环状RNA的产生、研究方法及功能

随着高通量测序技术的发展,环状RNA(circular RNAs,circRNAs)逐渐成为非编码RNA研究领域的热点。本文系统综述了环状RNA侧翼内含子自身互补配对驱动、RNA结合蛋白驱动以及套索驱动这3种环状RNA形成模型,并从高通量文库构建、生物信息学鉴别和常用的实验验证等3个方面对环状RNA的研究方法进行了介绍。同时,本文详细归纳了环状RNA作为microRNA(miRNA)或蛋白的海绵体、调控宿主基因的选择性剪接和表达、翻译成多肽等多种功能。最后通过系统综述植物环状RNA的特征及最新研究进展,为环状RNA在植物学中的进一步研究提供了新的视野。

Chromosomal-level genome and metabolome analyses of highly heterozygous allohexaploid Dendrocalamus brandisii elucidate shoot quality and developmental characteristics

Dendrocalamus brandisii(Munro)Kurz is a sympodial bamboo species with inimitable taste and flavorful shoots.Its rapid growth and use as high-quality material make this bamboo species highly valued for both food processing and wood applications.However,genome information for D.brandisii is lacking,primarily due to its polyploidy and large genome size.Here,we assembled a high-quality genome for hexaploid D.brandisii,which comprises 70chromosomes with a total size of 2,756 Mb,using long-read Hi Fi sequencing.Furthermore,we accurately separated the genome into its three constituent subgenomes.We used Oxford Nanopore Technologies long reads to construct a transcriptomic dataset covering 15 tissues for gene annotation to complement our genome assembly,revealing differential gene expression and post-transcriptional regulation.By integrating metabolome analysis,we unveiled that well-balanced lignin formation,as well as abundant flavonoid and fructose contents,contribute to the superior quality of D.brandisii shoots.Integrating genomic,transcriptomic,and metabolomic datasets provided a solid foundation for enhancing bamboo shoot quality and developing efficient gene-editing techniques.This study should facilitate research on D.brandisii and enhance its use as a food source and wood material by providing crucial genomic resources.

The Features and Regulation of Co-transcriptional Splicing in Arabidopsis

Precursor mRNA(pre-mRNA)splicing is essential for gene expression in most eukaryotic organisms.Previous studies from mammals,Drosophila,and yeast show that the majority of splicing events occurs co-transcriptionally.In plants,however,the features of co-transcriptional splicing(CTS)and its regulation still remain largely unknown.Here,we used chromatin-bound RNA sequencing to study CTS in Arabidopsis thaliana.We found that CTS is widespread in Arabidopsis seedlings,with a large proportion of alternative splicing events determined co-transcriptionally.CTS efficiency correlated with gene expression level,the chromatin landscape and,most surprisingly,the number of introns and exons of individual genes,but is independent of gene length.In combination with enhanced crosslinking and immunoprecipitation sequencing analysis,we further showed that the hnRNP-like proteins RZ-1B and RZ-1C promote efficient CTS globally through direct binding,frequently to exonic sequences.Notably,this general effect of RZ-1B/1C on splicing promotion is mainly observed at the chromatin level,not at the mRNA level.RZ-1C promotes CTS of multiple-exon genes in association with its binding to regions both proximal and distal to the regulated introns.We propose that RZ-1C promotes efficient CTS of genes with multiple exons through cooperative interactions with many exons,introns,and splicing factors.Our work thus reveals important features of CTS in plants and provides methodologies for the investigation of CTS and RNA-binding proteins in plants.

Development of an efficient expression system with large cargo capacity for interrogation of gene function in bamboo based on bamboo mosaic virus

Bamboo is one of the fastest growing plants among monocotyledonous species and is grown extensively in subtropical regions.Although bamboo has high economic value and produces much biomass quickly,gene functional research is hindered by the low efficiency of genetic transformation in this species.We therefore explored the potential of a bamboo mosaic virus(BaMV)-mediated expression system to investigate genotype-phenotype associations.We determined that the sites between the triple gene block proteins(TGBps)and the coat protein(CP)of BaMV are the most efficient insertion sites for the expression of exogenous genes in both monopodial and sympodial bamboo species.Moreover,we validated this system by individually overexpressing the two endogenous genes ACE1 and DEC1,which resulted in the promotion and suppression of intemode elongation,respectively.In particular,this system was able to drive the expression of three 2A-linked betalain biosynthesis genes(more than 4 kb in length)to produce betalain,indicating that it has high cargo capacity and may provide the prerequisite basis for the development of a DNA-free bamboo genome editing platform in the future.Since BaMV can infect multiple bamboo species,we anticipate that the system described in this study will greatly contribute to gene function research and further promote the molecular breeding of bamboo.

Multi-omics of Circular RNAs and Their Responses to Hormones in Moso Bamboo(Phyllostachys edulis)

Circular RNAs(circRNAs)are endogenous non-coding RNAs with covalently closed structures,which have important functions in plants.However,their biogenesis,degradation,and function upon treatment with gibberellins(GAs)and auxins(1-naphthaleneacetic acid,NAA)remain unknown.Here,we systematically identified and characterized the expression patterns,evolutionary conservation,genomic features,and internal structures of circRNAs using RNase R-treated libraries from moso bamboo(Phyllostachys edulis)seedlings.Moreover,we investigated the biogenesis of circRNAs dependent on both cis-and trans-regulation.We explored the function of circRNAs,including their roles in regulating microRNA(miRNA)-related genes and modulating the alternative splicing of their linear counterparts.Importantly,we developed a customized degradome sequencing approach to detect miRNA-mediated cleavage of circRNAs.Finally,we presented a comprehensive view of the participation of circRNAs in the regulation of hormone metabolism upon treatment of bamboo seedlings with GA and NAA.Collectively,our study provides insights into the biogenesis,function,and miRNA-mediated degradation of circRNAs in moso bamboo.

Allele-aware chromosome-scale assembly of the allopolyploid genome of hexaploid Ma bamboo(Dendrocalamus latiflorus Munro)

Dendrocalamus latiflorus Munro is a woody clumping bamboo with rapid shoot growth.Both genetic transformation and clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)gene editing techniques are available for D.latiflorus,enabling reverse genetic approaches.Thus,D.latiflorus has the potential to be a model bamboo species.However,the genome sequence of D.latiflorus has remained unreported due to its polyploidy and large genome size.Here,we sequenced the D.latiflorus genome and assembled it into three allele-aware subgenomes(AABBCC),representingthe largest genome of a major bamboo species.We assembled 70 allelic chromosomes(2,737 Mb)for hexaploid D.latiflorus using both singlemolecule sequencing from the Pacific Biosciences(Pac Bio)Sequel platform and chromosome conformation capture sequencing(Hi-C).Repetitive sequences comprised 52.65%of the D.latiflorus genome.We annotated 135231 protein-coding genes in the genome based on transcriptomes from eight different tissues.Transcriptome sequencing using RNA-Seq and Pac Bio singlemolecule real-time long-read isoform sequencing revealed highly differential alternative splicing(AS)between non-abortive and abortive shoots,suggesting that AS regulates the abortion rate of bamboo shoots.This high-quality hexaploid genome and comprehensive strand-specific transcriptome datasets for this Poaceae family member will pave the way for bamboo research using D.latiflorus as a model species.

Phytophthora Effectors Modulate Genome-wide Alternative Splicing of Host mRNAs to Reprogram Plant Immunity

Alternative splicing(AS)of pre-mRNAs increases transcriptome and proteome diversity,regulates gene expression through multiple mechanisms,and plays important roles in plant development and stress responses.However,the prevalence of genome-wide plant AS changes during infection and the mechanisms by which pathogens modulate AS remain poorly understood.Here,we examined the global AS changes in tomato leaves infected with Phytophthora infestans,the infamous Irish famine pathogen.We show that more than 2000 genes exhibiting significant changes in AS are not differentially expressed,indicating that AS is a distinct layer of transcriptome reprogramming during plant-pathogen interactions.Furthermore,our results show that P.infestans subverts host immunity by repressing the AS of positive regulators of plant immunity and promoting the AS of susceptibility factors.To study the underlying mechanism,we established a luminescence-based AS reporter system in Nicotiana benthamiana to screen pathogen effectors modulating plant AS.We identified nine splicing regulatory effectors(SREs)from 87 P.infestans effectors.Further studies revealed that SRE3 physically binds U1-70K to manipulate the plant AS machinery and subsequently modulates AS-mediated plant immunity.Our study not only unveils genome-wide plant AS reprogramming during infection but also establishes a novel AS screening tool to identify SREs from a wide range of plant pathogens,providing opportunities to understand the splicing regulatory mechanisms through which pathogens subvert plant immunity.

Profiling of circular RNA N^(6)-methyladenosine in moso bamboo(Phyllostachys edulis)using nanopore-based direct RNA sequencing

N6-methyladenosine(m^(6)A)is a prevalent modification in messenger RNAs and circular RNAs that play important roles in regulating various aspects of RNA metabolism.However,the occurrence of the m^(6)A modification in plant circular RNAs has not been reported.A widely used method to identify m^(6)A modifications relies on m^(6)A-specific antibodies followed by next-generation sequencing of precipitated RNAs(MeRIP-Seq).However,one limitation of MeRIP-Seq is that it does not provide the precise location of m^(6)A at single-nucleotide resolution.Although more recent sequencing techniques such as Nanopore-based direct RNA sequencing(DRS)can overcome such limitations,the technology does not allow sequencing of circular RNAs,as these molecules lack a poly(A)tail.Here,we developed a novel method to detect the precise location of m^(6)A modifications in circular RNAs using Nanopore DRS.We first enriched our samples for circular RNAs,which we then fragmented and sequenced on the Nanopore platform with a customized protocol.Using this method,we identified 470 unique circular RNAs from DRS reads based on the back-spliced junction region.Among exonic circular RNAs,about 10%contained m^(6)A sites,which mainly occurred around acceptor and donor splice sites.This study demonstrates the utility of our antibody-independent method in identifying total and methylated circular RNAs using Nanopore DRS.This method has the additional advantage of providing the exact location of m^(6)A sites at single-base resolution in circular RNAs or linear transcripts from non-coding RNA without poly(A)tails.

Degradome sequencing reveals endogenous small RNA targets in rice (Oryza sativa L. ssp. indica)

MicroRNAs(miRNAs)and small interfering RNAs(siRNAs)regulate gene expression in eukaryotes.Plant miRNAs modulate their targets mainly via messenger RNA(mRNA)cleavage.Small RNA(sRNA)targets have been extensively investigated in Arabidopsis using computational prediction,experimental validation,and degradome sequencing.However,small RNA targets are largely unknown in rice(Oryza sativa).Here,we report global identification of small RNA targets using high throughput degradome sequencing in the rice indica cultivar 93-11(Oryza sativa L.ssp.indica).One hundred and seventy-seven transcripts targeted by a total of 87 unique miRNAs were identified.Of targets for the conserved miRNAs between Arabidopsis and rice,transcription factors comprise around 70%(58 in 82),indicating that these miRNAs act as masters of gene regulatory nodes in rice.In contrast,non-conserved miRNAs targeted diverse genes which provide more complex regulatory networks.In addition,5 AUXIN RESPONSE FACTORs(ARFs)cleaved by the TAS3 derived ta-siRNAs were also detected.A total of 40 sRNA targets were further validated via RNA ligasemediated 5′rapid amplification of cDNA ends(RLM 5′-RACE).Our degradome results present a detailed sRNAtarget interaction atlas,which provides a guide for the study of the roles of sRNAs and their targets in rice.

Classification of Rice Yield Using UAV-Based Hyperspectral Imagery and Lodging Feature

High-yield rice cultivation is an effective way to address the increasing food demand worldwide.Correct classification of high-yield rice is a key step of breeding.However,manual measurements within breeding programs are time consuming and have high cost and low throughput,which limit the application in large-scale field phenotyping.In this study,we developed an accurate large-scale approach and presented the potential usage of hyperspectral data for rice yield measurement using the XGBoost algorithm to speed up the rice breeding process for many breeders.In total,13 japonica rice lines in regional trials in northern China were divided into different categories according to the manual measurement of yield.Using an Unmanned Aerial Vehicle(UAV)platform equipped with a hyperspectral camera to capture images over multiple time series,a rice yield classification model based on the XGBoost algorithm was proposed.Four comparison experiments were carried out through the intraline test and the interline test considering lodging characteristics at the midmature stage or not.The result revealed that the degree of lodging in the midmature stage was an important feature affecting the classification accuracy of rice.Thus,we developed a low-cost,high-throughput phenotyping and nondestructive method by combining UAV-based hyperspectral measurements and machine learning for estimation of rice yield to improve rice breeding efficiency.

Genome-wide profiling of circular RNAs,alternative splicing,and R-loops in stem-differentiating xylem of Populus trichocarpa

Circular RNAs(circRNAs)are a recently dis-covered type of non‐coding RNA derived from pre‐mRNAs.R‐loops consist of a DNA:RNA hybrid andthe associated single‐stranded DNA.InArabi-dopsis thaliana,circRNA:DNA R‐loops regulatealternative splicing(AS)ofSEPALLATA3(SEP3).However,the occurrence and functions ofcircRNAs and R‐loops inPopulus trichocarpaarelargely unexplored.Here,we performed circRNA‐enriched sequencing in the stem‐differentiatingxylem(SDX)ofP.trichocarpaand identified 2,742distinct circRNAs,including circ‐CESA4,circ‐IRX7,and circ‐GUX1,which are generated from genesinvolved in cellulose,and hemicellulose biosyn-thesis,respectively.To investigate the roles ofcircRNAs in modulating alternative splicing(AS),we detected 7,836 AS events using PacBio Iso‐Seq and identified 634 circRNAs that overlappedwith 699 AS events.Furthermore,using DNA:RNAhybrid immunoprecipitation followed by se-quencing(DRIP‐seq),we identified 8,932 R‐looppeaks that overlapped with 181 circRNAs and 672AS events.Notably,several SDX‐related circRNAsoverlapped with R‐loop peaks,pointing to theirpossible roles in modulating AS in SDX.Indeed,overexpressing circ‐IRX7increased the levels ofR‐loop structures and decreased the frequency ofintron retention in linearIRX7transcripts.Thisstudy provides a valuable R‐loop atlas resourceand uncovers the interplay between circRNAs andAS in SDX ofP.trichocarpa.