Staphylococcus aureus Small RNAs Possess Dephospho-CoA 5′-Caps, but No CoAlation Marks

Löcherer, Christian; Bühler, Nadja; Lafrenz, Pascal; Jäschke, Andres

doi:10.3390/ncrna8040046

Cited by 3 publications

(4 citation statements)

References 52 publications

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“…It is worth mentioning that small RNAs were removed from the liver samples, originally aiming to eliminate thiol-containing tRNAs, which may have led to the omission of small dpCoA-RNAs. Nevertheless, small RNAs to cap with dpCoA were previously found in bacteria . To address this issue, hybridization methods for removing tRNAs can be explored in future studies. , Moreover, in the case of RNA fragmentation occurring in ligating tag RNA with maleimide group using copper-catalyzed reaction, copper-free click reactions may also find uses in future research.…”

Section: Resultsmentioning

confidence: 99%

“…Nevertheless, small RNAs to cap with dpCoA were previously found in bacteria. 40 To address this issue, hybridization methods for removing tRNAs can be explored in future studies. 41 , 42 Moreover, in the case of RNA fragmentation occurring in ligating tag RNA with maleimide group using copper-catalyzed reaction, copper-free click reactions may also find uses in future research.…”

Section: Resultsmentioning

confidence: 99%

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DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction

et al. 2023

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Recent discoveries of noncanonical RNA caps, such as nicotinamide adenine dinucleotide (NAD + ) and 3′-dephospho-coenzyme A (dpCoA), have expanded our knowledge of RNA caps. Although dpCoA has been known to cap RNAs in various species, the identities of its capped RNAs (dpCoA-RNAs) remained unknown. To fill this gap, we developed a method called dpCoA tagSeq, which utilized a thiol-reactive maleimide group to label dpCoA cap with a tag RNA serving as the 5′ barcode. The barcoded RNAs were isolated using a complementary DNA strand of the tag RNA prior to direct sequencing by nanopore technology. Our validation experiments with model RNAs showed that dpCoA-RNA was efficiently tagged and captured using this protocol. To confirm that the tagged RNAs are capped by dpCoA and no other thiolcontaining molecules, we used a pyrophosphatase NudC to degrade the dpCoA cap to adenosine monophosphate (AMP) moiety before performing the tagSeq protocol. We identified 44 genes that transcribe dpCoA-RNAs in mouse liver, demonstrating the method's effectiveness in identifying and characterizing the capped RNAs. This strategy provides a viable approach to identifying dpCoA-RNAs that allows for further functional investigations of the cap.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction

et al. 2023

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“…Because D2* and D7* RNAs were expressed from strong constitutive promoters, they may accumulate at high levels that aided competition with intracellular ATP and allowed for detectable capping. Furthermore, while the in vivo existence of CoA-RNA was first reported in 2009 [ 2 ], CoA-RNA in living cells have been reported recently [ 45 , 46 ]. In addition, NudC, LudL and a number of other Nudix hydrolases, such as Nudt 2,7,8,12,15,16 and 19, possess hydrolytic activity to decap CoA-RNA in vitro [ 47 , 48 ] and in vivo [ 10 ].…”

Section: Discussionmentioning

confidence: 99%

Post-transcriptional capping generates coenzyme A-linked RNA

Sapkota,

Lucas,

Faulkner

et al. 2023

RNA Biology

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NAD can be inserted co-transcriptionally via non-canonical initiation to form NAD-RNA. However, that mechanism is unlikely for CoA-linked RNAs due to low intracellular concentration of the required initiator nucleotide, 3’-dephospho-CoA (dpCoA). We report here that phosphopantetheine adenylyltransferase (PPAT), an enzyme of CoA biosynthetic pathway, accepts RNA transcripts as its acceptor substrate and transfers 4’-phosphopantetheine to yield CoA-RNA post-transcriptionally. Synthetic natural (RNAI) and small artificial RNAs were used to identify the features of RNA that are needed for it to serve as PPAT substrate. RNAs with 4–10 unpaired nucleotides at the 5’ terminus served as PPAT substrates, but RNAs having <4 unpaired nucleotides did not undergo capping. No capping was observed when the +1A was changed to G or when 5’ triphosphate was removed by RNA pyrophosphohydrolase (RppH), suggesting the enzyme recognizes pppA-RNA as an ATP analog. PPAT binding affinities were equivalent for transcripts with +1A, +1 G, or 5’OH (+1A), indicating that productive enzymatic recognition is driven more by local positioning effects than by overall binding affinity. Capping rates were independent of the number of unpaired nucleotides in the range of 4–10 nucleotides. Capping was strongly inhibited by ATP, reducing CoA-RNA production ~70% when equimolar ATP and substrate RNA were present. Dual bacterial expression of candidate RNAs with different 5’ structures followed by CoA-RNA CaptureSeq revealed 12-fold enrichment of the better PPAT substrate, consistent with in vivo CoA-capping of RNA transcripts by PPAT. These results suggest post-transcriptional RNA capping as a possible mechanism for the biogenesis of CoA-RNAs in bacteria.

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“…NAD-RNA in human cell lines also responds to the infection by the human immunodeficiency virus (HIV-1). Specific transcripts such as the snRNA U1 lose the NAD cap and generally affect the NAD-RNA status of the cell [ 102 ]. Given the role of CD38 in antiviral signalling and HIV-1 infection, it is interesting to imagine NAD-RNA as a link connecting the two, however more research is necessary to test the possibility [ 101 ].…”

Section: Non-canonical Capsmentioning

confidence: 99%