Tomato (
) is an important vegetable and fruit crop. Its genome was completely sequenced and there are also a large amount of available expressed sequence tags (ESTs) and short reads generated by RNA ...sequencing (RNA-seq) technologies. Mapping transcripts including mRNA sequences, ESTs, and RNA-seq reads to the genome allows identifying pre-mRNA alternative splicing (AS), a post-transcriptional process generating two or more RNA isoforms from one pre-mRNA transcript. We comprehensively analyzed the AS landscape in tomato by integrating genome mapping information of all available mRNA and ESTs with mapping information of RNA-seq reads which were collected from 27 published projects. A total of 369,911 AS events were identified from 34,419 genomic loci involving 161,913 transcripts. Within the basic AS events, intron retention is the prevalent type (18.9%), followed by alternative acceptor site (12.9%) and alternative donor site (7.3%), with exon skipping as the least type (6.0%). Complex AS types having two or more basic event accounted for 54.9% of total AS events. Within 35,768 annotated protein-coding gene models, 23,233 gene models were found having pre-mRNAs generating AS isoform transcripts. Thus the estimated AS rate was 65.0% in tomato. The list of identified AS genes with their corresponding transcript isoforms serves as a catalog for further detailed examination of gene functions in tomato biology. The post-transcriptional information is also expected to be useful in improving the predicted gene models in tomato. The sequence and annotation information can be accessed at plant alternative splicing database (http://proteomics.ysu.edu/altsplice).
Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), is involved in amultitude of biological processes in eukaryotes. Symmetric arginine dimethylation mediated by ...PRMT5 modulates constitutive and alternative pre-mRNA splicing of diverse genes to regulate normal growth and development in multiple species; however, the underlying molecular mechanism remains largely unknown. A genetic screen for suppressors of an Arabidopsis symmetric arginine dimethyltransferase mutant, atprmt5, identified two gain-of-function alleles of pre-mRNA processing factor 8 gene (prp8-8 and prp8-9), the highly conserved core component of the U5 small nuclear ribonucleoprotein (snRNP) and the spliceosome. These two atprmt5 prp8 double mutants showed suppression of the developmental and splicing alterations of atprmt5 mutants. In atprmt5 mutants, the NineTeen complex failed to be assembled into the U5 snRNP to form an activated spliceosome; this phenotype was restored in the atprmt5 prp8-8 double mutants. We also found that loss of symmetric arginine dimethylation of Sm proteins prevents recruitment of the NineTeen complex and initiation of spliceosome activation. Together, our findings demonstrate that symmetric arginine dimethylation has important functions in spliceosome assembly and activation, and uncover a key molecular mechanism for arginine methylation in pre-mRNA splicing that impacts diverse developmental processes.
Moso bamboo (Phyllostachys edulis) is a well-known bamboo species of high economic value in the textile industry due to its rapid growth. Phytohormones, which are master regulators of growth and ...development, serve as important endogenous signals. However, the mechanisms through which phytohormones regulate growth in moso bamboo remain unknown to date.
Here, we reported that exogenous gibberellins (GA) applications resulted in a significantly increased internode length and lignin condensation. Transcriptome sequencing revealed that photosynthesis-related genes were enriched in the GA-repressed gene class, which was consistent with the decrease in leaf chlorophyll concentrations and the lower rate of photosynthesis following GA treatment. Exogenous GA applications on seedlings are relatively easy to perform, thus we used 4-week-old whole seedlings of bamboo for GA- treatment followed by high throughput sequencing. In this study, we identified 932 cis-nature antisense transcripts (cis-NATs), and 22,196 alternative splicing (AS) events in total. Among them, 42 cis-nature antisense transcripts (cis-NATs) and 442 AS events were differentially expressed upon exposure to exogenous GA
, suggesting that post-transcriptional regulation might be also involved in the GA
response. Targets of differential expression of cis-NATs included genes involved in hormone receptor, photosynthesis and cell wall biogenesis. For example, LAC4 and its corresponding cis-NATs were GA
-induced, and may be involved in the accumulation of lignin, thus affecting cell wall composition.
This study provides novel insights illustrating how GA alters post-transcriptional regulation and will shed light on the underlying mechanism of growth modulated by GA in moso bamboo.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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), representing the largest genome of a major bamboo species. We assembled 70 allelic chromosomes (2, 737 Mb) for hexaploid D. latiflorus using both single‐molecule sequencing from the Pacific Biosciences (PacBio) Sequel platform and chromosome conformation capture sequencing (Hi‐C). Repetitive sequences comprised 52.65% of the D. latiflorus genome. We annotated 135 231 protein‐coding genes in the genome based on transcriptomes from eight different tissues. Transcriptome sequencing using RNA‐Seq and PacBio single‐molecule 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.
The allele‐aware chromosome‐scale assembly of the genome of the hexaploid Ma bamboo Dendrocalamus latiflorus by Pacific Biosciences Sequel sequencing and chromosome conformation capture sequencing will facilitate overexpression and CRISPRCas9 gene editing studies.
Summary
In mammals, DNA methylation is associated with aging. However, age‐related DNA methylation changes during phase transitions largely remain unstudied in plants. Moso bamboo (Phyllostachys ...edulis) requires a very long time to transition from the vegetative to the floral phase. To comprehensively investigate the association of DNA methylation with aging, we present here single‐base‐resolution DNA methylation profiles using both high‐throughput bisulfite sequencing and single‐molecule nanopore‐based DNA sequencing, covering the long period of vegetative growth and transition to flowering in moso bamboo. We discovered that CHH methylation gradually accumulates from vegetative to reproductive growth in a time‐dependent fashion. Differentially methylated regions, correlating with chronological aging, occurred preferentially at both transcription start sites and transcription termination sites. Genes with CG methylation changes showed an enrichment of Gene Ontology (GO) categories in ‘vegetative to reproductive phase transition of meristem’. Combining methylation data with mRNA sequencing revealed that DNA methylation in promoters, introns and exons may have different roles in regulating gene expression. Finally, circular RNA (circRNA) sequencing revealed that the flanking introns of circRNAs are hypermethylated and enriched in long terminal repeat (LTR) retrotransposons. Together, the observations in this study provide insights into the dynamic DNA methylation and circRNA landscapes, correlating with chronological age, which paves the way to study further the impact of epigenetic factors on flowering in moso bamboo.
Significance Statement
Moso bamboo requires a very long time to transition from the vegetative to the floral phase. Here, we found that the DNA methylation level varies with chronological age in moso bamboo. This study also provides insights into flanking introns of circRNAs, which are hypermethylated and enriched in LTR retrotransposons.
Alternative polyadenylation （APA） is a widespread mechanism for gene regulation and has been implicated in flowering, but the molecular basis governing the choice of a specific poly（A） site during ...the vegetative-to-reproductive growth transition remains unclear. Here we characterize HLPI, an hnRNP A/B protein as a novel regulator for pre-mRNA 3＇-end processing in Arabidopsis. Genetic analysis reveals that HLP1 suppresses Flowering Locus C （FLC）, a key repressor of flowering in Arabidopsis. Genome-wide mapping of HLP1-RNA interactions indicates that HLP1 binds preferentially to A-rich and U-rich elements around cleavage and polyadenylation sites, implicating its role in 3＇-end formation. We show HLP1 is significantly enriched at transcripts involved in RNA metabolism and flowering. Comprehensive profiling of the poly（A） site usage reveals that HLP1 mutations cause thousands of poly（A） site shifts. A distal-to-proximal poly（A） site shift in the flowering regulator FCA, a direct target of HLP1, leads to upregulation of FLC and delayed flowering. Our results elucidate that HLP1 is a novel factor involved in 3＇-end processing and controls reproductive timing via targeting APA.
N6‐methyladenosine (m6A) 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 m6A ...modification in plant circular RNAs has not been reported. A widely used method to identify m6A modifications relies on m6A‐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 m6A 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 m6A 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 m6A 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 m6A sites at single‐base resolution in circular RNAs or linear transcripts from non‐coding RNA without poly(A) tails.
Nanopore‐based direct RNA sequencing cannot sequence circular RNAs, as they lack poly(A) tail. To overcome this limitation, we developed a novel method to detect circular RNAs and m6A modification using direct RNA sequencing. This method is also suitable for linear transcripts from non‐coding RNA without poly(A) tails.
The nucleosome assembly protein 1 (NAP1) family is the main histone chaperone of histone H2A–H2B. To explore the function of NAP1 family genes in moso bamboo (Phyllostachys edulis), characterized by ...extremely rapid growth and a long flowering cycle, we originally conducted a genome-wide analysis of the PheNAP1 gene. The phylogenetic relationship, gene expression pattern, DNA methylation, and histone modification were analyzed. Eventually, 12 PheNAP1 genes were recognized from the Phyllostachys edulis genome, divided into two sorts: the NRP subfamily (four members) and the NAP subfamily (eight members). Highly conserved motifs exist in each subfamily, which are distinct between subfamilies. PheNAP1 was distributed homogeneously on 10 out of 24 chromosomes, and gene duplication contributed significantly to the enhancement of the PheNAP1 gene in the genome. Cis-acting element analysis showed that PheNAP1 family genes are involved in light, hormone, and abiotic stress responses and may play an important role in the rapid growth and flowering. PheNAP1 exhibited the highest expression level in fast-growing shoots, indicating it is closely associated with the rapid growth of moso bamboo. Besides, PheNAP1 can rescue the early-flowering phenotype of nrp1-1 nrp2-2, and it affected the expression of genes related to the flowering pathway, like BSU1, suggesting the vital role that PheNAP1 may take in the flowering process of moso bamboo. In addition, histone modification results showed that PheNAP1 could bind to phosphorylation-, acetylation-, and methylation-modified histones to further regulate gene expression. A sketch appears: that PheNAP1 can accompany histones to regulate fast-growth- and flowering-related genes in moso bamboo. The consequences of this study enrich the understanding of the epigenetic regulation mechanism of bamboo plants and lays a foundation for further studies on the role of the NAP1 gene in Phyllostachys edulis and the function of chromatin regulation in forest growth and development.
Transposable elements (TEs) are ubiquitously present in plant genomes and often account for significant fractions of the nuclear DNA. For example, roughly 40% of the rice genome consists of TEs, many ...of which are retrotransposons, including 14% LTR- and ∼1% non-LTR retrotransposons. Despite their wide distribution and abundance, very few TEs have been found to be transpositional, indicating that TE activities may be tightly controlled by the host genome to minimize the potentially mutagenic effects associated with active transposition. Consistent with this notion, a growing body of evidence suggests that epigenetic silencing pathways such as DNA methylation, RNA interference, and H3K9me2 function collectively to repress TE activity at the transcriptional and posttranscriptional levels. It is not yet clear, however, whether the removal of histone modifications associated with active transcription is also involved in TE silencing. Here, we show that the rice protein JMJ703 is an active H3K4-specific demethylase required for TEs silencing. Impaired JMJ703 activity led to elevated levels of H3K4me3, the misregulation of numerous endogenous genes, and the transpositional reactivation of two families of non-LTR retrotransposons. Interestingly, loss of JMJ703 did not affect TEs (such as Tos17) previously found to be silenced by other epigenetic pathways. These results indicate that the removal of active histone modifications is involved in TE silencing and that different subsets of TEs may be regulated by distinct epigenetic pathways.
Protein arginine methylation, one of the most abundant and important posttranslational modifications, is involved in a multitude of biological processes in eukaryotes, such as transcriptional ...regulation and RNA processing. Symmetric arginine dimethylation is required for snRNP biogenesis and is assumed to be essential for pre-mRNA splicing; however, except for in vitro evidence, whether it affects splicing in vivo remains elusive. Mutation in an Arabidopsis symmetric arginine dimethyltransferase, AtPRMT5, causes pleiotropic developmental defects, including late flowering, but the underlying molecular mechanism is largely unknown. Here we show that AtPRMT5 methylates a wide spectrum of substrates, including some RNA binding or processing factors and U snRNP AtSmD1, D3, and AtLSm4 proteins, which are involved in RNA metabolism. RNA-seq analyses reveal that AtPRMT5 deficiency causes splicing defects in hundreds of genes involved in multiple biological processes. The splicing defects are identified in transcripts of several RNA processing factors involved in regulating flowering time. In particular, splicing defects at the flowering regulator FLOWERING LOCUS KH DOMAIN (FLK) in atprmt5 mutants reduce its functional transcript and protein levels, resulting in the up-regulation of a flowering repressor FLOWERING LOCUS C (FLC) and consequently late flowering. Taken together, our findings uncover an essential role for arginine methylation in proper pre-mRNA splicing that impacts diverse developmental processes.
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