Analysis of Domestication Loci in Wild Rice Populations.

The domestication syndrome is defined as a collection of domestication-related traits that have undergone permanent genetic changes during the domestication of cereals. Australian wild rice populations have not been exposed to gene flow from domesticated rice populations. A high level of natural variation of the sequences at domestication loci (e.g., seed shattering, awn development, and grain size) was found in Australian AA genome wild rice from the primary gene pool of rice. This natural variation is much higher than that found in Asian cultivated rice and wild Asian rice populations. The Australian Oryza meridionalis populations exhibit a high level of homozygous polymorphisms relative to domesticated rice, inferring the fixation of distinct wild and domesticated alleles. Alleles of the seed shattering genes (SH4/SHA1 and OsSh1/SH1) present in the shattering-prone O. meridionalis populations are likely to be functional, while the dysfunctional alleles of these seed shattering genes are found in domesticated rice. This confirms that unlike Asian wild rice populations, Australian wild rice populations have remained genetically isolated from domesticated rice, retaining pre-domestication alleles in their wild populations that uniquely allow the impact of domestication on the rice genome to be characterized. This study also provides key information about the domestication loci in Australian wild rice populations that will be valuable in the utilization of these genetic resources in crop improvement and de novo domestication.

Overexpressed Gα13 activates serum response factor through stoichiometric imbalance with Gßγ and mislocalization to the cytoplasm.

Gα13, a heterotrimeric G protein α subunit of the G12/13 subfamily, is an oncogenic driver in multiple cancer types. Unlike other G protein subfamilies that contribute to cancer progression via amino acid substitutions that abolish their deactivating, intrinsic GTPase activity, Gα13 rarely harbors such mutations in tumors and instead appears to stimulate aberrant cell growth via overexpression as a wildtype form. It is not known why this effect is exclusive to the G12/13 subfamily, nor has a mechanism been elucidated for overexpressed Gα13 promoting tumor progression. Using a reporter gene assay for serum response factor (SRF)-mediated transcription in HEK293 cells, we found that transiently expressed, wildtype Gα13 generates a robust SRF signal, approximately half the amplitude observed for GTPase-defective Gα13. When epitope-tagged, wildtype Gα13 was titrated upward in cells, a sharp increase in SRF stimulation was observed coincident with a "spillover" of Gα13 from membrane-associated to a soluble fraction. Overexpressing G protein ß and γ subunits caused both a decrease in this signal and a shift of wildtype Gα13 back to the membranous fraction, suggesting that stoichiometric imbalance in the αßγ heterotrimer results in aberrant subcellular localization and signalling by overexpressed Gα13. We also examined the acylation requirements of wildtype Gα13 for signalling to SRF. Similar to GTPase-defective Gα13, S-palmitoylation of the wildtype α subunit was necessary for SRF activation but could be replaced functionally by an engineered site for N-terminal myristoylation. However, a key difference was observed between wildtype and GTPase-defective Gα13: whereas the latter protein lacking palmitoylation sites was rescued in its SRF signalling by either an engineered polybasic sequence or a C-terminal isoprenylation site, these motifs failed to restore signalling by wildtype, non-palmitoylated Gα13. These findings illuminate several components of the mechanism in which overexpressed, wildtype Gα13 contributes to growth and tumorigenic signalling, and reveal greater stringency in its requirements for post-translational modification in comparison to GTPase-defective Gα13.

Gene Expression in the Developing Seed of Wild and Domesticated Rice.

The composition and nutritional properties of rice are the product of the expression of genes in the developing seed. RNA-Seq was used to investigate the level of gene expression at different stages of seed development in domesticated rice (Oryza sativa ssp. japonica var. Nipponbare) and two Australian wild taxa from the primary gene pool of rice (Oryza meridionalis and Oryza rufipogon type taxa). Transcriptome profiling of all coding sequences in the genome revealed that genes were significantly differentially expressed at different stages of seed development in both wild and domesticated rice. Differentially expressed genes were associated with metabolism, transcriptional regulation, nucleic acid processing, and signal transduction with the highest number of being linked to protein synthesis and starch/sucrose metabolism. The level of gene expression associated with domestication traits, starch and sucrose metabolism, and seed storage proteins were highest at the early stage (5 days post anthesis (DPA)) to the middle stage (15 DPA) and declined late in seed development in both wild and domesticated rice. However, in contrast, black hull colour (Bh4) gene was significantly expressed throughout seed development. A substantial number of novel transcripts (38) corresponding to domestication genes, starch and sucrose metabolism, and seed storage proteins were identified. The patterns of gene expression revealed in this study define the timing of metabolic processes associated with seed development and may be used to explain differences in rice grain quality and nutritional value.

The Long Read Transcriptome of Rice (Oryza sativa ssp. japonica var. Nipponbare) Reveals Novel Transcripts.

BACKGROUND: High-throughput next-generation sequencing technologies offer a powerful approach to characterizing the transcriptomes of plants. Long read sequencing has been shown to support the discovery of novel isoforms of transcripts. This approach enables the generation of full-length sequences revealing splice variants that may be important in regulating gene action. Investigation of the diversity of transcripts in the rice transcriptome including splice variants was conducted using PacBio long-read sequence data to improve the annotation of the rice genome. RESULTS: A cDNA library was prepared from RNA extracted from leaves, roots, seeds, inflorescences, and panicles of O. sativa ssp. japonica var Nipponbare and sequenced on a PacBio Sequel platform. This produced 346,190 non-redundant full-length non-chimeric reads (FLNC) resulting in 33,504 high-quality transcripts. Half of the transcripts were multi-exonic and entirely matched with the reference transcripts. However, 14,874 novel isoforms were also identified resulting predominantly from intron retention and at least one novel splice site. Intron retention was the prevalent alternative splicing event and exon skipping was the least observed. Of 73,659 splice junctions, 12,755 (17%) represented novel splice junctions with canonical and non-canonical intron boundaries. The complexity of the transcriptome was examined in detail for 19 starch synthesis-related genes, defining 276 spliced isoforms of which 94 splice variants were novel. CONCLUSION: The data reveal the great complexity of the rice transcriptome. The novel transcripts provide new insights that may be a key input in future research to improve the annotation of the rice genome.

Reticulate Evolution in AA-Genome Wild Rice in Australia.

The wild rice gene pool, i.e., AA-genome, in Australia is geographically and genetically distinct from that in Asia. Two distinct taxa are found growing together in northern Australia, Oryza meridionalis (including annual and perennial forms) and an Oryza rufipogon like taxa that have been shown to have a chloroplast genome sequence that is closer to that of O. meridionalis than to O. rufipogon from Asia. Rare plants of intermediate morphology have been observed in the wild despite a reported reproductive barrier between these two species. We now report the resequencing of plants from 26 populations including both taxa and putative hybrids. A comparison of chloroplast and nuclear genome sequences indicated re-combinations that demonstrated hybridisation in both directions. Individuals with intermediate morphology had high nuclear genome heterozygosity consistent with a hybrid origin. An examination of specific genes (e.g., starch biosynthesis genes) revealed the presence of heterozygotes with alleles from both parents suggesting that some wild plants were early generation hybrids. These plants may have low cross-fertility preserving the continuation of the two distinct species. Repeated backcrossing of these rare hybrids to one parent would explain the plants exhibiting chloroplast capture. These observations suggest that reticulate evolution is continuing in wild Oryza populations and may have been a key process in rice evolution and domestication.

Regulation of the Expression of the Myosin Heavy Chain (MYH) Gene myh14 in Zebrafish Development.

The human sarcomeric myosin heavy chain gene MYH14 contains an intronic microRNA, miR-499. Our previous studies demonstrated divergent genomic organization and expression patterns of myh14/miR-499 among teleosts; however, the regulatory mechanism is partly known. In this study, we report the regulation of myh14 expression in zebrafish, Danio rerio. Zebrafish myh14 has three paralogs, myh14-1, myh14-2, and myh14-3. Detailed promoter analysis suggested that a 5710-bp 5'-flanking region of myh14-1 and a 5641-bp region of myh14-3 contain a necessary regulatory region to recapitulate specific expression during embryonic development. The 5'-flanking region of zebrafish myh14-1 and its torafugu ortholog shared two distal and a single proximal conserved region. The two distal conserved regions had no effect on zebrafish myh14-1 expression, in contrast to torafugu expression, suggesting an alternative regulatory mechanism among the myh14 orthologs. Comparison among the 5'-flanking regions of the myh14 paralogs revealed two conserved regions. Deletion of these conserved regions significantly reduced the promoter activity of myh14-3 but had no effect on myh14-1, indicating different cis-regulatory mechanisms of myh14 paralogs. Loss of function of miR-499 resulted in a marked reduction in slow muscle fibers in embryonic development. Our study identified different cis-regulatory mechanisms controlling the expression of myh14/miR-499 and an indispensable role of miR-499 in muscle fiber-type specification in zebrafish.

m6 A-mediated alternative splicing coupled with nonsense-mediated mRNA decay regulates SAM synthetase homeostasis.

Alternative splicing of pre-mRNAs can regulate gene expression levels by coupling with nonsense-mediated mRNA decay (NMD). In order to elucidate a repertoire of mRNAs regulated by alternative splicing coupled with NMD (AS-NMD) in an organism, we performed long-read RNA sequencing of poly(A)+ RNAs from an NMD-deficient mutant strain of Caenorhabditis elegans, and obtained full-length sequences for mRNA isoforms from 259 high-confidence AS-NMD genes. Among them are the S-adenosyl-L-methionine (SAM) synthetase (sams) genes sams-3 and sams-4. SAM synthetase activity autoregulates sams gene expression through AS-NMD in a negative feedback loop. We furthermore find that METT-10, the orthologue of human U6 snRNA methyltransferase METTL16, is required for the splicing regulation in␣vivo, and specifically methylates the invariant AG dinucleotide at the distal 3' splice site (3'SS) in␣vitro. Direct RNA sequencing coupled with machine learning confirms m6 A modification of endogenous sams mRNAs. Overall, these results indicate that homeostasis of SAM synthetase in C. elegans is maintained by alternative splicing regulation through m6 A modification at the 3'SS of the sams genes.