Journal: bioRxiv

Article Title: Manganese-dependent microRNA trimming by 3’→5’ exonucleases generates 14-nucleotide or shorter tiny RNAs

doi: 10.1101/2022.10.06.511180

Figure Lengend Snippet: Specific 3’→5’ exonucleases convert miRNAs to tyRNAs. ( A ) Possible pathways towards AGO-associated tyRNAs. ( B ) In vivo stability of 14- and 23-nt ss miR-20a and their siRNA-like duplexes ( C ) Accumulation of tyRNAs upon expression of ISG20. ( D ) In vivo trimming of FLAG-AGO2-associated miR-20a by ISG20, TREX1, ERI1, PARN, and EXO5 and their catalytic mutants. A representative gel image (top) and western blots with antibodies for each protein (bottom). ( E ) tyRNA synthesis on four human AGOs by ISG20, TREX1, ERI1, and PARN.

Article Snippet: The membranes were incubated with primary antibodies: anti-FLAG antibody (1:2,000, Sigma), anti-ISG20 antibody (1 μg/mL, Proteintech), anti-TREX1 antibody (1:1,000, Thermo Fisher Scientific), anti-ERI1 antibody (1:1,000, Proteintech), anti-PARN antibody (1:2,000, FORTIS), anti-EXO5 antibody (0.4 μg/mL, Atlas Antibodies), and/or anti-alpha-tubulin antibody (1:000 μg/mL, Cell Signaling Technology), and secondary antibodies: anti-mouse or rabbit (1:15,000, LI-COR).

Techniques: In Vivo, Expressing, Western Blot

Journal: bioRxiv

Article Title: Manganese-dependent microRNA trimming by 3’→5’ exonucleases generates 14-nucleotide or shorter tiny RNAs

doi: 10.1101/2022.10.06.511180

Figure Lengend Snippet: ISG20, TREX1, and ERI1 generate tyRNAs autonomously. ( A ) In vitro guide trimming by 3’→5’ exonucleases in the presence of 2 mM MnCl 2 . ( Left ) A representative gel image for each AGO. ( Right ) The relative amount of each guide length after incubation with either 3’→5’ exonuclease. ( B ) In vitro trimming of AGO2-associated miR-20a by the catalytically dead exonuclease mutants at 2 mM MnCl 2 . ( C ) In vitro trimming of AGO2-associated miR-20a by 3’→5’ exonucleases in 2 mM MgCl 2 . ( D ) In vitro trimming of different miRNAs by ISG20. ( Left ) A representative gel image for each AGO. ( Right ) The relative amount of each guide length after incubation with ISG20. ( E ) Docking model of ISG20 (blue ribbon model) on a guide (red stick model)-bound AGO3 (surface model). ( F ) In vitro trimming of AGO3-associated different miRNAs, followed by target cleavage.

Article Snippet: The membranes were incubated with primary antibodies: anti-FLAG antibody (1:2,000, Sigma), anti-ISG20 antibody (1 μg/mL, Proteintech), anti-TREX1 antibody (1:1,000, Thermo Fisher Scientific), anti-ERI1 antibody (1:1,000, Proteintech), anti-PARN antibody (1:2,000, FORTIS), anti-EXO5 antibody (0.4 μg/mL, Atlas Antibodies), and/or anti-alpha-tubulin antibody (1:000 μg/mL, Cell Signaling Technology), and secondary antibodies: anti-mouse or rabbit (1:15,000, LI-COR).

Techniques: In Vitro, Incubation

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-125a-5p: a novel regulator of SLC26A6 expression in intestinal epithelial cells

doi: 10.1152/ajpcell.00068.2019

Figure Lengend Snippet: Mutation of miR-125a-5p binding region in putative anion transporter-1 (PAT-1) 3′-untranslated region (UTR) abrogated the effects of mimic-125a-5p transfection on relative luciferase activity. A: mutated miR-125a-5p seed sequence in 3′-UTR of PAT-1 Caco-2 cells (grown in T-75 flask for 3–5 days to achieve ~80% confluency) were cotransfected with miR-125a-5p and pmirGLO-PAT-1 or pmirGLO-PAT-1 mut125a-5p. B: firefly luciferase activities were measured and normalized with respective Renilla luciferase activities. miR-125a-5p binding region in 3′-UTR of PAT-1 is denoted in red color and respective mutated sequence is in blue color. Results are means ± SE of 3 independent experiments. Statistical analysis using one-way ANOVA followed by Tukey’s multiple comparison test showed a significant difference between pmirGLO-3′-UTR-PAT-1 + -ve control and pmirGLO-3′-UTR-PAT-1 + mimic-125a-5p (***P < 0.0001).

Article Snippet: A 263-bp fragment of the PAT-1 mRNA-3′-UTR was custom synthesized (GenScript, Piscataway, NJ).

Techniques: Mutagenesis, Binding Assay, Transfection, Luciferase, Activity Assay, Sequencing

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-125a-5p: a novel regulator of SLC26A6 expression in intestinal epithelial cells

doi: 10.1152/ajpcell.00068.2019

Figure Lengend Snippet: Map of potential microRNA binding sites in 3′-untranslated region (UTR) of putative anion transporter-1 (PAT-1): in silico analysis. Figure shows the Targetscan analysis of microRNAs that target 3′-UTR of PAT-1 and their respective location to where they bind and several microRNAs whose binding region in 3′-UTR of PAT-1 is broadly or poorly conserved among mammals and vertebrates. [Figure is based on the output from http://genome.ucsc.edu (hg19 assembly) and http://www.targetscan.org Release 7.1.]

Article Snippet: A 263-bp fragment of the PAT-1 mRNA-3′-UTR was custom synthesized (GenScript, Piscataway, NJ).

Techniques: Binding Assay, In Silico

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-125a-5p: a novel regulator of SLC26A6 expression in intestinal epithelial cells

doi: 10.1152/ajpcell.00068.2019

Figure Lengend Snippet: Transient transfection of putative anion transporter-1 (PAT-1) 3′-untranslated region (UTR) decreased relative luciferase activity in intestinal epithelial cells. A–D: relative luciferase activity after 48-h transient transfection of pmiRGLO and 3′-UTR PAT-1 in Caco-2 (A), T-84 cells (B), HT-29 (C), and SK-CO15 cells (D) (grown in T-75 flask for 3–5 days ~80% confluent). Results are shown as % control in response to pmirGLO-3′-UTR-PAT-1 transfection compared with pmirGLO empty vector transfection. All the results are means ± SE of 4 independent experiments. ***P < 0.0001, ****P < 0.00001 vs. pmiRGLO was considered as statistically significant using unpaired t-test.

Article Snippet: A 263-bp fragment of the PAT-1 mRNA-3′-UTR was custom synthesized (GenScript, Piscataway, NJ).

Techniques: Transfection, Luciferase, Activity Assay, Plasmid Preparation

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-125a-5p: a novel regulator of SLC26A6 expression in intestinal epithelial cells

doi: 10.1152/ajpcell.00068.2019

Figure Lengend Snippet: Cotransfection of microRNA mimics that target putative anion transporter-1 (PAT-1) 3′-untranslated region (UTR) decreased luciferase reporter activity. Caco-2 cells (grown to 80% confluency, normally achieved in 3–5 days depending on seeding density) were co-transfected with microRNA mimics (that target 3′-UTR of PAT-1) or negative control and PAT-1 3′UTR or pmiR-GLO. Forty-eight hours posttransfection, firefly luciferase activities were measured and normalized with respective Renilla luciferase activities. Results are means ± SE of 4 independent experiments. Difference between 3′-UTR-PAT-1 -ve control vs. pmirGLO-3′-UTR-PAT-1 + mimic-125a-5p (****P < 0.00001) and vs. pmirGLO-3′UTR-PAT-1 + mimic-423-5p (***P < 0.0001) was considered statistically significant using one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: A 263-bp fragment of the PAT-1 mRNA-3′-UTR was custom synthesized (GenScript, Piscataway, NJ).

Techniques: Cotransfection, Luciferase, Activity Assay, Transfection, Negative Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-125a-5p: a novel regulator of SLC26A6 expression in intestinal epithelial cells

doi: 10.1152/ajpcell.00068.2019

Figure Lengend Snippet: miR-125a-5p mimic transfection decreased putative anion transporter-1 (PAT-1) mRNA expression. RNA was extracted from Caco-2 cells (grown in T-75 flask for 3–5 days to achieve ~80% confluency) 48 h posttransfection with mimics of microRNAs that target PAT-1. PAT-1 mRNA levels were measured by quantitative real-time PCR as described in materials and methods. Values of mRNA levels for PAT-1 were normalized against GAPDH mRNA levels. Results are means ± SE of 6 independent experiments (**P < 0.01).

Article Snippet: A 263-bp fragment of the PAT-1 mRNA-3′-UTR was custom synthesized (GenScript, Piscataway, NJ).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-125a-5p: a novel regulator of SLC26A6 expression in intestinal epithelial cells

doi: 10.1152/ajpcell.00068.2019

Figure Lengend Snippet: Mimic-125a-5p transfection decreased putative anion transporter-1 (PAT-1) protein expression. A: cell lysates of Caco-2 cells (grown in T-75 flask for 3–5 days to achieve ~80% confluency) transiently transfected with negative control and mimic-125a-5p were subjected to 7.5% SDS-PAGE followed by transfer to nitrocellulose membrane. The blots were probed with anti-PAT-1 or anti-GAPDH antibody. B: densitometric analysis of the relative band intensities was performed using ImageJ software. Results represent means ± SE of 5 different experiments. Differences between negative control versus mimic-125a-5p transfected groups (**P < 0.001) were found to be statistically significant using unpaired t-test.

Article Snippet: A 263-bp fragment of the PAT-1 mRNA-3′-UTR was custom synthesized (GenScript, Piscataway, NJ).

Techniques: Transfection, Expressing, Negative Control, SDS Page, Software