The 5′ UTR intron-mediated enhancement of constitutive splicing of the tobacco microsome ω-3 fatty acid desaturase gene

Several plant genes have their first intron in the 5′ untranslated region (5′ UTR), and such 5′ UTR introns often show several biological functions, including the intron-mediated enhancement of protein expression through an increase of mRNA level (IME), intron-dependent spatial expression, and intron-mediated enhancement of translation. Here, we show another function of the 5′ UTR intron, i.e., the 5′ UTR intron-mediated enhancement of constitutive splicing. The NtFAD3 gene, which encodes a tobacco microsome ω-3 fatty acid desaturase, has a 552 nucleotide-long 5′ UTR intron (intron 1), and the other seven introns are located in the coding sequence. The splicing of the 5′ half region of the NtFAD3 was studied through an in vivo splicing assay using Arabidopsis leaf explants. The low splicing efficiency of intron 2 was much improved when the assay construct harbored intron 1. Deletion of intron 1 and the replacement of intron 1 to the NtFAD3 intron 8 decreased the splicing efficiency of intron 2. The splicing enhancers were redundant and dispersed in the 5′ splice site-proximal, 284-nucleotides region of intron 1. In addition, the interaction among the cis-elements, i.e., the splicing enhancers in the intron 1 and exon 2, were necessary for the efficient splicing of intron 2. The 5′ UTR intron-mediated constitutive splicing was partially inhibited when an SR-like protein, SR45, was deficient. These results indicated a novel function of the 5′ UTR intron, namely an enhancement of the constitutive splicing.     

The poplar ARGOS-LIKE gene promotes leaf initiation and cell expansion,and controls organ size

We identified a Populus nigra auxin-regulated gene involved in organ size (PnARGOS)-LIKE, encoding one organ size related protein in black poplar. It is homologous to AtARGOS and AtARGOS-LIKE genes of Arabidopsis thaliana. ABRE-like, G-box, GATA and I-box motifs were discovered in the promoter region of the poplar ARGOS-LIKE gene. In wild type aspen (Populus tremula) plants, an ortholog of the PnARGOS-LIKE gene (PtrARGOS-LIKE) was noticeably expressed in actively dividing and expanding young leaves and calli, whereas its mRNA content increased in response to exogenous 6-benzylaminopurine, 1-naphthaleneacetic acid, and 24-epibrassinolide. Expression of the PtrARGOS-LIKE gene was reduced under a salinity treatment. In addition, we generated transgenic tobacco and aspen plants with an up-regulated expression of the PnARGOS-LIKE gene. A constitutive expression of the gene contributed to an increase in size of stems and leaves of the transgenic tobacco plants. In the transgenic aspen, a constitutive expression of the PnARGOS-LIKE gene promoted an increase in the frequency of leaf initiations and in leaf length and area. The size of transgenic tobacco and aspen leaves increased due to the enlargement of individual cells. The results show the significance of the PnARGOS-LIKE gene for control of leaf initiation and organ growth by cell expansion in poplar.     

The role of expansin genes <Emphasis Type="Italic">PtrEXPA3</Emphasis> and <Emphasis Type="Italic">PnEXPA3</Emphasis> in the regulation of leaf growth in poplar

The genes of α-expansins of woody plants are of great interest for genetic engineering, since they can potentially be used to improve the tree growth parameters. In the flora of Russia, model woody plants for plant biotechnology are aspen (Populus tremula L.) and black poplar (Populus nigra L.). The objective of this study was to determine the role of α-expansin-encoding genes, aspen PtrEXPA3 and black poplar PnEXPA3, in the regulation and maintenance of woody plant growth. To achieve this goal, the PtrEXPA3 expression level were determined upon exogenous phytohormone treatment, the action of stress factors, and constitutive expression of the PnARGOS-LIKE gene. In addition, transgenic aspen plants with constitutive expression of the black poplar PnEXPA3 gene were generated, and their morphological analysis was carried out. The highest PtrEXPA3 mRNA level was detected in young intensely growing aspen leaves, and furthermore, expression of the gene was induced by exogenous cytokinins and auxins. In response to NaCl and constitutive expression of the PnARGOS-LIKE gene, the PtrEXPA3 mRNA level decreased. Transgenic aspen plants with constitutive PnEXPA3 expression were characterized by the decreased size of leaves, petioles, and internodes, as well as the increased size of leaf epidermal cells, while the stem size remained unchanged. Taken together, the data obtained enable the suggestion that the PtrEXPA3 and PnEXPA3 genes encode cytokinin- and auxin-regulated, leaf-specific expansins that are involved in the cell expansion.     

Chromosomal locations of U2 snDNA clusters in <Emphasis Type="Italic">Megaleporinus</Emphasis>, <Emphasis Type="Italic">Leporinus</Emphasis> and <Emphasis Type="Italic">Schizodon</Emphasis> (Characiformes: Anostomidae)

The U small nuclear RNA (U snRNA) genes comprise a multigene family and are required for splicing of pre-mRNA. In this paper, we aimed to study the chromosomal location of the U2 snRNA gene in Megaleporinus, Leporinus and Schizodon species, which constitute interesting models for the study of repetitive DNA and genomic evolution in fish once the group comprises species with and without heteromorphic sex chromosomes. The all six species showed 2n?=?54 chromosomes: Megaleporinus elongatus, Megaleporinus macrocephalus, Leporinus striatus, Leporinus friderici, Schizodon borelli and Schizodon isognathus. The U2 snDNA clusters were evident in only one medium-sized submetracentric pair in all analyzed species and this may represent a condition shared by Anostomidae family.     

Enhancers in the <Emphasis Type="Italic">Peril</Emphasis> lincRNA locus regulate distant but not local genes

Background

Recently, it has become clear that some promoters function as long-range regulators of gene expression. However, direct and quantitative assessment of enhancer activity at long intergenic noncoding RNA (lincRNA) or mRNA gene bodies has not been performed. To unbiasedly assess the enhancer capacity across lincRNA and mRNA loci, we performed a massively parallel reporter assay (MPRA) on six lincRNA loci and their closest protein-coding neighbors.

Results

For both gene classes, we find significantly more MPRA activity in promoter regions than in gene bodies. However, three lincRNA loci, Lincp21, LincEnc1, and Peril, and one mRNA locus, Morc2a, display significant enhancer activity within their gene bodies. We hypothesize that such peaks may mark long-range enhancers, and test this in vivo using RNA sequencing from a knockout mouse model and high-throughput chromosome conformation capture (Hi-C). We find that ablation of a high-activity MPRA peak in the Peril gene body leads to consistent dysregulation of Mccc1 and Exosc9 in the neighboring topologically associated domain (TAD). This occurs irrespective of Peril lincRNA expression, demonstrating this regulation is DNA-dependent. Hi-C confirms long-range contacts with the neighboring TAD, and these interactions are altered upon Peril knockout. Surprisingly, we do not observe consistent regulation of genes within the local TAD. Together, these data suggest a long-range enhancer-like function for the Peril gene body.

Conclusions

A multi-faceted approach combining high-throughput enhancer discovery with genetic models can connect enhancers to their gene targets and provides evidence of inter-TAD gene regulation.