Expression and processing of polycistronic artificial microRNAs and trans-acting siRNAs in Solanum lycopersicum and Nicotiana benthamiana

AbstractTargeted gene silencing using small regulatory RNAs is a widely used technique for genetic studies in plants. Artificial microRNAs are one common approach; they have the advantage of producing just a single functional small RNA which can be designed for high target specificity and low off-target effects. Simultaneous silencing of multiple targets with artificial microRNAs can be achieved by producing polycistronic microRNA precursors. Alternatively, specialized trans-acting short interfering RNA (tasiRNA) precursors can be designed to produce several specific tasiRNAs at once. Here we tested several artificial microRNA- and tasiRNA-based methods for multiplexed gene silencing in Solanum lycopersicum (tomato) and Nicotiana benthamiana. Small RNA sequencing analyses revealed that many previously described approaches resulted in poor small RNA processing. The 5’-most microRNA precursor hairpins on polycistronic artificial microRNA precursors were generally processed more accurately than precursors at the 3’ end. Polycistronic artificial microRNAs where the hairpin precursors were separated by transfer RNAs had the best processing precision. Strikingly, artificial tasiRNA precursors failed to be processed in the expected phased manner in our system. These results highlight the need for further development of multiplexed artificial microRNA and tasiRNA strategies. The importance of small RNA sequencing, as opposed to single-target assays such as RNA blots or real-time PCR, is also discussed.Significance statementSeveral strategies for multiplexed gene silencing using artificial microRNAs or tasiRNAs have been described. We find that many result in imprecise processing, and thus low accumulation of the intended small RNAs. Our findings highlight the importance of small RNA sequencing to fully analyze gene silencing experiments, and also the need for continued methodological development of these methods.

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Faculty Opinions recommendation of Identification of nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1161246.621733 ◽

2009 ◽

Author(s):

Alain Gojon

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Rna Sequencing ◽

Small Rna ◽

Small Rna Sequencing ◽

Chain Reaction ◽

Polymerase Chain

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Small RNA Sequencing to Discover Circulating MicroRNA Biomarkers of Testicular Toxicity in Dogs

International Journal of Toxicology ◽

10.1177/1091581820961515 ◽

2020 ◽

pp. 109158182096151

Author(s):

Jennifer C. Shing ◽

Kai Schaefer ◽

Shaun E. Grosskurth ◽

Andy H. Vo ◽

Tatiana Sharapova ◽

...

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

Exploratory Study ◽

Quantitative Polymerase Chain Reaction ◽

Small Rna Sequencing ◽

Circulating Microrna ◽

Testicular Toxicity ◽

Potential Candidate ◽

Drug Induced ◽

Organ Systems

Predictive indicators of testicular toxicity could improve drug development by allowing early in-life screening for this adverse effect before it becomes severe. We hypothesized that circulating microRNAs (miRNAs) could serve as testicular toxicity biomarkers in dogs. Herein, we describe the results of an exploratory study conducted to discover biomarkers of drug-induced testicular injury. Following a dose-selection study using the testicular toxicant ethylene glycol monomethyl ether (EGME), we chose a dose of 50 mg/kg/d EGME to avoid systemic toxicity and treated 2 groups of dogs (castrated, non-castrated) for 14 to 28 days. Castrated animals were used as negative controls to identify biomarkers specific for testicular toxicity because EGME can cause toxicity to organ systems in addition to the testis. Blood was collected daily during the dosing period, followed by recovery for 29 to 43 days with less frequent sampling. Dosing was well tolerated, resulting in mild-to-moderate degeneration in testes and epididymides. Global profiling of serum miRNAs at selected dosing and recovery time points was completed by small RNA sequencing. Bioinformatics data analysis using linear modeling demonstrated several circulating miRNAs that were differentially abundant during the dosing period compared with baseline and/or castrated control samples. Confirmatory reverse transcription quantitative polymerase chain reaction data in these animals was unable to detect sustained alterations of miRNAs in serum, except for 1 potential candidate cfa-miR-146b. Taken together, we report the results of a comprehensive exploratory study and suggest future directions for follow-up research to address the challenge of developing diagnostic biomarkers of testicular toxicity.

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Extensive Analysis of miRNA Trimming and Tailing Indicates that AGO1 Has a Complex Role in miRNA Turnover

Plants ◽

10.3390/plants10020267 ◽

2021 ◽

Vol 10 (2) ◽

pp. 267

Author(s):

Axel J. Giudicatti ◽

Ariel H. Tomassi ◽

Pablo A. Manavella ◽

Agustin L. Arce

Keyword(s):

Small Rna ◽

Stress Responses ◽

Cellular Localization ◽

Mirna Biogenesis ◽

Small Rna Sequencing ◽

Sequencing Data ◽

Extensive Analysis ◽

Small Regulatory Rnas ◽

Regulatory Rnas ◽

Argonaute 1

MicroRNAs are small regulatory RNAs involved in several processes in plants ranging from development and stress responses to defense against pathogens. In order to accomplish their molecular functions, miRNAs are methylated and loaded into one ARGONAUTE (AGO) protein, commonly known as AGO1, to stabilize and protect the molecule and to assemble a functional RNA-induced silencing complex (RISC). A specific machinery controls miRNA turnover to ensure the silencing release of targeted-genes in given circumstances. The trimming and tailing of miRNAs are fundamental modifications related to their turnover and, hence, to their action. In order to gain a better understanding of these modifications, we analyzed Arabidopsis thaliana small RNA sequencing data from a diversity of mutants, related to miRNA biogenesis, action, and turnover, and from different cellular fractions and immunoprecipitations. Besides confirming the effects of known players in these pathways, we found increased trimming and tailing in miRNA biogenesis mutants. More importantly, our analysis allowed us to reveal the importance of ARGONAUTE 1 (AGO1) loading, slicing activity, and cellular localization in trimming and tailing of miRNAs.

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Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽

10.3390/diagnostics11060964 ◽

2021 ◽

Vol 11 (6) ◽

pp. 964

Author(s):

Sarka Benesova ◽

Mikael Kubista ◽

Lukas Valihrach

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

High Sensitivity ◽

Small Rna Sequencing ◽

Rna Seq ◽

Liquid Biopsies ◽

Comprehensive Overview ◽

Rna Molecules ◽

Novel Mirna ◽

The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

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Identification of Zinc Deficiency-Responsive MicroRNAs in Brassica juncea Roots by Small RNA Sequencing

Journal of Integrative Agriculture ◽

10.1016/s2095-3119(13)60641-3 ◽

2013 ◽

Vol 12 (11) ◽

pp. 2036-2044 ◽

Cited By ~ 5

Author(s):

Dong-qing SHI ◽

Yuan ZHANG ◽

Jin-hu MA ◽

Yu-long LI ◽

Jin XU

Keyword(s):

Rna Sequencing ◽

Brassica Juncea ◽

Zinc Deficiency ◽

Small Rna ◽

Small Rna Sequencing

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Genome-wide identification of conserved and novel microRNAs in one bud and two tender leaves of tea plant (Camellia sinensis) by small RNA sequencing, microarray-based hybridization and genome survey scaffold sequences

BMC Plant Biology ◽

10.1186/s12870-017-1169-1 ◽

2017 ◽

Vol 17 (1) ◽

Cited By ~ 22

Author(s):

Anburaj Jeyaraj ◽

Xiao Zhang ◽

Yan Hou ◽

Mingzhu Shangguan ◽

Prabu Gajjeraman ◽

...

Keyword(s):

Rna Sequencing ◽

Camellia Sinensis ◽

Small Rna ◽

Tea Plant ◽

Small Rna Sequencing ◽

Genome Survey ◽

Genome Wide

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Comparison of small RNA profiles in Nicotiana benthamiana and Solanum lycopersicum infected by polygonum ringspot tospovirus reveals host-specific responses to viral infection

Virus Research ◽

10.1016/j.virusres.2015.09.019 ◽

2016 ◽

Vol 211 ◽

pp. 38-45 ◽

Cited By ~ 14

Author(s):

Paolo Margaria ◽

Laura Miozzi ◽

Marina Ciuffo ◽

Cristina Rosa ◽

Michael J. Axtell ◽

...

Keyword(s):

Viral Infection ◽

Solanum Lycopersicum ◽

Small Rna ◽

Nicotiana Benthamiana

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Abstract 46: High-Density Lipoproteins Control Monocyte Differentiation Through microRNA Intercellular Communication

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a46 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Kasey C Vickers ◽

Michael G Levin ◽

Michael P Anderson ◽

Qing Xu ◽

Joshua Anzinger ◽

...

Keyword(s):

Gene Expression ◽

Rna Sequencing ◽

High Throughput ◽

Small Rna ◽

Cellular Response ◽

Culture Media ◽

Recipient Cell ◽

Inflammatory Cells ◽

High Density Lipoproteins ◽

Small Rna Sequencing

Many HDL-microRNAs (miRNA) are well-characterized post-transcriptional regulators of inflammation, and are significantly increased on HDL with hypercholesterolemia and atherosclerosis in humans and mice. Therefore, we hypothesize that inflammatory cells uniquely control their own gene expression through cellular miRNA export to HDL and then regulate recipient cell gene expression through HDL-mediated miRNA delivery. To test this hypothesis, we used high-throughput proteomics, Open Arrays, small RNA sequencing, and gene expression microarrays. Human monocytes (plasma elutriation) were differentiated into dendritic cells and multiple macrophage phenotypes. Each cell-type was incubated with pure reconstituted HDL (rHDL), which was then purified from culture media by apolipoprotein A-I immunoprecipitation after 24 h, and both cellular and HDL-miRNAs were profiled using TaqMan Open Arrays. Macrophages were found to export high levels of miRNAs to HDL that inhibit monocyte/macrophage differentiation (miR-146a, miR-223); however, monocytes were also found to export many miRNAs associated with differentiation, including miR-92a, miR-222, miR-17, miR-20a, miR106a, and miR-21. Furthermore, many miRNAs were found to be transcribed in inflammatory cells, but completely exported to HDL and not retained in the cell. Most interestingly, HDL treatment was found to induce miR-223 transcription in monocytes, as determined by primary miR-223 transcript levels; however, intracellular levels of the mature form (miR-223) did not change. These results suggest that HDL induces the export of miRNAs it transports. PAR-CLIP with high-throughput small RNA sequencing was used to demonstrate that miRNAs are transferred from macrophages to endothelial cells and loaded onto cellular Argonaute 2-continaining RNA-induced silencing complexes. To demonstrate this in mice, human HDL, containing endogenous levels of miR-223, were injected into miR-223-null mice and inflammation-associated miRNA delivery was mapped in vivo. In summary, we found profound differences in the cellular response to HDL treatment and HDL-miRNA communication amongst inflammatory cell phenotypes that are physiologically relevant to cardiovascular disease.

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