Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

Abstract

21Understanding genome organization and gene regulation requires insight into RNA transcription, 22 processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a 23 wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA 24 methylation (m 6 A). Here we show that m 6 A can be mapped in full-length mRNAs transcriptome-wide 25 and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, 26 poly(A) site choice and poly(A) tail length. Loss of m 6 A from 3' untranslated regions is associated 27 with decreased relative transcript abundance and defective RNA 3′ end formation. A functional 28 consequence of disrupted m 6 A is a lengthening of the circadian period. We conclude that nanopore 29 direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length 30 single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying 31 this approach to less well-studied species could transform our understanding of what their genomes 32 encode. 33 34 misidentification of 3′ ends through internal priming 3 , spurious antisense and splicing events 46 produced by RT template switching 4,5 , and the inability to detect all base modifications in the 47 copying process 6 . The fragmentation of RNA prior to short-read sequencing makes it difficult to 48 interpret the combination of authentic RNA processing events and remains an unsolved problem 7 . 49We investigated whether long-read direct RNA sequencing (DRS) with nanopores 8 could 50 reveal the complexity of Arabidopsis mRNA processing and modifications. In nanopore DRS, the 51 protein pore (nanopore) sits in a membrane through which an electrical current is passed, and intact 52 RNA is fed through the nanopore by a motor protein 8 . Each RNA sequence within the nanopore 53 (5 bases) can be identified by the magnitude of signal it produces. Arabidopsis is a pathfinder model 54 in plant biology, and its genome annotation strongly influences the annotation and our 55 understanding of what other plant genomes encode. We applied nanopore DRS and Illumina RNAseq 56 to wild-type Arabidopsis (Col-0) and mutants defective in m 6 A 9 and exosome-mediated RNA decay 10 . 57We reveal m 6 A and combinations of RNA processing events (alternative patterns of 5′ capped 58 transcription start sites, splicing, 3′ polyadenylation and poly(A) tail length) in full-length Arabidopsis 59 mRNAs transcriptome-wide. 60 61 Results 62Nanopore DRS detects long, complex mRNAs and short, structured non-coding RNAs 63We purified poly (A)+ RNA from four biological replicates of 14-day-old Arabidopsis Col-0 seedlings. 64We incorporated synthetic External RNA Controls Consortium (ERCC) RNA Spike-In mixes into all 65 replicates 11,12 and carried out nanopore DRS. Illumina RNAseq was performed in parallel on similar 66 material. Using Guppy base-calling (Oxford Nanopore Technologies) and minimap2 alignment 67 software 13 , we identified around 1 mi...

Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing

“…It has been observed previously that about 12 nt are missing at the 5’-end of the sequenced RNAs. This observation can be explained by a lack of control of the RNA translocation speed after the motor protein falls off the 5’ end of the RNA (see Supplementary Figure 6a) 56,57 . Promoter analysis confirmed the presence of well-known sequence motifs of bacterial and archaeal promoters 27,31,58 .…”

Topologies of N6‐adenosine methylation (m6A) in land plant mitochondria and their putative effects on organellar gene expression

“…Global RNA‐seq analyses of the model plant A. thaliana indicated the presence of m 6 A modifications within many nuclear‐encoded mRNA transcripts (Zhong et al , ; Bodi et al , ; Shen et al , ; Růžička et al , ; Wei et al , ; Parker et al , ). These analyses further suggested that the organellar (i.e.…”

“…As in animals, the N 6 -methylation of adenosine residues in nuclear-encoded mRNAs in land plants is positioned predominantly towards the 3 0 -end of the transcript (Bodi et al, 2012;Dominissini et al, 2012;Chen and Liu, 2014;Luo et al, 2014;Ke et al, 2015;Wan et al, 2015;Burgess et al, 2016;Li et al, 2018;Scutenaire et al, 2018;Wei et al, 2018). This was also supported by Nanopore direct RNA sequencing of Arabidopsis plants (Parker et al, 2019). The significance of m 6 A is apparent in mutant plants with reduced levels of m 6 A that show embryogenesis defects and altered growth and developmental phenotypes (Zhong et al, 2008;Bodi et al, 2012;Wan et al, 2015;Shen et al, 2016;R u zi cka et al, 2017;Arribas-Hern andez et al, 2018;Chen et al, 2018;Li et al, 2018;Wei et al, 2018).…”

Occurrence and Functions of m6A and Other Covalent Modifications in Plant mRNA

“…(3) Differential RNA cleavage at modified versus unmodified nucleotides is used to identify modified sites based on the percentage of sequence read-throughs and stops at each nucleotide (Birkedal et al, 2015;Garcia-Campos et al, 2019;Zhang et al, 2019d). In addition to those three types of methods, direct RNA sequencing by Oxford Nanopore Technology (Garalde et al, 2018) has recently been used successfully to map N 6methyladenosine (m 6 A) sites based on systematic base-calling errors at modified sites (Liu et al, 2019;Parker et al, 2019;Yang et al, 2019). In general, methods in categories 2 and 3 produce modification maps with nucleotide resolution, while methods in category 1 produce intervals containing a modification.…”

“…In plants, there are now reports on mapping of three distinct covalent nucleotide modifications in the bodies of mRNA: m 6 A (Li et al, 2014c;Wan et al, 2015;Shen et al, 2016;Duan et al, 2017;Anderson et al, 2018;Miao et al, 2019;Parker et al, 2019), 5-methylcytidine (m 5 C; Cui et al, 2017;David et al, 2017;Yang et al, 2019), and pseudouridine (C; Sun et al, 2019). Other types of mRNA modifications, including C-U editing of mitochondrial and plastidial mRNAs (Shikanai, 2006), alternative 59-caps (Kiledjian, 2018;Wang et al, 2019), and untemplated addition of nucleotides to mRNA 39-ends (De Almeida et al, 2018), will not be covered here.…”