Paramutation at the maize pl1 locus is associated with RdDM activity at distal tandem repeats.

Exceptions to Mendelian inheritance often highlight novel chromosomal behaviors. The maize Pl1-Rhoades allele conferring plant pigmentation can display inheritance patterns deviating from Mendelian expectations in a behavior known as paramutation. However, the chromosome features mediating such exceptions remain unknown. Here we show that small RNA production reflecting RNA polymerase IV function within a distal downstream set of five tandem repeats is coincident with meiotically-heritable repression of the Pl1-Rhoades transcription unit. A related pl1 haplotype with three, but not one with two, repeat units also displays the trans-homolog silencing typifying paramutations. 4C interactions, CHD3a-dependent small RNA profiles, nuclease sensitivity, and polyadenylated RNA levels highlight a repeat subregion having regulatory potential. Our comparative and mutant analyses show that transcriptional repression of Pl1-Rhoades correlates with 24-nucleotide RNA production and cytosine methylation at this subregion indicating the action of a specific DNA-dependent RNA polymerase complex. These findings support a working model in which pl1 paramutation depends on trans-chromosomal RNA-directed DNA methylation operating at a discrete cis-linked and copy-number-dependent transcriptional regulatory element.

Locus-specific paramutation in Zea mays is maintained by a PICKLE-like chromodomain helicase DNA-binding 3 protein controlling development and male gametophyte function.

Paramutations represent directed and meiotically-heritable changes in gene regulation leading to apparent violations of Mendelian inheritance. Although the mechanism and evolutionary importance of paramutation behaviors remain largely unknown, genetic screens in maize (Zea mays) identify five components affecting 24 nucleotide RNA biogenesis as required to maintain repression of a paramutant purple plant1 (pl1) allele. Currently, the RNA polymerase IV largest subunit represents the only component also specifying proper development. Here we identify a chromodomain helicase DNA-binding 3 (CHD3) protein orthologous to Arabidopsis (Arabidopsis thaliana) PICKLE as another component maintaining both pl1 paramutation and normal somatic development but without affecting overall small RNA biogenesis. In addition, genetic tests show this protein contributes to proper male gametophyte function. The similar mutant phenotypes documented in Arabidopsis and maize implicate some evolutionarily-conserved gene regulation while developmental defects associated with the two paramutation mutants are largely distinct. Our results show that a CHD3 protein responsible for normal plant ontogeny and sperm transmission also helps maintain meiotically-heritable epigenetic regulatory variation for specific alleles. This finding implicates an intersection of RNA polymerase IV function and nucleosome positioning in the paramutation process.

Zebrafish rbm8a and magoh mutants reveal EJC developmental functions and new 3'UTR intron-containing NMD targets.

Many post-transcriptional mechanisms operate via mRNA 3'UTRs to regulate protein expression, and such controls are crucial for development. We show that homozygous mutations in two zebrafish exon junction complex (EJC) core genes rbm8a and magoh leads to muscle disorganization, neural cell death, and motor neuron outgrowth defects, as well as dysregulation of mRNAs subjected to nonsense-mediated mRNA decay (NMD) due to translation termination ≥ 50 nts upstream of the last exon-exon junction. Intriguingly, we find that EJC-dependent NMD also regulates a subset of transcripts that contain 3'UTR introns (3'UI) < 50 nts downstream of a stop codon. Some transcripts containing such stop codon-proximal 3'UI are also NMD-sensitive in cultured human cells and mouse embryonic stem cells. We identify 167 genes that contain a conserved proximal 3'UI in zebrafish, mouse and humans. foxo3b is one such proximal 3'UI-containing gene that is upregulated in zebrafish EJC mutant embryos, at both mRNA and protein levels, and loss of foxo3b function in EJC mutant embryos significantly rescues motor axon growth defects. These data are consistent with EJC-dependent NMD regulating foxo3b mRNA to control protein expression during zebrafish development. Our work shows that the EJC is critical for normal zebrafish development and suggests that proximal 3'UIs may serve gene regulatory function in vertebrates.

"Simple Tidy GeneCoEx": A gene co-expression analysis workflow powered by tidyverse and graph-based clustering in R.

Gene co-expression analysis is an effective method to detect groups (or modules) of co-expressed genes that display similar expression patterns, which may function in the same biological processes. Here, we present "Simple Tidy GeneCoEx", a gene co-expression analysis workflow written in the R programming language. The workflow is highly customizable across multiple stages of the pipeline including gene selection, edge selection, clustering resolution, and data visualization. Powered by the tidyverse package ecosystem and network analysis functions provided by the igraph package, the workflow detects gene co-expression modules whose members are highly interconnected. Step-by-step instructions with two use case examples as well as source code are available at https://github.com/cxli233/SimpleTidy_GeneCoEx.

Cold stress induces differential gene expression of retained homeologs in Camelina sativa cv Suneson.

Camelina sativa (L.) Crantz, a member of the Brassicaceae, has potential as a biofuel feedstock which is attributable to the production of fatty acids in its seeds, its fast growth cycle, and low input requirements. While a genome assembly is available for camelina, it was generated from short sequence reads and is thus highly fragmented in nature. Using long read sequences, we generated a chromosome-scale, highly contiguous genome assembly (644,491,969 bp) for the spring biotype cultivar 'Suneson' with an N50 contig length of 12,031,512 bp and a scaffold N50 length of 32,184,682 bp. Annotation of protein-coding genes revealed 91,877 genes that encode 133,355 gene models. We identified a total of 4,467 genes that were significantly up-regulated under cold stress which were enriched in gene ontology terms associated with "response to cold" and "response to abiotic stress". Coexpression analyses revealed multiple coexpression modules that were enriched in genes differentially expressed following cold stress that had putative functions involved in stress adaptation, specifically within the plastid. With access to a highly contiguous genome assembly, comparative analyses with Arabidopsis thaliana revealed 23,625 A. thaliana genes syntenic with 45,453 Suneson genes. Of these, 24,960 Suneson genes were syntenic to 8,320 A. thaliana genes reflecting a 3 camelina homeolog to 1 Arabidopsis gene relationship and retention of all three homeologs. Some of the retained triplicated homeologs showed conserved gene expression patterns under control and cold-stressed conditions whereas other triplicated homeologs displayed diverged expression patterns revealing sub- and neo-functionalization of the homeologs at the transcription level. Access to the chromosome-scale assembly of Suneson will enable both basic and applied research efforts in the improvement of camelina as a sustainable biofuel feedstock.

Nascent transcription affected by RNA polymerase IV in Zea mays.

All eukaryotes use three DNA-dependent RNA polymerases (RNAPs) to create cellular RNAs from DNA templates. Plants have additional RNAPs related to Pol II, but their evolutionary role(s) remain largely unknown. Zea mays (maize) RNA polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs), and transcriptional regulation of specific alleles. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based regulation. Comparisons of WT and rpd1 mutant GRO-seq profiles indicate that Pol IV globally affects transcription at both transcriptional start sites and immediately downstream of polyadenylation addition sites. We found no evidence of divergent transcription from gene promoters as seen in mammalian GRO-seq profiles. Statistical comparisons identify genes and TEs whose transcription is affected by RPD1. Most examples of significant increases in genic antisense transcription appear to be initiated by 3'-proximal long terminal repeat retrotransposons. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for specific regions of the maize genome including genes having developmental significance.