Project description:Small RNA diversity and function has been widely characterized in various tissues of the sporophytic generation of the angiosperm model Arabidopsis thaliana. In contrast, there is limited knowledge about small RNA diversity and their roles in developing male gametophytes. We thus carried out small RNA sequencing on RNA isolated from four stages of developing Arabidopsis thaliana pollen.
Project description:Small RNA diversity and function has been widely characterized in various tissues of the sporophytic generation of the angiosperm model Arabidopsis thaliana. In contrast, there is limited knowledge about small RNA diversity and their roles in developing male gametophytes. We thus carried out small RNA sequencing on RNA isolated from four stages of developing Arabidopsis thaliana pollen. Spores from 4 stages of pollen development (UNM: Uninucleate microspore M-bM-^@M-^S BCP: Bicellular pollen M-bM-^@M-^S TCP: Tricellular pollen M-bM-^@M-^S MP: Mature pollen) were isolated using a percoll gradient-based method (Honys and Twell, 2004) and the small RNA fraction for each sample was isolated and sequenced by Illumina technology. Reference: Honys, D. and Twell, D. (2004) Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biol. 5/11/R85.
Project description:Pollen grains of Arabidopsis thaliana contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein coding genes expressed in pollen, including 289 assayed only by non-specific probe sets. Additional exons and previously unannotated 5’ and 3’ UTRs for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 confirmed by PCR. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of on-going annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser.
Project description:Here we use bisulfite conversion of RNA combined with high-throughput IIlumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites in ribosomal RNAs of all three sub-cellular transcriptomes in Arabidopsis thaliana. m5C sites in rRNAs were also anlyzed in Arabidopsis T-DNA knockouts for the RNA methyltransferases TRM4A, TRM4B, TRDMT1, NSUN5, NOP2A, NOP2B and NOP2C.