ms2 stem loop sequence  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: RSRC2 is a novel RNA-binding protein that safeguards mitotic fidelity by interacting with the lncRNA C1QTNF1-AS1

doi: 10.1093/nar/gkag229

Figure Lengend Snippet: C1QNTF1–AS1 interacts with RSRC2, an RBP of unknown function. ( A ) Schematic representation of the C1QTNF1–AS1 and C1QTNF1/CTRP1 genomic landscape ( C1QTNF1–AS1 annotated as NR_040 018/NR_040 019 in RefSeq; Gencode gene ENSG00000265096 ; chr17:79019209-79027601, hg38). ( B ) Maximum intensity projections of representative images of C1QTNF1–AS1 exon smRNA FISH in HCT116 showing its nuclear localization. Nuclei were stained with DAPI (magenta) and outlined with a dashed circle. Right panel: quantification of total transcript in the nucleus (N) and cytoplasm (C), solid line represents the mean. N = 3 ( n (HCT116) =398). ( C ) Expression levels of C1QTNF1–AS1 in HCT116 cells following depletion with three LNA gapmers targeting either exon 3 (LNA 1) or first intron of C1QTNF1–AS1 (LNA 2, 3), as measured by qPCR. Primers spanning mature (ex2-3) C1QTNF1–AS1 were used. Results are presented relative to negative control (Ctl A) LNA; N = 3. ( D ) Schematic representation of workflow for the ASO pulldown of C1QTNF1–AS1 in HCT116 cells. Five different ASOs targeting different regions of the C1QTNF1–AS1 locus were used, with luciferase (Luc) ASOs as a negative control. Pulldown efficacy was assessed by qPCR , and proteins were identified using LC-MS analysis (see Materials and methods). ( E ) The volcano plot highlighting proteins enriched in C1QTNF1–AS1 pulldown using ASO 1, 3, and 5 versus Luc. Significant C1QTNF1–AS1 protein interactors are highlighted in red (FDR 5%). ( F ) RIP-qPCR from HCT116 extracts. Left panel: Western blot of RSRC2 in the input and IP samples to show RSRC2 IP efficiency compared to IgG. Right panel: RIP-qPCR showing association of RSRC2 with C1QTNF1–AS1 transcript. GAPDH was used as negative control RNA for RSRC2 RIP. RIP enrichments are presented as % of input RNA (normalized to IgG); N = 3. ( G ) Relative expression of RSRC2 in HCT116 cells following siRNA-mediated depletion of RSRC2, as measured by qPCR. Results are presented relative to control siRNA (Ctl); N = 3. ( H ) Representative western blot showing RSRC2 protein expression in HCT116 cells following siRNA-mediated depletion of RSRC2. β-Tubulin and Ponceau staining were used as loading controls. An asterisk indicates an unspecific RSRC2 protein band. ( I ) Densitometric analysis of RSRC2 levels from panel (H) relative to control siRNA (Ctl); N = 3. ( J ) Interaction intensities between C1QTNF1–AS1 and the indicated proteins show that C1QTNF1–AS1 interacts with RSRC2. In-cell interactions were measured in an incPRINT experiment where MS2-tagged C1QTNF1–AS1 RNA was co-expressed with a set of FLAG-tagged proteins in HEK293T cells harbouring a luciferase detector fused to the MS2 coat protein (MS2CP). Upon the formation of FLAG–protein–RNA–MS2–MS2CP ternary complexes, RNA–protein interactions were measured by luciferase activity . eGFP (enhanced green fluorescent protein) was used as a negative control and PABPC3 (a polyadenylated RBP) was used to control for RNA expression. Xist(C) -MS2 vector was used alongside C1QTNF1AS1 –MS2 as a positive control for RNA–protein interactions. RLU are relative light unit; N = 4. ( K ) Protein expression levels were estimated from horseradish peroxidase ELISA of FLAG-tagged proteins in the same experiment as in panel (J). Error bars in all panels are shown as mean ± S.E.M.; scale bar: 5 μm; N = number of cells analysed. An unpaired t -test with Welch’s correction was applied in panels (C), (G), and (I). Unpaired t -test was used in panel (F). * <0.05, **<0.01, and ****<0.0001.

Article Snippet: C1QTNF1–AS1 insert was cloned upstream of the MS2 stem-loop sequence of the 10xMS2 vector using a Gibson assembly cloning kit (E5510S, NEB) at 50°C for 15 min.

Techniques: Staining, Expressing, Negative Control, Luciferase, Liquid Chromatography with Mass Spectroscopy, Western Blot, Control, Activity Assay, RNA Expression, Plasmid Preparation, Positive Control, Enzyme-linked Immunosorbent Assay