To cap it all off, again: dynamic capping and recapping of coding and non-coding RNAs to control transcript fate and biological activity

Figures & data

Figure 1. Chemical structures of different forms of 5' RNA caps. The methylation or NAD modifications are highlighted by blue circles. Only cap-types described in the text are shown. Thus, this is not an exhaustive list of cap types

Figure 2. Biochemical pathways for m⁷G RNA capping. Capping is completed by two enzymes in mammals, RNGTT carries out the removal of the 5' phosphate (Step 1) and subsequent addition of the guanosine via the unique 5'-5' pyrophosphate linage (Step 2). This G-capped RNA is now a substrate for the RNMT, which uses SAM as the methyl donor to generate m⁷G capped RNA and S-adenosyl homocysteine (SAH). Additional methylation events depicted in Figure 1 are carried out by other enzymes and not described here

Table 1. Methods to study capping on a per RNA basis

Methods	Description	Types of RNAs identified	References
CapIP (normal)	Using an anti-m^7G cap antibody to identify m^7G capped RNAs	RNAs that have m^7G or with less affinity TMG caps. Addition of m^7GpppG competition can demonstrate m^7G specificity	Cowling and Cole, 2007; Cole and Cowling, 2009; Bochnig et al, 1987; Moteki and Price, 2002 [10,48,73,74]
CapIP (quantitative)	RNAs and amount of anti-m^7G antibody are carefully titrated to quantitatively immunoprecipitate transcripts with parallel analysis of supernatants to monitor depletion of RNAs to assess percent capping	RNAs that have m^7G or with less affinity TMG caps. Addition of m^7GpppG competition can demonstrate m^7G specificity	CulljkovicKraljacic et al, 2020 [9]
CapTure/eIF4E pulldown	Pulldown with the cap-binding protein eIF4E. Purified eIF4E protein is immobilized on a solid support (such as GST-eIF4E on glutathione beads), and purified RNAs incubated with the resin (and with a negative control such as GST). In CapTure, an eIF4E mutant that binds the m^7G cap more tightly (the eIF4E K119A mutant) is used for more efficient capture of transcripts. Human eIF4E can bind to TMG and G-capped transcripts albeit with much lower affinity. Competition assays with m^7GpppG is important for validating interactions are m^7G dependent.	m^7G RNAs, with potential for weak binding to TMG and/or G-capped RNAs	Choi and Hagedorn, 2003 [45]
Cap Quantitation (CapQ)	Enzymatic method using alkaline phosphatase (AP) leading to OH-groups on the 5‘ end of uncapped RNAs followed by tobacco acid pyrophosphatase (TAP) leaving a 5‘ monophosphate which is then ligated to a biotinylated oligonucleotide to isolate capped RNAs for analysis. In parallel, total RNA levels are monitored using TAP, AP and PNK. In this way percentages of capped RNAs can be directly calculated on a per RNA basis or coupled with RNA-Seq	RNAs with a 5’-5’ pyrophosphate bond (e.g. m^7G, G, NAD, TMG)	Culljkovic-Kraljacic et al, 2020 [9]
Oligocapping	AP removes 5‘ phosphatases of non-capped RNAs followed by TAP to remove pyrophosphate leaving a 5‘ monophosphate used for ligation. To identify RNAs, the ligated oligonucleotide provides the 5' primer and the 3‘ primer is used to quantitate the gene of interest by RT-qPCR	RNAs with a 5‘ pyrophosphate bond (m^7G, G, TMG, NAD, other).	Maruyama and Sugano 1994 [50]
CapSMART	Switching Mechanism at the 5' end of RNA Transcripts (SMART): AP to leave OH on uncapped RNAs, with PNK to add single 5‘ phosphate on previously uncapped RNAs which are then ligated to STOP oligonucleotides to block template switching. Then capped RNAs are captured	RNAs with a 5‘ pyrophosphate bond (m^7G, G, TMG, NAD, other)	Machida and Lin, 2014 [49]
NonCapSMART	RNAs were treated with AP, followed by TAP leaving 5‘ phosphates on the RNAs that were capped and 5‘ OH on the RNAs that were not capped. STOP oligonucleotides are ligated onto the previously capped RNAs to block template switching while uncapped RNAs captured using template switching.	Uncapped RNAs (RNAs without a 5'pyrophosphate)	Machida and Lin 2014 [49]
XRN sensitivity assays	XRN is a 5' to 3' nuclease that will degrade RNAs that are not capped	Uncapped RNAs (those without a 5'-5' pyrophosphate linkage: m^7G, G, TMG, NAD, etc)	Mukherjee et al, 2012 [14]
RLM 5'RACE	Uncapped RNAs have 5' phosphate that is ligated to an adaptor that is subsequently hybridized to complementary biotinylated adaptor which allowed isolation of only not capped RNAs	Uncapped RNAs	Jiao et al., 2008 [7]
Cap Trapper	Biotinylation of diol residues (5' cap structure and 3' terminal nucleotide)	RNAs with a 5‘-5'pyrophosphate (e.g. m^7G, G, TMG, NAD)	Carninci et al 1996 [51]
Cap analysis of gene expression (CAGE)	Original CAGE used CapTrapper for finding 5' ends, and olido-dT primers for synthesizing cDNAs [52]. Later incarnations of this method with template switching are no longer cap-specific for the 5' end.	RNAs with a 5‘-5'pyrophosphate (e.g. m^7G, G, TMG, NAD)	Shiraki et al 2003 [52]; Kodzius et al, 2006 [54]

Figure 3. Model for the role of dynamic capping in RNA activity. Only nuclear RNA export and translation are shown for simplicity, but cap dynamics could impact many other facets of RNA metabolism such as splicing and polyadenylation. Dynamic capping has a major impact on transcription and has been described as a transcriptional checkpoint. NPC indicates nuclear pore complex used for RNAs to export the nucleus. Red balls indicate m⁷G caps

Cowling VH, Cole MD. The Myc transactivation domain promotes global phosphorylation of the RNA polymerase II carboxy-terminal domain independently of direct DNA binding. Mol Cell Biol. 2007;27:2059–2073.

 PubMed Web of Science ®Google Scholar

Cole MD, Cowling VH. Specific regulation of mRNA cap methylation by the c-Myc and E2F1 transcription factors. Oncogene. 2009;28(9):1169–1175.

 PubMed Web of Science ®Google Scholar

Bochnig P, Reuter R, Bringmann P, et al. A monoclonal antibody against 2,2,7-trimethylguanosine that reacts with intact, class U, small nuclear ribonucleoproteins as well as with 7-methylguanosine-capped RNAs. Eur J Biochem. 1987;168(2):461–467.

 PubMedGoogle Scholar

Moteki S, Price D. Functional coupling of capping and transcription of mRNA. Mol Cell. 2002;10(3):599–609.

 PubMed Web of Science ®Google Scholar

Culjkovic-Kraljacic B, Skrabanek L, Revuelta MV, et al. The eukaryotic translation initiation factor eIF4E elevates steady-state m 7 G capping of coding and noncoding transcripts. Proc Natl Acad Sci U S A. 2020;117(43):26773–26783.

 PubMed Web of Science ®Google Scholar

Choi YH, Hagedorn CH. Purifying mRNAs with a high-affinity eIF4E mutant identifies the short 3’ poly(A) end phenotype. Proc Natl Acad Sci U S A. 2003;100(12):7033–7038.

 PubMed Web of Science ®Google Scholar

Kazuo M, Sumio S. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene. 1994;138(1–2):171–174.

 PubMed Web of Science ®Google Scholar

Machida RJ, Lin -Y-Y, Oudejans C. Four Methods of Preparing mRNA 5' End Libraries Using the Illumina Sequencing Platform. PLoS One. 2014;9(7):e101812.

 PubMed Web of Science ®Google Scholar

Mukherjee C, Patil D, Kennedy B, et al. Identification of cytoplasmic capping targets reveals a role for cap homeostasis in translation and mRNA stability. Cell Rep. 2012;2(3):674–684.

 PubMed Web of Science ®Google Scholar

Jiao Y, Riechmann JL, Meyerowitz EM. Transcriptome-Wide Analysis of Uncapped mRNAs in Arabidopsis Reveals Regulation of mRNA Degradation. Plant Cell. 2008;20(10):2571–2585.

 PubMed Web of Science ®Google Scholar

Carninci P, Kvam C, Kitamura A, et al. High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics. 1996;37(3):327–336.

 PubMed Web of Science ®Google Scholar

Shiraki T, Kondo S, Katayama S, et al. Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage. Proc Natl Acad Sci U S A. 2003;100(26):15776–15781.

 PubMed Web of Science ®Google Scholar

Kodzius R, Kojima M, Nishiyori H, et al. CAGE: cap analysis of gene expression. Nat Methods. 2006;3(3):211–222.

 PubMed Web of Science ®Google Scholar