Full-length annotation with multistrategy RNA-seq uncovers transcriptional regulation of lncRNAs in cotton

Abstract

Long noncoding RNAs (lncRNAs) are crucial factors during plant development and environmental responses. To build an accurate atlas of lncRNAs in the diploid cotton Gossypium arboreum, we combined Isoform-sequencing, strand-specific RNA-seq (ssRNA-seq), and cap analysis gene expression (CAGE-seq) with PolyA-seq and compiled a pipeline named plant full-length lncRNA to integrate multi-strategy RNA-seq data. In total, 9,240 lncRNAs from 21 tissue samples were identified. 4,405 and 4,805 lncRNA transcripts were supported by CAGE-seq and PolyA-seq, respectively, among which 6.7% and 7.2% had multiple transcription start sites (TSSs) and transcription termination sites (TTSs). We revealed that alternative usage of TSS and TTS of lncRNAs occurs pervasively during plant growth. Besides, we uncovered that many lncRNAs act in cis to regulate adjacent protein-coding genes (PCGs). It was especially interesting to observe 64 cases wherein the lncRNAs were involved in the TSS alternative usage of PCGs. We identified lncRNAs that are coexpressed with ovule- and fiber development–associated PCGs, or linked to GWAS single-nucleotide polymorphisms. We mapped the genome-wide binding sites of two lncRNAs with chromatin isolation by RNA purification sequencing. We also validated the transcriptional regulatory role of lnc-Ga13g0352 via virus-induced gene suppression assay, indicating that this lncRNA might act as a dual-functional regulator that either activates or inhibits the transcription of target genes.

The Conservation of Long Intergenic Non-Coding RNAs and Their Response to Verticillium dahliae Infection in Cotton

“…More investigations involving diverse cotton accessions and pathogens (also different strains of the same pathogen) are, thus, required to have an in-depth understanding of the landscape of the cotton lncRNAs responding to disease infection and the function of lncRNAs in the interaction between cotton and pathogens. Ideally, such study could combine multiple strategies, such as strand-specific RNA-seq, SMRT full-length RNA-seq, and cap analysis of gene expression, in the integration of lncRNAs, so it can simultaneously investigate the alternative splicing, transcription start, termination site, expression level, and change of lncRNAs [ 47 ].…”

Assembly and annotation of the Gossypium barbadense L. ‘Pima-S6’ genome raise questions about the chromosome structure and gene content of Gossypium barbadense genomes

“…This conservative filtering approach used by us, but also by others, for example Chen and colleagues (2020), could potentially lead to discarding loci without protein-coding regions. Long non-coding RNA loci are an example of loci without protein-coding potential, and in fact 8,514 long noncoding RNAs have been identified in G. hirsutum [ 33 ] and 8,113 in G. arboreum [ 34 ] to cite two reports. In addition, 1,965 Pima-S6 and 2,163 Pima 3–79 RagTag genes whose proteins lack homology to know proteins had evidence of expression in an RNA-seq dataset obtained from roots and leaves, and 5,272 Pima 3–79 RagTag genes had evidence of expression in a more comprehensive RNA-seq dataset from BioProject PRJNA266265.…”

“…We identified 62,760 phylogenetic hierarchical orthogroups across all 11 assemblies. Diploid assemblies were represented in 37,090 (G. herbaceum; A 1 ), 34 4a). This indicates that, while sharing over 90% of their orthogroups, there are still proteins from one genome that have no ortholog in the other genome.…”

Opportunities and Challenges of Predictive Approaches for the Non-coding RNA in Plants

“…Furthermore, 5′-RNA ligase mediated amplification of cDNA ends (5′RLM-RACE) assays are directly used for verifying the predicted binding sites (Cui et al, 2020 ). The detection of novel lncRNAs can be completed by multiple RNA-Seq strategies, such as isoform-sequencing, strand-specific RNA-Seq (ssRNA-Seq), cap analysis gene expression with polyA-Seq technologies (CAGE-Seq) (Zheng et al, 2021 ). Although lncRNAs are considered as a part of ncRNAs, some of them still remain a weak ability of translating small peptides.…”