Extensive 5′-surveillance guards against non-canonical NAD-caps of nuclear mRNAs in yeast

Abstract

The ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) acts as a non-canonical cap structure on prokaryotic and eukaryotic ribonucleic acids. Here we find that in budding yeast, NAD-RNAs are abundant (>1400 species), short (<170 nt), and mostly correspond to mRNA 5′-ends. The modification percentage of transcripts is low (<5%). NAD incorporation occurs mainly during transcription initiation by RNA polymerase II, which uses distinct promoters with a YAAG core motif for this purpose. Most NAD-RNAs are 3′-truncated. At least three decapping enzymes, Rai1, Dxo1, and Npy1, guard against NAD-RNA at different cellular locations, targeting overlapping transcript populations. NAD-mRNAs are not translatable in vitro. Our work indicates that in budding yeast, most of the NAD incorporation into RNA seems to be disadvantageous to the cell, which has evolved a diverse surveillance machinery to prematurely terminate, decap and reject NAD-RNAs.

Use of NAD tagSeq II to identify growth phase-dependent alterations in E. coli RNA NAD + capping

“…SPAAC tagging resulted in much longer reads and a much higher efficiency in identifying NAD-RNAs than CuAAC tagging. In budding yeast, it was recently reported that most NAD-mRNAs identified using NAD captureSeq were 5′ fragments from thousands of genes, which might be incidentally produced ( 21 ). NAD-mRNAs identified from E. coli using NAD captureSeq also showed enrichment in 5′ fragments ( 7 ).…”

“…A large majority of NAD-RNAs of the high-confidence NAD-RNAs identified in this study are from protein-coding genes, but some genes coding for small regulatory RNAs produced a high proportion of their transcripts as NAD-RNAs. NAD-RNAs from Bacillus subtilis , yeast, mammals, and Arabidopsis were also found to be mainly from protein-coding genes ( 10 , 11 , 13 , 14 , 34 ). It has been reported that in vitro-synthesized NAD-mRNAs were not translated in a yeast extract or in mammalian cells ( 11 , 34 ).…”

“…NAD-RNAs from Bacillus subtilis , yeast, mammals, and Arabidopsis were also found to be mainly from protein-coding genes ( 10 , 11 , 13 , 14 , 34 ). It has been reported that in vitro-synthesized NAD-mRNAs were not translated in a yeast extract or in mammalian cells ( 11 , 34 ). However, in Arabidopsis , NAD-mRNAs were found enriched in the actively translating polysomal fraction, suggesting that NAD-mRNAs could be translated ( 13 ).…”

“…The method was later used for the identification of over 37 NAD-RNAs from yeast and a wider range of NAD-RNAs from mammalian cells and Arabidopsis plants ( 11 – 13 ). A recent study using NAD captureSeq identified thousands of NAD-RNAs in yeast, mostly from 5′ regions of protein-coding genes ( 21 ).…”

Synthesis of 5′-Thiamine-Capped RNA

“…An adaptation of the aforementioned protocol also led to the detection of NAD-capped RNA sequences in Gram-positive bacteria, such as Bacillus subtilis and Staphylococcus aureus [ 20 , 21 ], as well as in eukaryotes, like Saccharomyces cerevisiae , Arabidopsis thaliana and Homo sapiens [ 22 , 23 , 24 , 25 ]. In addition, the existence of enzymes that specifically target 5′-NAD-, dephospho-CoA- or FAD-capped RNAs, such as the nudix hydrolase NudC and proteins of the DXO family, supports the idea of regulated capping and decapping processes [ 24 , 26 , 27 ].…”