An official website of the United States government
Abstract Small RNAs play important roles during plant development by regulating transcript levels of target mRNAs, maintaining genome integrity, and reinforcing DNA methylation.Dicer-like 5(Dcl5) is proposed to be responsible for precise slicing in many monocots to generate diverse 24-nt phased, secondary small interfering RNAs (phasiRNAs), which are exceptionally abundant in meiotic anthers of diverse flowering plants. The importance and functions of these phasiRNAs remain unclear. Here, we characterized several mutants ofdcl5, including alleles generated by the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9system and a transposon-disrupted allele. We report thatdcl5mutants have few or no 24-nt phasiRNAs, develop short anthers with defective tapetal cells, and exhibit temperature-sensitive male fertility. We propose that DCL5 and 24-nt phasiRNAs are critical for fertility under growth regimes for optimal yield.
more »
« less
Pervasive misannotation of microexons that are evolutionarily conserved and crucial for gene function in plants
Abstract It is challenging to identify the smallest microexons (≤15-nt) due to their small size. Consequently, these microexons are often misannotated or missed entirely during genome annotation. Here, we develop a pipeline to accurately identify 2,398 small microexons in 10 diverse plant species using 990 RNA-seq datasets, and most of them have not been annotated in the reference genomes. Analysis reveals that microexons tend to have increased detained flanking introns that require post-transcriptional splicing after polyadenylation. Examination of 45 conserved microexon clusters demonstrates that microexons and associated gene structures can be traced back to the origin of land plants. Based on these clusters, we develop an algorithm to genome-wide model coding microexons in 132 plants and find that microexons provide a strong phylogenetic signal for plant organismal relationships. Microexon modeling reveals diverse evolutionary trajectories, involving microexon gain and loss and alternative splicing. Our work provides a comprehensive view of microexons in plants.
more »
« less
- Award ID(s):
- 1818082
- PAR ID:
- 10362550
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 13
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract BackgroundCas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants. ResultsTo improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis inE. coliand identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency inT0plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites. ConclusionsOur results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.more » « less
-
Abstract Single-cell RNA sequencing is increasingly used to investigate cross-species differences driven by gene expression and cell-type composition in plants. However, the frequent expansion of plant gene families due to whole-genome duplications makes identification of one-to-one orthologues difficult, complicating integration. Here we demonstrate that coexpression can be used to trim many-to-many orthology families down to identify one-to-one gene pairs with proxy expression profiles, improving the performance of traditional integration methods and reducing barriers to integration across a diverse array of plant species.more » « less
-
IntroductionGene expression is often controlled via cis-regulatory elements (CREs) that modulate the production of transcripts. For multi-gene genetic engineering and synthetic biology, precise control of transcription is crucial, both to insulate the transgenes from unwanted native regulation and to prevent readthrough or cross-regulation of transgenes within a multi-gene cassette. To prevent this activity, insulator-like elements, more properly referred to as transcriptional blockers, could be inserted to separate the transgenes so that they are independently regulated. However, only a few validated insulator-like elements are available for plants, and they tend to be larger than ideal. MethodsTo identify additional potential insulator-like sequences, we conducted a genome-wide analysis ofUtricularia gibba(humped bladderwort), one of the smallest known plant genomes, with genes that are naturally close together. The 10 best insulator-like candidates were evaluated in vivo for insulator-like activity. ResultsWe identified a total of 4,656 intergenic regions with expression profiles suggesting insulator-like activity. Comparisons of these regions across 45 other plant species (representing Monocots, Asterids, and Rosids) show low levels of syntenic conservation of these regions. Genome-wide analysis of unmethylated regions (UMRs) indicates ~87% of the targeted regions are unmethylated; however, interpretation of this is complicated becauseU. gibbahas remarkably low levels of methylation across the genome, so that large UMRs frequently extend over multiple genes and intergenic spaces. We also could not identify any conserved motifs among our selected intergenic regions or shared with existing insulator-like elements for plants. Despite this lack of conservation, however, testing of 10 selected intergenic regions for insulator-like activity found two elements on par with a previously published element (EXOB) while being significantly smaller. DiscussionGiven the small number of insulator-like elements currently available for plants, our results make a significant addition to available tools. The high hit rate (2 out of 10) also implies that more useful sequences are likely present in our selected intergenic regions; additional validation work will be required to identify which will be most useful for plant genetic engineering.more » « less
-
Summary Pre‐mRNA splicing is an essential step for the regulation of gene expression. In order to specifically capture splicing variants in plants for genome‐wide association studies (GWAS), we developed a software tool to quantify and visualise Variations of Splicing in Population (VaSP).VaSP can quantify splicing variants from short‐read RNA‐seq datasets and discover genotype‐specific splicing (GSS) events, which can be used to prioritise causal pre‐mRNA splicing events in GWAS. We applied our method to an RNA‐seq dataset with 328 samples from 82 genotypes from a rice diversity panel exposed to optimal and saline growing conditions.In total, 764 significant GSS events were identified in salt stress conditions. GSS events were used as markers for a GWAS with the shoot Na+accumulation, which identified six GSS events in five genes significantly associated with the shoot Na+content. Two of these genes,OsNUC1andOsRAD23emerged as top candidate genes with splice variants that exhibited significant divergence between the variants for shoot growth under salt stress conditions.VaSP is a versatile tool for alternative splicing analysis in plants and a powerful tool for prioritising candidate causal pre‐mRNA splicing and corresponding genomic variations in GWAS.more » « less
