An atlas of plant full-length RNA reveals tissue-specific and monocots-dicots conserved regulation of poly(A) tail length.

Poly(A) tail is a hallmark of eukaryotic messenger RNA and its length plays an essential role in regulating mRNA metabolism. However, a comprehensive resource for plant poly(A) tail length has yet to be established. Here, we applied a poly(A)-enrichment-free, nanopore-based method to profile full-length RNA with poly(A) tail information in plants. Our atlas contains over 120 million polyadenylated mRNA molecules from seven different tissues of Arabidopsis, as well as the shoot tissue of maize, soybean and rice. In most tissues, the size of plant poly(A) tails shows peaks at approximately 20 and 45 nucleotides, while the poly(A) tails in pollen exhibit a distinct pattern with strong peaks centred at 55 and 80 nucleotides. Moreover, poly(A) tail length is regulated in a gene-specific manner-mRNAs with short half-lives in general have long poly(A) tails, while mRNAs with long half-lives are featured with relatively short poly(A) tails that peak at ~45 nucleotides. Across species, poly(A) tails in the nucleus are almost twice as long as in the cytoplasm. Our comprehensive dataset lays the groundwork for future functional and evolutionary studies on poly(A) tail length regulation in plants.

Landscape of transcription termination in Arabidopsis revealed by single-molecule nascent RNA sequencing.

BACKGROUND: The dynamic process of transcription termination produces transient RNA intermediates that are difficult to distinguish from each other via short-read sequencing methods. RESULTS: Here, we use single-molecule nascent RNA sequencing to characterize the various forms of transient RNAs during termination at genome-wide scale in wildtype Arabidopsis and in atxrn3, fpa, and met1 mutants. Our data reveal a wide range of termination windows among genes, ranging from ~ 50 nt to over 1000 nt. We also observe efficient termination before downstream tRNA genes, suggesting that chromatin structure around the promoter region of tRNA genes may block pol II elongation. 5' Cleaved readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. Consistent with previous reports, we also observe long chimeric transcripts with cryptic splicing in fpa mutant; but loss of CG DNA methylation has no obvious impact on termination in the met1 mutant. CONCLUSIONS: Our method is applicable to establish a comprehensive termination landscape in a broad range of species.

FLEP-seq: simultaneous detection of RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale by single-molecule nascent RNA sequencing.

Elongation, splicing and polyadenylation are fundamental steps of transcription, and studying their coordination requires simultaneous monitoring of these dynamic processes on one transcript. We recently developed a full-length nascent RNA sequencing method in the model plant Arabidopsis that simultaneously detects RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale. This method allows calculation of the kinetics of cotranscriptional splicing and detects polyadenylated transcripts with unspliced introns retained at specific positions posttranscriptionally. Here we describe a detailed protocol for this method called FLEP-seq (full-length elongating and polyadenylated RNA sequencing) that is applicable to plants. Library production requires as little as one nanogram of nascent RNA (after rRNA/tRNA removal), and either Nanopore or PacBio platforms can be used for sequencing. We also provide a complete bioinformatic pipeline from raw data processing to downstream analysis. The minimum time required for FLEP-seq, including RNA extraction and library preparation, is 36 h. The subsequent long-read sequencing and initial data analysis ranges between 31 and 40 h, depending on the sequencing platform.

Post-transcriptional splicing of nascent RNA contributes to widespread intron retention in plants.

In eukaryotes, genes are transcribed by RNA polymerase-II (Pol-II) and introns are removed by the spliceosome largely cotranscriptionally1-3; analysis using long-read sequencing revealed that splicing occurs immediately after Pol-II passes introns in yeast4,5. Here, we developed a Nanopore-based method to profile chromatin-bound RNA that enables the simultaneous detection of splicing status, Pol-II position and polyadenylation at the genome-wide scale in Arabidopsis. We found that more than half of the introns remain unspliced after Pol-II transcribes 1 kb past the 3' splice site, which is much slower than the rate of splicing reported in yeast4,5. Many of the full-length chromatin-bound RNA molecules are polyadenylated, yet still contain unspliced introns at specific positions. These introns are nearly absent in the cytoplasm and are resistant to nonsense-mediated decay, suggesting that they are post-transcriptionally spliced before the transcripts are released into the cytoplasm; we therefore termed these introns post-transcriptionally spliced introns (pts introns). Analysis of around 6,500 public RNA-sequencing libraries found that the splicing of pts introns requires the function of splicing-related proteins such as PRMT5 and SKIP, and is also influenced by various environmental signals. The majority of the intron retention events in Arabidopsis are at pts introns, suggesting that chromatin-tethered post-transcriptional splicing is a major contributor to the widespread intron retention that is observed in plants, and could be a mechanism to produce fully spliced functional mRNAs for rapid response.

Functional Analysis of RNA Interference-Related Soybean Pod Borer (Lepidoptera) Genes Based on Transcriptome Sequences.

RNA interference (RNAi) is useful for controlling pests of agriculturally important crops. The soybean pod borer (SPB) is the most important soybean pest in Northeastern Asia. In an earlier study, we confirmed that the SPB could be controlled via transgenic plant-mediated RNAi. Here, the SPB transcriptome was sequenced to identify RNAi-related genes, and also to establish an RNAi-of-RNAi assay system for evaluating genes involved in the SPB systemic RNAi response. The core RNAi genes, as well as genes potentially involved in double-stranded RNA (dsRNA) uptake were identified based on SPB transcriptome sequences. A phylogenetic analysis and the characterization of these core components as well as dsRNA uptake related genes revealed that they contain conserved domains essential for the RNAi pathway. The results of the RNAi-of-RNAi assay involving Laccase 2 (a critical cuticle pigmentation gene) as a marker showed that genes encoding the sid-like (Sil1), scavenger receptor class C (Src), and scavenger receptor class B (Srb3 and Srb4) proteins of the endocytic pathway were required for SPB cellular uptake of dsRNA. The SPB response was inferred to contain three functional small RNA pathways (i.e., miRNA, siRNA, and piRNA pathways). Additionally, the SPB systemic RNA response may rely on systemic RNA interference deficient transmembrane channel-mediated and receptor-mediated endocytic pathways. The results presented herein may be useful for developing RNAi-mediated methods to control SPB infestations in soybean.