Journal: Nature Communications

Article Title: SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing

doi: 10.1038/ncomms10734

Figure Lengend Snippet: ( a ) SIRT7 CLIP-seq reads mapped to a custom annotation file of a human rDNA repeat (middle) or the transcribed region (bottom). The region encoding 18S, 5.8S and 28S rRNA is highlighted. SIRT7 reads after subtraction of IgG reads were normalized to input reads ( y axis). ( b ) Gene ontology categories of SIRT7 CLIP-seq peaks. The most representative clusters are shown according to the ajusted P value (−log 10 ). ( c ) SIRT7-bound snoRNAs comprise C/D box, H/ACA box snoRNAs and scaRNAs. The number ( n ) and relative abundance (%) of each snoRNA class associated with SIRT7 is presented. ( d ) U3, SNORA73A and 73B snoRNAs are overrepresented among SIRT7-associated snoRNAs. SIRT7 reads mapped to corresponding snoRNAs are indicated as percentage of all snoRNAs identified by CLIP-seq. ( e ) Comparison of SIRT7-associated RNAs under native and denaturing conditions. His/V5-tagged SIRT7 expressed in HEK293T cells was affinity-purified on Ni-NTA-agarose under native or denaturing conditions, and associated RNAs were detected by RT–qPCR. Lysates from non-transfected HEK293T cells were used for control (Ctrl). Associated pre-RNA was monitored by RT–qPCR using primer H1 . Bars represent means±s.d. from three experiments. See also . ( f ) ChIP assays showing association of endogenous SIRT7 (left panel) or transiently overexpressed Flag-SIRT7 (right panel) with the indicated gene loci in HEK293T cells. rDNA was amplified using primers H4 (coding) and H18 (IGS; ). Bars represent means±s.d. from three experiments. See also .

Article Snippet: After sonication (Bioruptor, Diagenode) to yield 250–500 bp fragments chromatin was diluted fivefold with IP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.0 and 167 mM NaCl), precleared with protein A and G Sepharose (GE Healthcare) in the presence of 200 μg ml −1 of sonicated E. coli DNA, and incubated overnight with the respective antibodies.

Techniques: Comparison, Affinity Purification, Quantitative RT-PCR, Transfection, Control, Amplification

Journal: bioRxiv

Article Title: Synthetic circRNAs employ IRES activity for translation in cells and in cell-free translation systems

doi: 10.64898/2026.03.28.715045

Figure Lengend Snippet: (A) Experimental outline of the in vitro generated circRNA-based reporter assay based on the mRuby3_circ_EGFP_ScaI_v3-4 plasmid (pKL480) for evaluation of IRES activity of different IRES inserts, including inverse sequences as controls for circRNA translation dependent on insert length and GC-content. Following DNA template synthesis (PCR amplification from plasmid backbone using poly(A)- and poly(T)-including primers), circRNA was generated by T7 in vitro transcription. Linear RNA species were removed by RNase R-treatment and column purification. Resulting circRNA was subsequently used for in-cell transfection or for in vitro translation systems. EGFP and 3xHA-Nluc reporter systems were used throughout the following analyses. The plasmid-encoded mRuby was not used. (B) Illustration of the circularization reaction mediated by group I dt introns leading to self-spliced circRNA. The observed sequence scar formed by the remaining parts of the td introns are highlighted in light and dark red. Remaining split introns were generated as side products and need to be removed. (C) Overview of the tested IRES sequences indicating length and viral or cellular origin of the IRESes. (D) Quality control of the generated EGFP reporter including circRNAs using 1% FA-agarose gel. Linear side products (upper band) disappear by RNase R digestion. Only the circular RNase R-resistant band remains (lower band). 500 ng total RNA was loaded per lane. The RiboRuler High Range RNA Ladder (Thermo, #SM1821) serves as a reference. (E) Quality control of the generated 3xHA-Nluc encoding circRNAs using 1% FA-agarose gel. Linear side products (upper band) are mainly degraded by RNase R digestion. Only the circular RNase R-resistant band remains (lower band). Only Dlx1 shows less efficient circularization leading to a higher concentration of side products. Therefore, the RNase R incubation time was increased to 90 min. 500 ng total RNA was loaded per lane. RNA species: linear (L), circular (C), introns (I). (F) Quality control, using the High Sensitivity RNA ScreenTape, of the generated circRNA before purification (upper panel) and after RNase R digestion (lower panel). Remaining contaminants can be observed as light grey bands.

Article Snippet: The PCR products were purified using the Monarch PCR & DNA Cleanup kit (NEB, T1030L) and 1 μg DNA was subsequently used for in vitro transcription using the High Scribe T7 High Yield RNA synthesis Kit (NEB, E2040S) according to manufacturer’s protocol (20 μL total reaction volume) supplemented with 40 U RiboLock RNase inhibitor (Thermo, EO0381) and incubated for 2 hrs at 37°C and 800 rpm.

Techniques: In Vitro, Generated, Reporter Assay, Plasmid Preparation, Activity Assay, Amplification, Purification, Transfection, Sequencing, Control, Agarose Gel Electrophoresis, Concentration Assay, Incubation