Maize experienced substantial metabolic divergence from its wild ancestor, teosinte.

Alternative splicing (AS) enhances transcriptome diversity and plays important roles in regulating plant processes. Although widespread natural variation in AS has been observed in plants, how AS is regulated and contribute to phenotypic variation is poorly understood. Here, we report a population-level transcriptome assembly and genome-wide association study to identify splicing quantitative trait loci (sQTLs) in developing maize (Zea mays) kernels from 368 inbred lines. We detected 19,554 unique sQTLs for 6570 genes. Most sQTLs showed small isoform usage changes without involving major isoform switching between genotypes. The sQTL-affected isoforms tend to display distinct protein functions. We demonstrate that nonsense-mediated mRNA decay, microRNA-mediated regulation, and small interfering peptide-mediated peptide interference are frequently involved in sQTL regulation. The natural variation in AS and overall mRNA level appears to be independently regulated with different cis-sequences preferentially used. We identified 214 putative trans-acting splicing regulators, among which ZmGRP1, encoding an hnRNP-like glycine-rich RNA binding protein, regulates the largest trans-cluster. Knockout of ZmGRP1 by CRISPR/Cas9 altered splicing of numerous downstream genes. We found that 739 sQTLs colocalized with previous marker-trait associations, most of which occurred without changes in overall mRNA level. Our findings uncover the importance of AS in diversifying gene function and regulating phenotypic variation.

Significance: The m ⁷ G cap is the canonical RNA cap in eukaryotes, but other noncanonical RNA caps exist, including the NAD ⁺ cap. NAD captureSeq has been widely used to profile NAD ⁺ -capped RNAs (NAD-RNAs) in prokaryotes and eukaryotes. However, NAD captureSeq reacts at a low level with m ⁷ G-RNAs and introduces copper ions that cause RNA fragmentation, resulting in reduced sensitivity and loss of full-length sequence information. To address these issues, we developed the copper-free SPAAC-NAD-seq, which utilizes the strain-promoted azide–alkyne cycloaddition reaction to capture NAD-RNAs followed by high-throughput sequencing. Compared to NAD captureSeq, SPAAC-NAD-seq is more sensitive and retains full-length sequence information. This optimized technique provides a useful tool to profile NAD-RNAs in prokaryotes and, when combined with m ⁷ G-RNA depletion, in eukaryotes.

Background: Small RNAs (sRNAs) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) serve as core players in gene silencing at transcriptional and post-transcriptional levels in plants, but their subcellular localization has not yet been well studied, thus limiting our mechanistic understanding of sRNA action.Results: We investigate the cytoplasmic partitioning of sRNAs and their targets globally in maize (Zea mays, inbred line “B73”) and rice (Oryza sativa, cv. “Nipponbare”) by high-throughput sequencing of polysome-associated sRNAs and 3′ cleavage fragments, and find that both miRNAs and a subset of 21-nucleotide (nt)/22-nt siRNAs are enriched on membrane-bound polysomes (MBPs) relative to total polysomes (TPs) across different tissues. Most of the siRNAs are generated from transposable elements (TEs), and retrotransposons positively contributed to MBP overaccumulation of 22-nt TE-derived siRNAs (TE-siRNAs) as opposed to DNA transposons. Widespread occurrence of miRNA-mediated target cleavage is observed on MBPs, and a large proportion of these cleavage events are MBP-unique. Reproductive21PHAS(21-nt phasiRNA-generating) and24PHAS(24-nt phasiRNA-generating) precursors, which were commonly considered as noncoding RNAs, are bound by polysomes, and high-frequency cleavage of21PHASprecursors by miR2118 and24PHASprecursors by miR2275 is further detected on MBPs. Reproductive 21-nt phasiRNAs are enriched on MBPs as opposed to TPs, whereas 24-nt phasiRNAs are nearly completely devoid of polysome occupancy.Conclusions: MBP overaccumulation is a conserved pattern for cytoplasmic partitioning of sRNAs, and endoplasmic reticulum (ER)-bound ribosomes function as an independent regulatory layer for miRNA-induced gene silencing and reproductive phasiRNA biosynthesis in maize and rice.

Significance: Some RNAs in both prokaryotes and eukaryotes were recently found to contain the NAD ⁺ cap, indicating a novel mechanism in gene regulation through noncanonical RNA capping. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry has been used to label NAD ⁺ -capped RNAs (NAD-RNAs) for their identification. However, copper-caused RNA fragmentation/degradation interferes with the analysis. We developed the NAD tagSeq II method for transcriptome-wide NAD-RNA analysis based on copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry. This method was used to compare NAD-RNA and total transcriptome profiles in Escherichia coli . Our study reveals genome-wide alterations in E. coli RNA NAD ⁺ capping in different growth phases and indicates that NAD-RNAs could be the primary form of transcripts from some genes under certain environments.