rapidly evolving collection of RNAs, which have no apparent protein-coding capacity. Their biogenesis is similar to mRNAs including transcriptional control and post-transcriptional processing. Diverse molecular and biological roles were assigned to lncRNAs although most of them probably did not acquire a detectable biological role. Since some lncRNAs serve as precursors for small noncoding regulatory RNAs in RNA silencing pathways, both types of noncoding RNA are reviewed together.

scholarly journals Methyltransferase-like protein 16 binds the 3′-terminal triple helix of MALAT1 long noncoding RNA

2016 ◽  
Vol 113 (49) ◽  
pp. 14013-14018 ◽  
Author(s):  
Jessica A. Brown ◽  
Charles G. Kinzig ◽  
Suzanne J. DeGregorio ◽  
Joan A. Steitz
Keyword(s):  
Protein Binding ◽  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Triple Helix ◽  
Rna Binding ◽  
Hek293t Cell ◽  
Proximity Ligation Assay ◽  
Triple Helices ◽  
Gel Shift ◽  
In Cells

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a cancer-promoting long noncoding RNA, accumulates in cells by using a 3′-triple-helical RNA stability element for nuclear expression (ENE). The ENE, a stem-loop structure containing a U-rich internal loop, interacts with a downstream A-rich tract (ENE+A) to form a blunt-ended triple helix composed of nine U•A-U triples interrupted by a C•G-C triple and C-G doublet. This unique structure prompted us to explore the possibility of protein binding. Native gel-shift assays revealed a shift in radiolabeled MALAT1 ENE+A RNA upon addition of HEK293T cell lysate. Competitive gel-shift assays suggested that protein binding depends not only on the triple-helical structure but also its nucleotide composition. Selection from the lysate using a biotinylated-RNA probe followed by mass spectrometry identified methyltransferase-like protein 16 (METTL16), a putative RNA methyltransferase, as an interacting protein of the MALAT1 ENE+A. Gel-shift assays confirmed the METTL16–MALAT1 ENE+A interaction in vitro: Binding was observed with recombinant METTL16, but diminished in lysate depleted of METTL16, and a supershift was detected after adding anti-METTL16 antibody. Importantly, RNA immunoprecipitation after in vivo UV cross-linking and an in situ proximity ligation assay for RNA–protein interactions confirmed an association between METTL16 and MALAT1 in cells. METTL16 is an abundant (∼5 × 105 molecules per cell) nuclear protein in HeLa cells. Its identification as a triple-stranded RNA binding protein supports the formation of RNA triple helices inside cells and suggests the existence of a class of triple-stranded RNA binding proteins, which may enable the discovery of additional cellular RNA triple helices.

scholarly journals Of rodents and ruminants: a comparison of small noncoding RNA requirements in mouse and bovine reproduction

2021 ◽  
Vol 99 (3) ◽  
Author(s):  
Lauren G Chukrallah ◽  
Aditi Badrinath ◽  
Kelly Seltzer ◽  
Elizabeth M Snyder
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Noncoding Rna ◽  
Reproductive Potential ◽  
Noncoding Rnas ◽  
Model Organism ◽  
Cell Physiology ◽  
Male Germ Cells ◽  
Small Noncoding Rna ◽  
Small Noncoding Rnas

Abstract Ruminants are major producers of meat and milk, thus managing their reproductive potential is a key element in cost-effective, safe, and efficient food production. Of particular concern, defects in male germ cells and female germ cells may lead to significantly reduced live births relative to fertilization. However, the underlying molecular drivers of these defects are unclear. Small noncoding RNAs, such as piRNAs and miRNAs, are known to be important regulators of germ-cell physiology in mouse (the best-studied mammalian model organism) and emerging evidence suggests that this is also the case in a range of ruminant species, in particular bovine. Similarities exist between mouse and bovids, especially in the case of meiotic and postmeiotic male germ cells. However, fundamental differences in small RNA abundance and metabolism between these species have been observed in the female germ cell, differences that likely have profound impacts on their physiology. Further, parentally derived small noncoding RNAs are known to influence early embryos and significant species-specific differences in germ-cell born small noncoding RNAs have been observed. These findings demonstrate the mouse to be an imperfect model for understanding germ-cell small noncoding RNA biology in ruminants and highlight the need to increase research efforts in this underappreciated aspect of animal reproduction.

scholarly journals Paternal environmental exposure-induced spermatozoal small noncoding RNA alteration meditates the intergenerational epigenetic inheritance of multiple diseases

2021 ◽  
Author(s):  
Xin Yin ◽  
Azhar Anwar ◽  
Yanbo Wang ◽  
Huanhuan Hu ◽  
Gaoli Liang ◽  
...  
Keyword(s):  
Environmental Exposure ◽  
Noncoding Rna ◽  
Relevant Literature ◽  
Epigenetic Inheritance ◽  
Rna Regulation ◽  
Small Noncoding Rna ◽  
Small Noncoding Rnas ◽  
Male Line ◽  
The Veil

AbstractStudies of human and mammalian have revealed that environmental exposure can affect paternal health conditions as well as those of the offspring. However, studies that explore the mechanisms that meditate this transmission are rare. Recently, small noncoding RNAs (sncRNAs) in sperm have seemed crucial to this transmission due to their alteration in sperm in response to environmental exposure, and the methodology of microinjection of isolated total RNA or sncRNAs or synthetically identified sncRNAs gradually lifted the veil of sncRNA regulation during intergenerational inheritance along the male line. Hence, by reviewing relevant literature, this study intends to answer the following research concepts: (1) paternal environmental factors that can be passed on to offspring and are attributed to spermatozoal sncRNAs, (2) potential role of paternal spermatozoal sncRNAs during the intergenerational inheritance process, and (3) the potential mechanism by which spermatozoal sncRNAs meditate intergenerational inheritance. In summary, increased attention highlights the hidden wonder of spermatozoal sncRNAs during intergenerational inheritance. Therefore, in the future, more studies should focus on the origin of RNA alteration, the target of RNA regulation, and how sncRNA regulation during embryonic development can be sustained even in adult offspring.

scholarly journals The small noncoding RNA sr8384 determines solvent synthesis and cell growth in industrial solventogenic clostridia

2019 ◽  
Author(s):  
Yunpeng Yang ◽  
Nannan Lang ◽  
Huan Zhang ◽  
Lu Zhang ◽  
Changsheng Chai ◽  
...  
Keyword(s):  
Cell Growth ◽  
Clostridium Acetobutylicum ◽  
Noncoding Rna ◽  
Large Scale ◽  
Target Genes ◽  
Biological Processes ◽  
Small Noncoding Rna ◽  
Small Noncoding Rnas ◽  
Solventogenic Clostridia

ABSTRACTSmall noncoding RNAs (sncRNAs) are crucial regulatory molecules in organisms and are well known not only for their roles in the control of diverse essential biological processes but also for their value in genetic modification. However, to date, in gram-positive anaerobic solventogenic clostridia (which are a group of important industrial bacteria with exceptional substrate and product diversity), sncRNAs remain minimally explored, leading to a lack of detailed understanding regarding these important molecules and their use as targets for genetic improvement. Here, we performed large-scale phenotypic screens of a transposon-mediated mutant library ofClostridium acetobutylicum, a typical solventogenic clostridial species, and discovered a novel sncRNA (sr8384) that functions as a determinant positive regulator of growth and solvent synthesis. Comparative transcriptomic data combined with genetic and biochemical analyses revealed that sr8384 acts as a pleiotropic regulator and controls multiple targets that are associated with crucial biological processes, through direct or indirect interactions. Notably, modulation of the expression level of either sr8384 or its core target genes significantly increased the growth rate, solvent titer and productivity of the cells, indicating the importance of sr8384-mediated regulatory network inC. acetobutylicum. Furthermore, a homolog of sr8384 was discovered and proven to be functional in another importantClostridiumspecies,C. beijerinckii, suggesting the potential broad role of this sncRNA in clostridia. Our work showcases a previously unknown potent and complex role of sncRNAs in clostridia, providing new opportunities for understanding and engineering these anaerobes, including pathogenicClostridiumspecies.IMPORTANCEThe discovery of sncRNAs as new resources for functional studies and strain modifications are promising strategies in microorganisms. However, these crucial regulatory molecules have hardly been explored in industrially important solventogenic clostridia. Here, we identified sr8384 as a novel determinant sncRNA controlling cellular performance of solventogenicClostridium acetobutylicumand performed detailed functional analysis, which is the most in-depth study of sncRNAs in clostridia to date. We reveal the pleiotropic function of sr8384 and its multiple direct and indirect crucial targets, which represents a valuable source for understanding and optimizing this anaerobe. Of note, manipulation of these targets leads to improved cell growth and solvent synthesis. Our findings provide a new perspective for future studies on regulatory sncRNAs in clostridia.

scholarly journals Small noncoding RNA expression during extreme anoxia tolerance of annual killifish (Austrofundulus limnaeus) embryos

2017 ◽  
Vol 49 (9) ◽  
pp. 505-518 ◽  
Author(s):  
Claire L. Riggs ◽  
Jason E. Podrabsky
Keyword(s):  
Noncoding Rna ◽  
Expression Profiles ◽  
Global Analysis ◽  
Differentially Expressed ◽  
Anoxia Tolerance ◽  
Small Noncoding Rna ◽  
Death Studies ◽  
Small Noncoding Rnas ◽  
Annual Killifish ◽  
Austrofundulus Limnaeus

Small noncoding RNAs (sncRNA) have recently emerged as specific and rapid regulators of gene expression, involved in a myriad of cellular and organismal processes. MicroRNAs, a class of sncRNAs, are differentially expressed in diverse taxa in response to environmental stress, including anoxia. In most vertebrates, a brief period of oxygen deprivation results in severe tissue damage or death. Studies on sncRNA and anoxia have focused on these anoxia-sensitive species. Studying sncRNAs in anoxia-tolerant organisms may provide insight into adaptive mechanisms supporting anoxia tolerance. Embryos of the annual killifish Austrofundulus limnaeus are the most anoxia-tolerant vertebrates known, surviving over 100 days at their peak tolerance at 25°C. Their anoxia tolerance and physiology vary over development, such that both anoxia-tolerant and anoxia-sensitive phenotypes comprise the species. This allows for a robust comparison to identify sncRNAs essential to anoxia-tolerance. For this study, RNA sequencing was used to identify and quantify expression of sncRNAs in four embryonic stages of A. limnaeus in response to an exposure to anoxia and subsequent aerobic recovery. Unique stage-specific patterns of expression were identified that correlate with anoxia tolerance. In addition, embryos of A. limnaeus appear to constitutively express stress-responsive miRNAs. Most differentially expressed sncRNAs were expressed at higher levels during recovery. Many novel groups of sncRNAs with expression profiles suggesting a key role in anoxia tolerance were identified, including sncRNAs derived from mitochondrial tRNAs. This global analysis has revealed groups of candidate sncRNAs that we hypothesize support anoxia tolerance.

scholarly journals Identification of a Functional Small Noncoding RNA of African Swine Fever Virus

2020 ◽  
Vol 94 (21) ◽  
Author(s):  
Laura E. M. Dunn ◽  
Alasdair Ivens ◽  
Christopher L. Netherton ◽  
David A. G. Chapman ◽  
Philippa M. Beard
Keyword(s):  
Small Rnas ◽  
Noncoding Rna ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Hemorrhagic Disease ◽  
Small Noncoding Rna ◽  
Small Noncoding Rnas ◽  
Log Reduction ◽  
Complex Interactions

ABSTRACT African swine fever virus (ASFV) causes a lethal hemorrhagic disease of domestic pigs, against which no vaccine is available. ASFV has a large, double-stranded DNA genome that encodes over 150 proteins. Replication takes place predominantly in the cytoplasm of the cell and involves complex interactions with host cellular components, including small noncoding RNAs (sncRNAs). A number of DNA viruses are known to manipulate sncRNA either by encoding their own or disrupting host sncRNA. To investigate the interplay between ASFV and sncRNAs, a study of host and viral small RNAs extracted from ASFV-infected primary porcine macrophages (PAMs) was undertaken. We discovered that ASFV infection had only a modest effect on host miRNAs, with only 6 miRNAs differentially expressed during infection. The data also revealed 3 potential novel small RNAs encoded by ASFV, ASFVsRNA1-3. Further investigation of ASFVsRNA2 detected it in lymphoid tissue from pigs with ASF. Overexpression of ASFVsRNA2 led to an up to 1-log reduction in ASFV growth, indicating that ASFV utilizes a virus-encoded small RNA to disrupt its own replication. IMPORTANCE African swine fever (ASF) poses a major threat to pig populations and food security worldwide. The disease is endemic to Africa and Eastern Europe and is rapidly emerging into Asia, where it has led to the deaths of millions of pigs in the last 12 months. The development of safe and effective vaccines to protect pigs against ASF has been hindered by lack of understanding of the complex interactions between ASFV and the host cell. We focused our work on characterizing the interactions between ASFV and sncRNAs. Although comparatively modest changes to host sncRNA abundances were observed upon ASFV infection, we discovered and characterized a novel functional ASFV-encoded sncRNA. The results from this study add important insights into ASFV host-pathogen interactions. This knowledge may be exploited to develop more effective ASFV vaccines that take advantage of the sncRNA system.

scholarly journals Mammalian antiviral systems directed by small RNA

2021 ◽  
Vol 17 (12) ◽  
pp. e1010091
Author(s):  
Tomoko Takahashi ◽  
Steven M. Heaton ◽  
Nicholas F. Parrish
Keyword(s):  
Small Rna ◽  
Noncoding Rna ◽  
Current Knowledge ◽  
Genetic Material ◽  
Human Populations ◽  
Small Interfering Rnas ◽  
Transfer Rnas ◽  
Immune Systems ◽  
Small Noncoding Rna ◽  
Endogenous Genes

There are strong incentives for human populations to develop antiviral systems. Similarly, genomes that encode antiviral systems have had strong selective advantages. Protein-guided immune systems, which have been well studied in mammals, are necessary for survival in our virus-laden environments. Small RNA–directed antiviral immune systems suppress invasion of cells by non-self genetic material via complementary base pairing with target sequences. These RNA silencing-dependent systems operate in diverse organisms. In mammals, there is strong evidence that microRNAs (miRNAs) regulate endogenous genes important for antiviral immunity, and emerging evidence that virus-derived nucleic acids can be directly targeted by small interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), and transfer RNAs (tRNAs) for protection in some contexts. In this review, we summarize current knowledge of the antiviral functions of each of these small RNA types and consider their conceptual and mechanistic overlap with innate and adaptive protein-guided immunity, including mammalian antiviral cytokines, as well as the prokaryotic RNA-guided immune system, CRISPR. In light of recent successes in delivery of RNA for antiviral purposes, most notably for vaccination, we discuss the potential for development of small noncoding RNA–directed antiviral therapeutics and prophylactics.

scholarly journals Identification of a σB-Dependent Small Noncoding RNA in Listeria monocytogenes

2008 ◽  
Vol 190 (18) ◽  
pp. 6264-6270 ◽  
Author(s):  
Jesper Sejrup Nielsen ◽  
Anders Steno Olsen ◽  
Mette Bonde ◽  
Poul Valentin-Hansen ◽  
Birgitte H. Kallipolitis
Keyword(s):  
Listeria Monocytogenes ◽  
Stress Tolerance ◽  
Sigma Factor ◽  
Noncoding Rna ◽  
Noncoding Rnas ◽  
Alternative Sigma Factor ◽  
Dependent Manner ◽  
Small Noncoding Rna ◽  
Small Noncoding Rnas ◽  
Regulatory Circuits

ABSTRACT In Listeria monocytogenes, the alternative sigma factor σB plays important roles in stress tolerance and virulence. Here, we present the identification of SbrA, a novel small noncoding RNA that is produced in a σB-dependent manner. This finding adds the σB regulon to the growing list of stress-induced regulatory circuits that include small noncoding RNAs.

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