miR172 regulates stem cell fate and defines the inner boundary of APETALA3 and PISTILLATA expression domain in Arabidopsis floral meristems

In Arabidopsis, two floral homeotic genes APETALA2 (AP2) and AGAMOUS (AG) specify the identities of perianth and reproductive organs, respectively, in flower development. The two genes act antagonistically to restrict each other to their proper domains of action within the floral meristem. In addition to AG, which antagonizes AP2, miR172, a microRNA, serves as a negative regulator of AP2. In this study, we showed that AG and miR172 have distinct functions in flower development and that they largely act independently in the negative regulation of AP2. We uncovered functions of miR172-mediated repression of AP2 in the regulation of floral stem cells and in the delineation of the expression domain of another class of floral homeotic genes. Given the antiquity of miR172 in land plants, our findings have implications for the recruitment of a microRNA in the building of a flower in evolution.     

The human P2X4 receptor gene is alternatively spliced

ATP acts as a fast excitatory neurotransmitter by binding to a large family of membrane proteins, P2X receptors, that have been shown to be ligand-gated, non-selective cation channels. We report the cloning of a full-length and alternatively spliced form of the human P2X₄ gene. Clones were identified from a human stomach cDNA library using a rat P2X₄ probe. Nucleotide sequence analysis of positive clones identified the full-length human P2X₄ cDNA, which codes for a 388-residue protein that is highly homologous (82%) to the rat gene, and an alternatively spliced cDNA. In the alternatively spliced cDNA, the 5′-untranslated region and the first 90 amino acids in the coding region of full-length human P2X₄ are replaced by a 35 amino acid coding sequence that is highly homologous with a region of chaparonin proteins in the hsp-90 family. The open reading frames of the full-length and splice variant clones were confirmed by in vitro translation. Northern analysis indicated expression of the full-length P2X₄ message in numerous human tissues including smooth muscle, heart, and skeletal muscles. Alternatively spliced RNAs were identified in smooth muscle and brain by RT–PCR and confirmed by RNAse protection assays using a 710 bp anti-sense RNA probe that spanned the alternatively spliced and native P2X₄ regions. Injection of full-length, but not alternatively spliced, cRNA into Xenopus oocytes resulted in the expression of ATP gated non-selective cation currents.     

Sulf2 gene is alternatively spliced in mammalian developing and tumour tissues with functional implications

SULF2 enzyme regulates the activities of a number of signalling pathways that in many tissues are up-regulated during development and disease. As we recently showed for avian Sulf1, the present study demonstrates that mammalian Sulf2 gene can also generate functionally distinct splice variants that would regulate normal development and tumour growth differentially. It is thus important to distinguish SULF1/SULF2 isoforms in mammalian tissues to understand their functional and clinical relevance to disease. This study demonstrates that unlike normal adult lung with little or no SULF2 expression, this enzyme is expressed at high levels in most lung tumours showing differential cellular distribution of full length and shorter SULF2 variants in such tumours. Furthermore, we show that the short SULF2 splice variants are associated with those signalling pathways that are inhibited by full length SULF1/SULF2 variants and therefore could promote growth in such lung tumours.     

The Drosophila Ret gene is transcribed in multiple alternatively spliced forms

We have cloned and sequenced the 5' and 3' ends of the Drosophila homolog of the vertebrate c-ret gene, Ret, and have derived from it the predicted protein sequence of Ret. The extracellular domain of Ret is very widely diverged from that of its vertebrate counterparts but the cadherin motif present in vertebrate c-ret proteins can also be discerned in Ret. As with the vertebrate gene, multiple splice variants were detected at the 5'-end of Ret, one of which inserts an exon with a protein-terminating frameshift into the cDNA. In contrast to human c-ret, which may vary its signalling specificity by using splicing-derived, alternative C-terminal sequences, Ret cDNAs showed no variation at their 3'-ends.     

Pre-miR172及miR172调控油菜AP2基因表达的规律分析

为探究油菜miR172前体(pre-miR172)及成熟体(miR172)对AP2基因的调控功能,该研究通过生物信息学方法对miR172和AP2启动子进行调控元件预测,分析6条油菜AP2基因的进化关系及miR172与AP2的靶向关系; 通过qRT-PCR方法检测AP2、miR172和pre-miR172在早熟和晚熟油菜不同组织的表达规律; 比较分析miR172丰度和AP2表达量间的相关关系,以及比较分析 pre-miR172和miR172在表达水平上的相关关系; 通过过表达pre-miR172,再次验证pre-miR172对成熟体miR172及AP2的作用。结果表明:(1)miR172和AP2启动子区均存在调控花发育的顺式元件。(2)6条AP2序列均经历了强烈的纯化选择,均具备miR172的结合位点,属miR172的靶基因。(3)miR172家族成员均可促进早熟油菜AP2表达,但miR172d作用不明显。在晚熟油菜中,miR172a和miR172c作用微弱,miR172b和miR172d二者共同发挥作用降低AP2的表达水平。(4)pre-miR172家族对于早熟油菜中miR172家族的表达水平均有促进作用; 在晚熟油菜中pre-miR172a和pre-miR172b对其成熟序列的形成发挥正调控作用,pre-miR172c和pre-miR172d则对于其成熟序列的形成发挥负调控作用。过表达pre-miR172后,miR172和AP2表达规律与上述结果保持一致,证实pre-miR172对miR172及AP2的调控功能。该研究结果丰富了油菜AP2基因的功能调控路径,为基因的调控功能研究提供了新的思路。     

The MUC3 gene encodes a transmembrane mucin and is alternatively spliced.

Epithelial mucins are a family of secreted and cell surface glycoproteins expressed by epithelial tissues and implicated in epithelial cell protection, adhesion modulation and signaling. The gene encoding human MUC3 (hMUC3), localised to chromosome 7q22, is most highly expressed in the small intestine. It has previously been reported to be a non-transmembrane mucin with minimal homology to its suggested orthologues from rat (rMuc3) and mouse (mMuc3). RT-PCR was performed to investigate the carboxyl terminus of the published sequence of hMUC3 from normal colon and small intestine tissues and also from a series of 10 colorectal cancer cell lines. Two distinct PCR products were identified. In contrast to the previously published hMUC3 sequence, which terminates shortly after a single cysteine-rich EGF-like domain, conceptual protein translation of the dominant and largest PCR product identified two extracellular cysteine-rich EGF-like domains separated by an N-glycosylation-rich domain and a potential coiled-coil region, followed by a putative transmembrane region and a 75 amino acid cytoplasmic tail. The smaller of the two PCR products was found to be an alternative splice variant of MUC3 including the first EGF-like domain but lacking part of the second EGF-like domain and the transmembrane region. Nine out of 10 colorectal cancer cell lines were found to express MUC3. Interestingly, one of the cell lines, LoVo, expressed predominantly the alternative splice form lacking a transmembrane domain. Structural homology of the new protein sequence of hMUC3 with rMuc3 and mMuc3 indicates it is closely related to the rodent proteins and is likely to be involved in ligand-binding and intracellular signaling. The new finding that MUC3 encodes a transmembrane molecule presents a new paradigm for the structure of this mucin and the manner in which it may function.     

Multiple cardiovascular defects caused by the absence of alternatively spliced segments of fibronectin

Alternatively spliced variants of fibronectin (FN) containing exons EIIIA and EIIIB are expressed around newly forming vessels in development and disease but are downregulated in mature vasculature. The sequences and patterns of expression of these splice variants are highly conserved among vertebrates, suggestive of their biological importance; however the functions of EIIIA and EIIIB-containing FNs are unknown. To understand the role(s) of these splice variants, we deleted both EIIIA and EIIIB exons from the FN gene and observed embryonic lethality with incomplete penetrance by embryonic day 10.5. Deletion of both EIIIA and EIIIB exons did not affect synthesis or cell surface deposition of FN, indicating that embryonic lethality was due specifically to the absence of EIIIA and EIIIB exons from FN. EIIIA/EIIIB double-null embryos displayed multiple embryonic cardiovascular defects, including vascular hemorrhage, failure of remodeling embryonic and yolk sac vasculature, defective placental angiogenesis and heart defects. In addition, we observed defects in coverage and association with dorsal aortae of alpha-smooth-muscle-actin-positive cells. Our studies indicate that the presence or absence of EIIIA and EIIIB exons alters the function of FN and demonstrate the requirement for these alternatively spliced exons in cardiovascular development.     

A novel role for the floral homeotic gene APETALA2 during Arabidopsis fruit development

The majority of the Arabidopsis fruit comprises an ovary with three primary tissue types: the valves, the replum and the valve margins. The valves, which are derived from the ovary walls, are separated along their entire length by the replum. The valve margin, which consists of a separation layer and a lignified layer, forms as a narrow stripe of cells at the valve-replum boundaries. The valve margin identity genes are expressed at the valve-replum boundary and are negatively regulated by FUL and RPL in the valves and replum, respectively. In ful rpl double mutants, the valve margin identity genes become ectopically expressed, and, as a result, the entire outer surface of the ovary takes on valve margin identity. We carried out a genetic screen in this sensitized genetic background and identified a suppressor mutation that restored replum development. Surprisingly, we found that the corresponding suppressor gene was AP2, a gene that is well known for its role in floral organ identity, but whose role in Arabidopsis fruit development had not been previously described. We found that AP2 acts to prevent replum overgrowth by negatively regulating BP and RPL, two genes that normally act to promote replum formation. We also determined that AP2 acts to prevent overgrowth of the valve margin by repressing valve margin identity gene expression. We have incorporated AP2 into the current genetic network controlling fruit development in Arabidopsis.     

Discovery of gene families and alternatively spliced variants by RecA-mediated cloning

Probing the functional complexity of the human genome will require new gene cloning techniques, not only to discover intraspecies gene homologs and interspecies gene orthologs, but also to identify alternatively spliced gene variants. We report homologous cDNA cloning methods that allow cloning of gene family members, genes from different species, and alternatively spliced gene variants. We cloned human 14-3-3 gene family members using DNA probes with as much as 35% sequence divergence, cloned alternatively spliced gene forms of Rad51D, and cloned a novel splice form of the human 14-3-3 theta gene with a unique expression pattern. Interspecies gene cloning was demonstrated for the mouse Rad51C and mouse beta-actin genes using human gene probes. The gene family cloning method is fast, efficient, and free from PCR errors; moreover, it exploits the abilities of RecA protein to pair homologous or partially homologous DNA sequences stably in kinetically trapped, multistranded DNA hybrids that can be used for subsequent gene clone enrichment.