Identification of a Selective Polymerase Enables Detection of N⁶-Methyladenosine in RNA

Abstract

N6-methyladenosine (m6A) is the most abundant mRNA modification, and has important links to human health. While recent studies have successfully identified thousands of mammalian RNA transcripts containing the modification, it is extremely difficult to identify the exact location of any specific m6A. Here we have identified a polymerase with reverse transcriptase activity (from Thermus thermophilus) that is selective by up to 18-fold for incorporation of thymidine opposite unmodified A over m6A. We show that the enzyme can be used to locate and quantify m6A in synthetic RNAs by analysis of pausing bands, and have used the enzyme in tandem with a nonselective polymerase to locate the presence and position of m6A in high-abundance cellular RNAs. By this approach we demonstrate that the long-undetermined position of m6A in mammalian 28S rRNA is nucleotide 4190.

Dynamics of the human and viral m6A RNA methylomes during HIV-1 infection of T cells

“…As shown in Fig 3c, Tth pol failed to extend the probes for both A7877 and A7883 sites, indicating that these A residues are methylated in vivo. In control experiments using ribosomal RNA, Tth pol extended oliogs for A4189 (unmethylated site) and did not extend to A4190 (methylated site), which are consistent with previously reported results 37 . Altogether, these results show that the both A in the RRE hairpin are methylated in vivo.…”

“…In order to define which adenosine in the RRE hairpin structure is methylated in vivo, we performed primer extension experiments using AMV RT and Tth DNA polymerase 37 . A7877 and A7883 modifications were analysed by sequence-specific complementary ssDNA probes and enzymatic extensions.…”

“…These experiments were performed as previously described 37 with the following modifications. ssDNA oligos were 5′ radio-labelled with 32 P. For 28S 4189 and 28S 4190 (negative and positive controls) probing 10 µg total RNA from HIV-1 infected MT4 cells was used.…”

Entwicklung einer DNA‐Polymerase für die direkte m6A‐Sequenzierung

“…Kürz-lich wurde festgestellt, dass manche RT-aktive DNA-Polymerasen m 6 Av on unmodifiziertem Au nterscheiden kçnnen. [18] Wirhaben diese Eigenschaft nun weiterentwickelt und eine DNA-Polymerase erzeugt, die signifikant erhçhte Fehlerraten gegenüber m 6 A( und nicht unmodifiziertem A) aufweist. Das Enzym wurde ausgehend von einer thermostabilen KlenTaq-DNA-Polymerase mit RT-Aktivität( KlenTaqL459M S515R I638F M747K, im Folgenden als RT-KTQ bezeichnet) evolviert.…”

“…[5] Zur Erstellung solcher Tr ainingsdatensätze kçnnten Sequenzdaten von modifizierten RNA-Oligonukleotiden und/oder von validierten m 6 A-Stellen in rRNA, mRNAu nd lncRNAg enutzt werden. [18,25] Abbildung 3. Analyse einer bekannten m 6 A-Stelle in E.-coli-tRNA-Val durch RT-KTQ G668Y Y671A.…”