Revealing Missing Human Protein Isoforms Based on Ab Initio Prediction, RNA-seq and Proteomics
Zhiqiang HuHamish S. ScottGuangrong QinGuangyong ZhengXixia ChuLu XieDavid L. AdelsonBergithe E OftedalParvathy VenugopalMilena BabicChristopher N HahnBing ZhangXiaojing WangNan LiChaochun Wei
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In this project, we design a methods to detect novel transcripts integrating ab initio prediction on genomic sequences and filtration with various RNA-seq data. 100 splice sites were selected and RT-PCR on 48 human tissues and cell lines (including a blank negative control) were conducted. The product of the same tissue or cell line were mixed and sequenced with Illumina Mi-seq.Keywords:
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Metallothioneins (MTs) are proteins known to be involved in defense mechanisms against heavy metals and reactive oxygen species. In human, more than ten MT isoform genes have been identified, in contrast to much fewer isoforms in other mammalian species. The increased number of isoforms in human may have some biological significance; for example, isoforms may have been functionally differentiated to deal with various environmental factors in the evolutional process. However, we know little about the functions of the individual MT isoforms. To clarify functional differences between human MT isoforms, we developed a method to determine individual isoform mRNA levels using real-time polymerase chain reaction (PCR), and studied responses of the isoform genes against heavy metals (Zn, Cd, Cu) and As in HeLa cells. These metals induced all MT isoforms except for MT-1A by Cu, though their induced levels were different. Furthermore, these metals preferentially induced isoforms MT-2A and MT-1X suggesting that these isoforms may be important in protecting from their cytotoxicity.
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Paxillin, a focal adhesion protein, exists as multiple isoforms in humans (α, β, and γ). To understand more about the physiological role of each isoform, we have employed the mouse system. We found that although the α and β isoforms are present in the mouse, the γ isoform is not. The α isoform protein was detected clearly in most adult tissues, whereas the β isoform protein was almost undetectable except in spleen, testis, thymus, and lung. On the other hand, mRNAs of both isoforms were detectable in all tissues we examined. High levels of the β isoform protein was detected in peritoneal exudate macrophage cells in adult mouse as well as in cultured fibroblasts, together with the α isoform. The α isoform was expressed at a constant level throughout the embryonic stages we examined, whereas the β isoform protein was detected at the mid-stages of development and increased to levels almost equal to those of the α isoform during the late stages of embryogenesis. Therefore, unlike the α isoform, expression of the β isoform protein is restricted in adult tissues. Moreover, we showed that α and β isoforms were colocalized within the same focal adhesion plaques, and cytoplasmic pools of both isoforms exist in the perinuclear area, colocalized with the Golgi apparatus.
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This chapter contains sections titled: Introduction α Isoforms β Isoforms Physiological Role of Na,K-ATPase Isoforms Duplication and Divergence of α and β Isoform Genes Enzymatic Properties of α and β Isoform Genes Differential Expression of α and β Isoform Genes Non-transport Function of the Na,K-ATPase In Vivo Studies of Differential Isoform Function Gene Knock-out of the β2 Isoform Gene Replacement of the β2 Isoform with β1 Overexpression of the α2 Isoform Gene Knock-out of the α1 and α2 Isoform Genes Model for Differential Function of the α1 Isoform and the α2, α3 and α4 Isoforms Na,K-ATPase Isoforms and Disease Migraine Headache and the α2 Isoform Acknowledgments References
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The transcription factor p73 belongs to the p53 family of tumour suppressors and similar to other family members, transcribed as different isoforms with opposing pro- and anti-apoptotic functions. Unlike p53, p73 mutations are extremely rare in cancers. Instead, the pro-apoptotic activities of transcriptionally active p73 isoforms are commonly inhibited by over-expression of the dominant negative p73 isoforms. Therefore the relative ratio of different p73 isoforms is critical for the cellular response to a chemotherapeutic agent. Here, we analysed the expression of N-terminal p73 isoforms in cell lines and mouse tissues. Our data showed that the transcriptionally competent TAp73 isoform is abundantly expressed in cancer cell lines compared to the dominant negative ΔNp73 isoform. Interestingly, we detected higher levels of ΔNp73 in some mouse tissues, suggesting that ΔNp73 may have a physiological role in these tissues.
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A bstract : The Na,K‐ATPase is composed of two subunits, α and β, and each subunit consists of multiple isoforms. In the case of α, four isoforms, α1, α2, α3, and α4 are present in mammalian cells. The distribution of these isoforms is tissue‐ and developmental‐specific, suggesting that they may play specific roles, either during development or coupled to specific physiological processes. In order to understand the functional properties of each of these isoforms, we are using gene targeting, where animals are produced lacking either one copy or both copies of the corresponding gene or have a modified gene. To date, we have produced animals lacking the α1 and α2 isoform genes. Animals lacking both copies of the α1 isoform gene are not viable, while animals lacking both copies of the α2 isoform gene make it to birth, but are either born dead or die very soon after. In the case of animals lacking one copy of the α1 or α2 isoform gene, the animals survive and appear healthy. Heart and EDL muscle from animals lacking one copy of the α2 isoform exhibit an increase in force of contraction, while there is reduced force of contraction in both muscles from animals lacking one copy of the α1 isoform gene. These studies indicate that the α1 and α2 isoforms carry out different physiological roles. The α2 isoform appears to be involved in regulating Ca 2+ transients involved in muscle contraction, while the α1 isoform probably plays a more generalized role. While we have not yet knocked out the α3 or α4 isoform genes, studies to date indicate that the α4 isoform is necessary to maintain sperm motility. It is thus possible that the α2, α3, and α4 isoforms are involved in specialized functions of various tissues, helping to explain their tissue‐ and developmental‐specific regulation.
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Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus and is implicated in a variety of processes in both compartments. We recently identified a novel isoform of myosin IC and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C) that differ only in the addition of short isoform-specific N-terminal peptides. The expression pattern of the isoforms and the mechanisms of expression regulation remain unknown. To determine the expression patterns of myosin IC isoforms, we performed a comprehensive expression analysis of the two myosin IC isoforms (isoform A and B) that contain isoform-specific sequences. By immunoblotting with isoform-specific antibodies and by qRT-PCR with isoform-specific primer we demonstrate that myosin IC isoforms A and B have distinct expression patterns in mouse tissues. Specifically, we show that myosin IC isoform A is expressed in a tissue specific pattern, while myosin IC isoform B is ubiquitously expressed at comparable levels in mouse tissues. The differences in the expression profile of the myosin IC isoforms indicate a tissue-specific MYOIC gene regulation and further suggest that the myosin IC isoforms, despite their high sequence homology, might have tissue-specific and isoform-specific functions.
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Na+/K+-ATPase is composed of two subunits named α and β. Each subunit has three isoforms of α1, α2 and α3 for α, β1, β2 and β3 for β (4,11,18,25). Expression of each isoform gene is regulated tissue specifically and developmentally (5,20,21). Each isoform has its unique affinities for ligands including Na+, K+ and ATP and for an inhibitor ouabain (6,9,10). It is believed that any of the α isoforms could be assembled with any of the β isoforms, suggesting that the gene expression of each isoform gene is involved in the production of functional difference of the assembled enzyme. Each isoform gene has been isolated from various species and characterized (12,15,17,26,27, 28,30,32).
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