Chez Charles/ tags/ pages tagged cap

RNA caps

Methods for enriching capped RNAs

(work in progress)

  • See the Wikipedia for CAGE methods (Cap Analysis Gene Expression).

  • Thiessen and coll., 1986 cloned the full-length cDNA by affinity purification with m7G antibodies, and RNAse A digestion of the incomplete cDNA/RNA duplexes. They cite Schneider 1986 for the method, but I could not find this reference.

  • A method similar to oligo-capping was reported by Sekine and Kato in 1993.

  • Oligo-capping (Maruyama et al., 1994): dephosphorylate, uncover functional phosphate with decapping enzyme, and ligate RNA adaptor with T4 RNA ligase.

(wip: template-switching: find original publication. Described in deeper details in the template switching tag page.

(wip: Fromont-racine et al.)

  • CAPture (Edery, Chu, Sonenberg and Pelletier, 1995): a protein fusion of mouse eIF4E (for cap binding) and protein A (for IgG binding) is used to capture capped RNA/cDNA duplexes on a sepharose IgG column. Completion of first-strand synthesis is ascertained by RNAse A digestion.

  • CapSelect (Schmidt et al., 1999): Add a poly-A tail to the first-strand cDNA, ligate a double-stranded adapter ending with a single-strand (T)TTTGGG overhang, and prime second-strand synthesis.

  • Cap-jumping (Efimov et al., 2001): oxidize diols, covalently bind a 3′-amine oligonucleotide. The reverse transcriptase reads through the cap structure and chemical bond, and integrates the reverse complement of the oligonucleotide to the first-strand cDNA.

  • Choi and Hagedorn (2003) improved the CAPture method by using a K119A mutant of eIF4E. It is fused to GST instead of protein A.

  • Clepet et al., 2004 modified oligo-capping, to use T4 DNA ligase and a double-stranded adapter with NNNNNN overhang instead of T4 RNA ligase and a single-stranded linker.

  • RNA captor (Clepet, 2011): a modified oligo-capping using T4 RNA ligase 2 and a double-stranded adaptor. The shorter oligonucleotide in the adaptor is blocked by an amine group on its 3′ end, and protrudes with 3 random bases (NNN) on its 5′ end.

  • Kwak et al., 2013 modified oligo-capping to create Pro-cap, a method for nuclear run-on analysis at single-nucleotide resulution.

  • CapSMART (Machida and Lin, 2014): non-capped RNAs are dephosphorylated, rephoshporylated and a blocking linker containing isoguanosines and isocytidines (so that it can not be reverse-transcribed) is ligated to them. A classical SMART protocol follows. cDNAs are amplified with biotinylated forward primers, so that the 5′ end can be recovered after mechanical fragmentation.

  • capCLIP (Jensen and coll., 2021): eIF4E is flagged by gene editing, mRNA crosslinked to eIF4E and the whole is immunoprecipitated. This circumvents the problem of access to good eIF4E antibodies for immunoprecipitation.

Atypical caps (work in progress)

Cap physiology (work in progress)

Some RNAs have more than half of their molecules non-capped. Increasing expression of eIF4E increases their capped proportion to similar levels as other genes. Culjkovic-Kraljacic and coll, 2020

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Hepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide

Sherwood AV, Rivera-Rangel LR, Ryberg LA, Larsen HS, Anker KM, Costa R, Vågbø CB, Jakljevič E, Pham LV, Fernandez-Antunez C, Indrisiunaite G, Podolska-Charlery A, Grothen JER, Langvad NW, Fossat N, Offersgaard A, Al-Chaer A, Nielsen L, Kuśnierczyk A, Sølund C, Weis N, Gottwein JM, Holmbeck K, Bottaro S, Ramirez S, Bukh J, Scheel TKH, Vinther J. Hepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.

Nature. 2023 Jul;619(7971):811-818. doi:10.1038/s41586-023-06301-3.

Hepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.

Arabidopsis thaliana Nudix pyrophosphohydrolase 23 (AtNUDX23) decapping followed with adapter ligation results in libraries enriched in HCV(+) and HCV(-) RNAs. 75% of the HCV RNAs are capped and most of the rest is 5′ triphosphate. FAD capping was not found in “bovine viral diarrhea virus (BVDV) and the ortho flavivirus tick-borne encephalitis virus (TBEV), both from the Flaviviridae family, as well as chikungunya virus (CHIKV), an alphavirus of the Togaviridae family“. “no replication of JFH1-SGR-Feo was observed upon riboflavin depletion, whereas replication was rescued by supplementing exogenous riboflavin or FAD” “inclusion of FAD as the priming nucleotide resulted in efficient de novo initiation of replication and production of an initiation product consisting of FAD linked to CMP” “Incubation of 5′-ppp RNA with recombinant NS5B and flavin mononucleotide (FMN) did not result in 5′-FAD capping”

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capCLIP: a new tool to probe translational control in human cells through capture and identification of the eIF4E-mRNA interactome.

Jensen KB, Dredge BK, Toubia J, Jin X, Iadevaia V, Goodall GJ, Proud CG.

Nucleic Acids Res. 2021 Oct 11;49(18):e105. doi:10.1093/nar/gkab604

capCLIP: a new tool to probe translational control in human cells through capture and identification of the eIF4E-mRNA interactome.

eIF4E flagged via CRISPR/Cas9 gene editing. mRNAs were crosslinked to eIF4E and immunoprecipitated with anti-flag antibodies. First-strand cDNAs primed via a linker ligated in 3′. This method (capCLIP) was used to study the effect of rapamycin treatment and eIF4E phosphorylation.

The eukaryotic translation initiation factor eIF4E elevates steady-state m7G capping of coding and noncoding transcripts.

Culjkovic-Kraljacic B, Skrabanek L, Revuelta MV, Gasiorek J, Cowling VH, Cerchietti L, Borden KLB.

Proc Natl Acad Sci U S A. 2020 Oct 27;117(43):26773-26783. doi:10.1073/pnas.2002360117

The eukaryotic translation initiation factor eIF4E elevates steady-state m7G capping of coding and noncoding transcripts.

Some RNAs have more than half of their molecules non-capped. Increasing expression of eIF4E increases their capped proportion to similar levels as other genes. Enzymatic (cap quantification; CapQ) and quantitative cap immunoprecipitation (CapIP) methods. Uses the mouse IgG2aκ anti-7-methylguanosine (m7G)-Cap antibody of MBL (RN016M). The maker's site notes that “This antibody (Clone 150-15) reacts with 5′-terminal 7-methylguanosine (m7G) cap structure of RNA and partially cross-reacts with m7G within RNA”. Overexpression of eIF4E-Flag in RNA immunoprecipitation experiments increased enrichment of RNA export targets such as RNMT or RNGTT, but export-deficient S53A-eIF4E-Flag mutants did not. Overexpression also shifted RNA translation to heavier polysomal fractions and elevated m7G levels in the nuclear and cytoplasmic compartments. This elevation is reduced by RNMT knockdown and stronger on specific target, for instance from the capping machinery and the Myc pathway. A CapQ assay (sequencing an oligo-capping library and a control PNK library) confirmed the observations made by immunoprecipitation. In control conditions, the capping rate of the targets of eIF4E capping are lower than those of the non-targets.

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Dinucleoside polyphosphates act as 5'-RNA caps in bacteria.

Hudeček O, Benoni R, Reyes-Gutierrez PE, Culka M, Šanderová H, Hubálek M, Rulíšek L, Cvačka J, Krásný L, Cahová H.

Nat Commun. 2020 Feb 26;11(1):1052. doi:10.1038/s41467-020-14896-8

Dinucleoside polyphosphates act as 5'-RNA caps in bacteria.

T7 RNAP and E. coli RNAP efficiently initiate transcription with N(p)nN cap analogs at mM concentrations. LC-MS analysis of sRNAs digested with Nuclease P1 detects N(p)nN caps. Existence of the internal polyphosphate chain demonstrated by the fact that the N(p)nN caps are not fragmented by ionization, like GppppG and unlike pppGpG. m7Gp4Gm and m6Ap3A were further identified using synthetic standards. The RppH and ApaH enzymes cleaved caps, leaving 5′-p or 5′-ppp ends respectively. Cap methylation protects from RppH cleavage but not from ApaH.

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Transcription of the 5'-terminal cap nucleotide by RNA-dependent DNA polymerase: possible involvement in retroviral reverse transcription.

Volloch VZ, Schweitzer B, Rits S.

DNA Cell Biol. 1995 Dec;14(12):991-6 doi:10.1089/dna.1995.14.991

Transcription of the 5'-terminal cap nucleotide by RNA-dependent DNA polymerase: possible involvement in retroviral reverse transcription.

Utilises a 5′ hairpin in the beta-globin mRNA to design a cDNA extension assay where the reverse-transcribed cap creates a terminal mismatch that inhibits extension. De-capping the RNA removes the inhibition. 40 mM Tris 8.3, 40 mM KCl, 5 mM MgCl2, 2 mM DTT. AMV, 45°C

Determination of the site of first strand transfer during Moloney murine leukemia virus reverse transcription and identification of strand transfer-associated reverse transcriptase errors.

EMBO J. 1997 Feb 17;16(4):856-65 doi:10.1093/emboj/16.4.856

Kulpa D, Topping R, Telesnitsky A.

Determination of the site of first strand transfer during Moloney murine leukemia virus reverse transcription and identification of strand transfer-associated reverse transcriptase errors.

“The results presented here support the model that +1 substitutions arise during reverse transcription via non‐templated addition followed by mismatch extension upon strand transfer. Another possibility we considered was that +1G could potentially be templated by the 7‐methyl‐G cap present on mRNAs and viral genomic RNAs (Coffin, 1996). Avian myeloblastosis virus reverse transcriptase can add a cap‐complementary C residue during cDNA synthesis on mRNA in vitro, but not when the RNA has been de‐capped (Volloch et al., 1995). However, studies with purified enzymes and model primer–templates have demonstrated that +1G can arise at template switch junctions in the absence of a 7‐methyl‐G cap (Peliska and Benkovic, 1994), and our detection of +1C mutants demonstrates that not all additions to −ssDNA could be cap‐templated.”

Cloning of the human cDNA for the U1 RNA-associated 70K protein.

Theissen H, Etzerodt M, Reuter R, Schneider C, Lottspeich F, Argos P, Lührmann R, Philipson L.

EMBO J. 1986 Dec 1;5(12):3209-17.

Cloning of the human cDNA for the U1 RNA-associated 70K protein.

Clones the full-length cDNA by affinity purification with m7G antibodies, and RNAse A digestion of the incomplete cDNA/RNA duplexes. Cites Schneider 1986 for the method, but could not find this reference.

mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation.

Rydzik AM, Warminski M, Sikorski PJ, Baranowski MR, Walczak S, Kowalska J, Zuberek J, Lukaszewicz M, Nowak E, W Claridge TD, Darzynkiewicz E, Nowotny M, Jemielity J.

Nucleic Acids Res. 2017 Jun 28. doi:10.1093/nar/gkx569

mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation.

Different conformation in eIF4E. Resists to hydrolysis by DcpS and Dcp2.

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An efficient strategy to isolate full-length cDNAs based on an mRNA cap retention procedure (CAPture).

Mol Cell Biol. 1995 Jun;15(6):3363-71.

Edery I, Chu LL, Sonenberg N, Pelletier J.

An efficient strategy to isolate full-length cDNAs based on an mRNA cap retention procedure (CAPture).

An eIF-4E fusion protein is used to bind the caps of RNA/DNA duplexes. RNase A cuts single stranded RNAs, thus separating the complexes from the cDNA if they are not full length.

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Insight into the mechanism of nonenzymatic RNA primer extension from the structure of an RNA-GpppG complex.

Proc Natl Acad Sci U S A. 2017 Jul 18;114(29):7659-7664. doi:10.1073/pnas.1704006114

Zhang W, Tam CP, Walton T, Fahrenbach AC, Birrane G, Szostak JW.

Insight into the mechanism of nonenzymatic RNA primer extension from the structure of an RNA-GpppG complex.

« The formation of two Watson–Crick base pairs leads to a much higher affinity of GpppG than GMP for a CC template (Kd of ∼0.2 mM vs. ∼20 mM, respectively). » « The tight binding of GpppG to a CC template suggests that GpppG might be an ideal primer for the initiation of template copying by primer extension. The resulting RNAs would begin with a 5ʹ cap-like GpppG moiety, suggesting a potential evolutionary origin for the eukaryotic mRNA 5ʹ-cap structure. »

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Structural basis for m7G recognition and 2'-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I.

Devarkar SC, Wang C, Miller MT, Ramanathan A, Jiang F, Khan AG, Patel SS, Marcotrigiano J.

Proc Natl Acad Sci U S A. 2016 Jan 5. pii: 201515152 doi:10.1073/pnas.1515152113

Structural basis for m7G recognition and 2'-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I.

5′ppp and Cap-0, but not Cap-1 dsRNA bind RIG-I and activate signalling.

Four methods of preparing mRNA 5' end libraries using the illumina sequencing platform.

Machida RJ, Lin YY.

PLoS One. 2014 Jul 8;9(7):e101812. doi: 10.1371/journal.pone.0101812

Four methods of preparing mRNA 5' end libraries using the illumina sequencing platform.

Capture of 5′ ends via biotinylated forward PCR primers. CapSMART: ligation of modified oligonucleotides that block RT on 5′-phoshphorylated RNAs (after kinase treatment). The signal is not much different from standard SMART.

CLP1 links tRNA metabolism to progressive motor-neuron loss.

Hanada T, Weitzer S, Mair B, Bernreuther C, Wainger BJ, Ichida J, Hanada R, Orthofer M, Cronin SJ, Komnenovic V, Minis A, Sato F, Mimata H, Yoshimura A, Tamir I, Rainer J, Kofler R, Yaron A, Eggan KC, Woolf CJ, Glatzel M, Herbst R, Martinez J, Penninger JM

Nature. 2013 Mar 28;495(7442):474-80. doi: 10.1038/nature11923

CLP1 links tRNA metabolism to progressive motor-neuron loss.

5ʹ-triphosphate tRNA ends (with leader), found in TAP-treated sRNA libraries.

Box H/ACA snoRNAs are preferred substrates for the trimethylguanosine synthase in the divergent unicellular eukaryote Trichomonas vaginalis.

Simoes-Barbosa A, Chakrabarti K, Pearson M, Benarroch D, Shuman S, Johnson PJ.

RNA. 2012 Sep;18(9):1656-65. doi: 10.1261/rna.034249.112.

Box H/ACA snoRNAs are preferred substrates for the trimethylguanosine synthase in the divergent unicellular eukaryote Trichomonas vaginalis.

In Trichomonas vaginalis, snRNAs are not capped, but non-intronic snoRNAs are (with three methyls).

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CapSeq and CIP-TAP identify Pol II start sites and reveal capped small RNAs as C. elegans piRNA precursors.

Gu W, Lee HC, Chaves D, Youngman EM, Pazour GJ, Conte D Jr, Mello CC.

Cell. 2012 Dec 21;151(7):1488-500. doi: 10.1016/j.cell.2012.11.023

CapSeq and CIP-TAP identify Pol II start sites and reveal capped small RNAs as C. elegans piRNA precursors.

Capped short RNAs identified by oligo-capping at the promoter of C. elegans transcripts.

CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs.

Nucleic Acids Res. 1999 Nov 1;27(21):e31.

Schmidt WM, Mueller MW.

CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs.

Extra cytosine are more frequently added in presence of the 5′ cap. Increasing Mg2+ to 6 mM increases the frequency of addition of more than 1 C, but only moderately. Instead, when adding BSA in the reaction, supplementing it with 1 or 2 mM Mn2+ after 1h, 3-4 extra dC residues are added to most of the first strand cDNAs. Standard reaction: 0.75 μM oligo dT (or 0.25 μM gene-specific RT primer); 50 mM Tris-HCl (pH 8.3); 75 mM KCl; 3 mM MgCl2; 5 mM DTT; dNTPs 1mM each; 0.1 mg BSA; 20 U RNAse inhibitor (Roche), 200 U SuperScript II; 1h at 42 °C.

Detection of the 5'-cap structure of messenger RNAs with the use of the cap-jumping approach.

Efimov VA, Chakhmakhcheva OG, Archdeacon J, Fernandez JM, Fedorkin ON, Dorokhov YL, Atabekov JG.Efimov VA1, Chakhmakhcheva OG, Archdeacon J, Fernandez JM, Fedorkin ON, Dorokhov YL, Atabekov JG.

Nucleic Acids Res. 2001 Nov 15;29(22):4751-9.

Detection of the 5'-cap structure of messenger RNAs with the use of the cap-jumping approach.

Binds an oligonucleotide to the cap using its free diol group, and integrates its sequence to the full-length cDNA using template-switching.

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The human L1 promoter: variable transcription initiation sites and a major impact of upstream flanking sequence on promoter activity.

Lavie L, Maldener E, Brouha B, Meese EU, Mayer J.

Genome Res. 2004 Nov;14(11):2253-60 doi:10.1101/gr.2745804

The human L1 promoter: variable transcription initiation sites and a major impact of upstream flanking sequence on promoter activity.

The presence of an extra G in pseudogenes suggests that many reverse-transcriptases can reverse transcribe the cap.

Determination of messenger RNA 5'-ends by reverse transcription of the cap structure.

Nucleic Acids Res. 1993 Jul 25;21(15):3597-8

Hirzmann J, Luo D, Hahnen J, Hobom G.

Determination of messenger RNA 5'-ends by reverse transcription of the cap structure.

The mRNA cap can template an extra C in the first strand cDNA. “50% of the cDNAs of capped mRNA molecules had an extra G”

“poly(A)+ RNA was obtained using Hybond-mAP paper (Amersham). 1 µg of poly(A)+ -RNA in 9.65 µL of water was heated to 60°C for 3 min, cooled on ice, added to 4 µL of 5 × RT buffer (1 × RT buffer is 40 mM Tris-HCl, pH 8.3, 5 mM MgCl2, 40 mM KCl, 2 mM DTE), 3 µL dNTPs (10 mM each), 0.25 µL (10 units) of RNasin (Promega), 2.5 µL of XhoI-(dT)17 oligonucleotide primer (300 pmol, 0.5 µg/µL). and 0.6 µL (16 units) of avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer). The reaction was continued at 42 °C for 2 hours, and excess XhoI-(dT)17 primer and substrates were removed by glass powder purification followed by ethanol precipitation.”