Validation strategies for antibodies targeting modified ribonucleotides

Abstract

Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of m6A on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including m1A, m5C, m7G, 2′-OMe, and Ψ were detected. However, since their abundances are low and tools used for their corroboration are often not well characterized, their physiological relevance remains largely elusive. Antibodies targeting modified nucleotides are often used but have limitations such as low affinity or specificity. Moreover, they are not always well characterized and due to the low abundance of the modification, particularly on mRNAs, generated data sets might resemble noise rather than specific modification patterns. Therefore, it is critical that the affinity and specificity is rigorously tested using complementary approaches. Here, we provide an experimental toolbox that allows for testing antibody performance prior to their use.

Defining the RBPome of primary T helper cells to elucidate higher-order Roquin-mediated mRNA regulation

“…To investigate the temporal impact on Icos expression by Roquin-1/2, Regnase-1, m6A, and miRNA regulation we analyzed inducible, CD4-specific inactivation of Roquin-1 together with Roquin-2 ( Rc3h1-2 ) or of Regnase-1 ( Zc3h12a ). We also analyzed the inactivation of Wtap, an essential component of the m6A methyltransferase complex 47 , or of Dgcr8, which is required for pre-miRNA biogenesis 48 . To this end, we performed tamoxifen gavage on mice expressing a Cre-ERT2 knockin allele from the CD4 locus 49 together with the floxed, Roquin-1/2 paralogs encoding, Rc3h1 and Rc3h2 alleles (Fig.…”

“…For in vitro culture analysis, deletion of Roquin-1/2 ( Rc3h1 fl/fl ;Rc3h2 fl/fl ; Cd4-Cre-ERT2 ) 62 , Regnase-1 ( Zc3h12a fl/fl ; Cd4-Cre-ERT2 ) 63 , Wtap ( Wtap fl/fl ; Cd4-Cre-ERT2 ) 47 , and Dgcr8 ( Dgcr8 fl/fl ; Cd4-Cre-ERT2 ) 48 encoding alleles in Cd4-Cre-ERT2 mice was induced in vivo by oral transfer of 5 mg tamoxifen (Sigma) in corn oil. Two doses of tamoxifen each day were given on 2 consecutive days (total of 20 mg tamoxifen per mouse).…”

Comprehensive analysis of translation from overexpressed circular RNAs reveals pervasive translation from linear transcripts

“…Following antibodies were used for western blot: mouse-anti-HA (monoclonal, 1:1000, Covance, clone HA.11), rabbit-anti-GAPDH (polyclonal, clone FL-335, 1:500, Santa Cruz Biotechnology), rabbit-anti-GAPDH (monoclonal, clone 14C10, Cell Signaling mAb #2118, 1:1000), mouse-anti-beta-Actin (monoclonal, clone AC15, 1:10 000, Abcam), Rat-anti-m 6 A (monoclonal, clone 9B7 ( 36 )), mouse-anti-Flag (monoclonal, clone M2, 1:1000, Sigma-Aldrich), mouse-anti-beta-Tubulin (polyclonal, 1:1000, Abcam), rabbit-anti-METTL3 (polyclonal, 15073-1-AP, 1:1000, proteintech). Goat-anti-rabbit/mouse/rat IRDye 680RD or goat-anti-rabbit/mouse/rat/guinea pig IRDye 800CW antibodies (Li-Cor Biosciences) were used as secondary antibodies.…”

“…Total RNA was isolated from C643 WT and C643 M3-KO cells by TRIzol. 20 μg of anti-m 6 A antibody (Clone 9B7 ( 36 )) was pre-bound to protein G magnetic beads (Pierce-Thermo Scientific) in 500 μl IP buffer (25 mM Tris pH 7.5, 150 mM KCl, 0.5% NP-40, 2 mM EDTA) for 1 h at room temperature or at least 2 h at 4°C. Afterward, the beads were washed two time with IP buffer before adding 10 μg of total RNA in a final volume of 500 μl of IP buffer.…”

Advances in mRNA 5-methylcytosine modifications: Detection, effectors, biological functions, and clinical relevance

“…70 The limitations of m 5 C-RIP-seq include its high dependence on specific antibodies and the risk of nonspecific binding to RNA. 93 To address these issues, Weichmann et al 96 developed an experimental toolbox capable of verifying antibody performance, while improving the accuracy and credibility of m 5 C-RIP-seq data. However, m 5 C-RIP-seq cannot accurately identify the location of single nucleoside sites, as the length of the sequence reads it produces is generally 100-150 nt and is hindered by the RNA secondary structure.…”