Hydroxypyruvate Reductase with a Carboxy-Terminal Targeting Signal to Microbodies is Expressed in Arabidopsis

Five Arabidopsis EST cDNA clones of hydroxypyruvate reductase(HPR), a photorespiratory enzyme in leaf peroxisomes, were sequenced.Deduced amino acid sequences revealed that HPR in Arabidopsiscontained the carboxy-terminal targeting signal to microbodies.Nucle-otide sequence analysis showed that the cDNA with thelongest insert contained an open reading frame of 1,158 bp whichencoded a polypeptide with 386 amino acids with a calculatedmolecular mass of 42,251 Da. A Southern blot analysis suggestedthat the Arabidopsis HPR gene, like that of the pumpkin HPRgene, exists as a single copy. Two kinds of pumpkin HPR mRNAmight be produced from a single gene by alternative splicing,but the structure of the genomic DNA indicated that the ArabidopsisHPR gene did not undergo alternative splicing. We detected apolypeptide with a molecular mass of 42 kDa in green leavesof Arabidopsis using an HPR-specific antibody. Immunoelectronmicroscopy revealed that Arabidopsis HPR protein was exclusivelylocalized in leaf peroxisomes in green leaves. These resultsindicate that HPR is expressed in a form with a carboxy-terminaltargeting signal to microbodies and is localized in microbodiesin Arabidopsis, suggesting that the differences in the genestructure and the regulation of gene expression of HPR are probablydue to species-specific differences in plants. (Received November 11, 1996; Accepted February 1, 1997)     

Alternative splicing in ascomycetes

Alternative splicing is a complex and regulated process, which results in mRNA with different coding capacities from a single gene. Extend and types of alternative splicing vary greatly among eukaryotes. In this review, I focus on alternative splicing in ascomycetes, which in general have significant lower extend of alternative splicing than mammals. Yeast-like species have low numbers of introns and consequently alternative splicing is lower compared to filamentous fungi. Several examples from single studies as well as from genomic scale analysis are presented, including a survey of alternative splicing in Neurospora crassa. Another focus is regulation by riboswitch RNA and alternative splicing in a heterologous system, along with putative protein factors involved in regulation.     

Identification of alternatively spliced dab1 isoforms in zebrafish

We have investigated the genomic organization, the occurrence of alternative splicing and the differential expression of the zebrafish disabled1 (dab1) gene. Dab1 is a key effector of the Reelin pathway, which regulates neuronal migration during brain development in vertebrates. The coding region of the zebrafish dab1 gene spans over 600 kb of genomic DNA and is composed of 15 exons. Alternative splicing in a region enriched for tyrosine residues generates at least three different isoforms. These isoforms are developmentally regulated and show differential tissue expression. Comparison with mouse and human data shows an overall conservation of the genomic organization with different alternative splicing events generating species-specific isoforms. Because these alternative splicing events give rise to isoforms with different numbers of phosphorylateable tyrosines, we speculate that alternative splicing of the dab1 gene in zebrafish and in other vertebrates regulates the nature of the cellular response to the Reelin signal.Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users.     

Cloning of the Pumpkin Ascorbate Oxidase Gene and Analysis of a Cis-Acting Region Involved in Induction by Auxin

A genomic clone encoding ascorbate oxidase was isolated frompumpkin (Cucurbita sp.)- This gene is consisted of four exonsand three introns. Analyses of the promoter fusion to ß-glucuronidasereporter gene by transient expression assay in pumpkin fruittissues suggested the existence of a cis-acting region responsiblefor auxin regulation. (Received November 28, 1996; Accepted March 8, 1997)     

ERK-regulated differential expression of the Mitf 6a/b splicing isoforms in melanoma

The master regulator of the melanocyte lineage Mitf is intimately involved in development as well as melanoma, controlling cell survival, differentiation, proliferation and metastasis/migration. Consistent with its central role, Mitf expression and Mitf post-translational modifications are tightly regulated. An additional potential level of regulation is afforded by differential splicing of Mitf exon-6 leading to the generation of two isoforms that differ by the presence of six amino-acids in the Mitf (+) isoform and which have differential effects on cell cycle progression. However, whether the ratio of the two isoforms is regulated and whether their expression correlates with melanoma progression is not known. Here, we show that the differential expression of the Mitf 6a/b isoforms is dependent on the MAPKinase signalling, being linked to the activation of MEK1-ERK2, but not to N-RAS/B-RAF mutation status. In addition, quantification of Mitf 6a/b splicing forms in 86 melanoma samples revealed substantially increased levels of the Mitf (−) form in a subset of metastatic melanomas. The results suggest that differential expression of the Mitf 6a/b isoforms may represent an additional mechanism for regulating Mitf function and melanoma biology.     

Analysis of the role of Caenorhabditis elegans GC-AG introns in regulated splicing

GC-AG introns represent 0.7% of total human pre-mRNA introns. To study the function of GC-AG introns in splicing regulation, 196 cDNA-confirmed GC-AG introns were identified in Caenorhabditis elegans. These represent 0.6% of the cDNA- confirmed intron data set for this organism. Eleven of these GC-AG introns are involved in alternative splicing. In a comparison of the genomic sequences of homologous genes between C.elegans and Caenorhabditis briggsae for 26 GC-AG introns, the C at the +2 position is conserved in only five of these introns. A system to experimentally test the function of GC-AG introns in alternative splicing was developed. Results from these experiments indicate that the conserved C at the +2 position of the tenth intron of the let-2 gene is essential for developmentally regulated alternative splicing. This C allows the splice donor to function as a very weak splice site that works in balance with an alternative GT splice donor. A weak GT splice donor can functionally replace the GC splice donor and allow for splicing regulation. These results indicate that while the majority of GC-AG introns appear to be constitutively spliced and have no evolutionary constraints to prevent them from being GT-AG introns, a subset of GC-AG introns is involved in alternative splicing and the C at the +2 position of these introns can have an important role in splicing regulation.     

A computational approach for genome-wide mapping of splicing factor binding sites

Alternative splicing is regulated by splicing factors that serve as positive or negative effectors, interacting with regulatory elements along exons and introns. Here we present a novel computational method for genome-wide mapping of splicing factor binding sites that considers both the genomic environment and the evolutionary conservation of the regulatory elements. The method was applied to study the regulation of different alternative splicing events, uncovering an interesting network of interactions among splicing factors.     

Structure and Expression of the Tobacco Nuclear Gene Encoding RNA-binding Protein RZ-1: The Existence of an Intron in the 3'-Untranslated Region

We have previously characterized a tobacco cDNA encoding a noveltype RNA-binding protein (RZ-1), which contains a zinc fingermotif in addition to a consensus sequence-type RNA-binding domainand is localized in the nucleus. Here we isolated its genomicclone from a Nicotiana sylvestris genomic library. Southernblot analysis suggested that RZ-1 is coded for by a single locusper haploid genome. Comparison of the cDNA and genomic sequencesindicated that the RZ-1 gene contains two introns, one in thecoding region and another in the 3'-untranslated region. RT-PCRand ribonuclease protection analyses showed that splicing ofRZ-1 pre-mRNA occurs efficiently. The RZ-1 protein is activelysynthesized in rapidly dividing tobacco cells, as demonstratedby immunoblot analysis.     

Molecular cloning and analysis of the mouse gicerin gene

Gicerin is a cell adhesion molecule, which has five immunoglobulin-like loop structures in an extracellular domain followed by a single transmembrane domain and a short cytoplasmic tail. We have reported that gicerin participates in neurite extension and structural organization of the nervous system, and its expression in the nervous system is high during the development and dramatically decreased after birth. To elucidate the mechanism how the expression of gicerin is regulated, we performed a genomic cloning of a mouse gicerin. A fragment of 16 kbp genomic clone contained 8 kbp gicerin gene composed of 16 exons with 6 kbp upstream region. Genomic cloning revealed that two isoforms of gicerin were generated by an alternative splicing of exon 15 results in cytoplasmic domains composed of either 63 or 21 amino acids. As for an expressional regulation of gicerin, we found that the mRNA content of gicerin in PC12 cells was regulated by cAMP. Quantitative-PCR analysis revealed that forskolin induced four-fold increase of gicerin mRNA. To characterize the involvement of its promoter region, we examined the promoter activity in PC12 cells by a luciferase-reporter assay. We found that a CRE site located at 60 bp upstream of gicerin gene was responsible for the increase of its mRNA induced by forskolin.     

Tau isoforms which contain the domain encoded by exon 6 and their role in neurite elongation

The regulation of tau protein expression during different stages of cellular differentiation and development as well as its functional role in morphogenesis, neurofibrillary tangle formation, and neurodegeneration have been topics of extensive study but have not been completely clarified yet. Tau undergoes complex regulated splicing in the mammalian nervous system. Our previous study with tau exon 6 demonstrated that it shows a splicing regulation profile which is distinct from that of the other tau exons as well as a unique expression pattern which is spatially and temporally regulated. In this study, we investigated the expression, localization, and effects of tau isoforms which contain exon 6 in neuroblastoma cells which stably overexpress them. We found that expression of one particular combination of tau exons (the longest adult isoform plus the domain of exon 6) significantly inhibits neurite elongation.     

转录辅调节因子在剪接过程中的作用

细胞通过基因表达调控来应对外界刺激,其中对基因转录起始和pre-mRNA剪接的调控是基因表达调控的重要环节。越来越多的实验显示基因转录和pre-mRNA剪接这两个过程在时空上密切相关。基因转录能调节剪接模式的选择性,反之剪接过程也影响基因转录。近年来研究发现转录辅调节因子在联系转录和剪接过程中扮演着重要角色。转录辅调节因子对基因表达的调控不仅在于影响转录产物的量,还可以调控pre-mRNA的选择性剪接并产生不同的剪接体,从而翻译出具有不同生物学功能的蛋白质。本文主要阐述了基因转录与剪接之间的关系以及它们之间相互作用的机制,有利于更深入理解基因表达调控的过程。