EpiCentre TAPs out
After more than a year of backorder status, EpiCentre Technologies has decided to discontinue all Tobacco Acid Pyrophosphatase (TAP) products and kits containing them. Normally a company discontinuing a product is not major news, but TAP is an important enzyme for RNA biology work and EpiCentre was the only company making it in the world.
TAP has a fairly unique activity in that it can remove the 5′ Guanosine cap from mRNAs leaving a 5′ monophosphate. In protocols like 5′ RACE, this allows researchers to then use RNA ligase to attach an oligonucleotide to the 5′ end of decapped mRNAs for transcriptional start site determination.
Apparently, making a recombinant enzyme is not possible and one must purify it from tobacco plants via a difficult process… too difficult.
So what are the alternatives available?
RNA 5′ polyphosphatase as an alternative to TAP?
RNA 5′ polyphosphatase at first glance looks like a reasonable candidate to be a TAP replacement, however this enzyme removes gamma and beta phosphates from a 5′ triphosphorylated RNA, such as a primary transcript.
This enzyme has zero activity on the m7 GTP cap of an mRNA. Similarly bacterial RppH removes the 5′ triphosphate/pyrophosphate from bacterial transcripts (see Deana et al. 2008: doi:10.1038/nature06475).
But what about DCP2, the primary eukaryotic enzyme implicated in mRNA decapping?
According to published reports, DCP2 catalyzes the conversion of m7 GTP caps to m7 GDP and 5′ monophosphorylated mRNA (see van Dijk et al. 2002 doi:10.1093/emboj/cdf678). But isn’t that what TAP does?
Indeed researchers have used TAP and DCP2 interchangeably to render mRNAs sensitive to 5′ monophosphate-dependent exonuclease, for example (see Otsuka et al. 2009 doi:10.1128/MCB.01325-08).
A quick look suggests that no company is currently offering active DCP2. Recombinant Human DCP2 is available as an off-the-shelf protein shipped on dry ice, but biological activity has not been assessed. Also a variety of tools exist that would allow facilitated expression of DCP2 such as yeast DCP2 expression Adenoviruses and Gateway constructs for human DCP2. It remains to be determined if DCP2 is the answer to the world’s TAP needs. Yeast DCP2, for example, requires other P-body proteins for maximal activity (see Wang et al 2013 doi:10.1128/MCB.01583-12). These co-factors include DCP1, Edc1-3, Lsm1-7, Dhh1 and Pat1.
The surprising part is that DCP2 hypomorph mice have no apparent phenotype, probably because there are multiple decapping proteins in mammalian cells, including Nudt16 (see Song et al. 2010 doi: 10.1016/j.molcel.2010.10.010).
Another enzyme that has some potential is human NUDT16. Like DCP2, this protein is a member of the Nudix family of proteins and is able to remove caps from both mRNAs and snoRNAs (see Lu et al 2011 doi: 10.1007/s13238-011-1009-2). According to a recent report (Song et al. 2013 doi:10.1261/rna.037309.11), multiple Nudix family members have decapping activity, however NUDT16, NUDT12, and NUDT15 most potently convert m7GTP capped mRNAs to the m7GMP form. As with the case of DCP2, recombinant human proteins are commercially-available, but the enzymatic activity has not been determined and Nudix ORF expression constructs will facilitate recombinant protein production.
So just what enzyme RNA biologists will turn to now that TAP is unavailable remains to be determined. It will be interesting to watch this development.
My selection of reagents for RNA studies
You will find below my selection of the most popular catalog mRNAs, chemically synthesized mRNAs with any modication and long mRNAs is available.
5′-capped and poly(A) tailed mRNAs:
80+ 5′ methylcytidine, and pseudouridine-containing mRNAs
80+ methylcytidine and pseudouridine-containing mRNAs encoding for EGFP, mCherry, Factor IX, Cas9 and Cas9 Nickase…
Another alternative is to design custom oligos (with appropriate modifications and dyes) for optimal experimental designs. Online forms can now assist you when submitting your requests for custom oligo production.
What about you?
What are your favorite RNA research reagents? Leave your comments here!