生物谷報(bào)道：美國(guó)科學(xué)家通過對(duì)一種被稱作PMR1的蛋白進(jìn)行長(zhǎng)期研究,，發(fā)現(xiàn)這種蛋白可能在癌癥的產(chǎn)生機(jī)制中起關(guān)鍵的作用,。這些發(fā)現(xiàn)使人能夠了解Src究竟是怎樣使癌癥產(chǎn)生的。這項(xiàng)由俄亥俄州大學(xué)綜合癌癥中心的研究人員們進(jìn)行的研究發(fā)表在《分子細(xì)胞》期刊的3月9日刊上。
研究人員們發(fā)現(xiàn),，PMR1可被另一種分子—一種被稱作Src的富含能量的蛋白—所激活,。Src于1977年被發(fā)現(xiàn)，是頭一個(gè)被發(fā)現(xiàn)的“腫瘤基因”,。在健康的細(xì)胞中,，Src可幫助控制細(xì)胞的分裂繁殖、分化,、存活及運(yùn)動(dòng),。變異的Src可在大約一半的結(jié)腸癌、肝癌,、肺癌,、乳腺癌及胰腺腫瘤中發(fā)現(xiàn)，并且與正常的細(xì)胞相比,，癌細(xì)胞中的Src數(shù)量可顯著增大,。“Src 與癌癥之間的聯(lián)系30年前就被發(fā)現(xiàn)了，但直到今天,，我們?nèi)匀徊恢榔湓谀[瘤發(fā)展中所起的確切作用,，”首席研究員，分子及細(xì)胞生物化學(xué)教授 Daniel R. Schoenberg說道,。
研究表明,，Src可能是通過PMR1而起作用的，后者能使腫瘤抑制蛋白或其它生長(zhǎng)調(diào)節(jié)蛋白停止產(chǎn)生,，通常這些蛋白能使細(xì)胞停止生長(zhǎng),。之前由Schoenberg領(lǐng)導(dǎo)的研究發(fā)現(xiàn)，PMR1可摧毀某些特定的信使RNA,，從而幫助對(duì)蛋白的制造加以控制,。信使RNA是一些攜帶有用于組裝蛋白的信息的分子。那項(xiàng)研究還表明,，PMR1可連接到信使RNA上,，并呆在那里充當(dāng)沉默信使。 但是,，當(dāng)其接受到恰當(dāng)?shù)男盘?hào)時(shí),，PMR1將切下并摧毀信使RNA，使蛋白停止制造,。細(xì)胞使用這一機(jī)制來對(duì)諸如生長(zhǎng)因子等蛋白的制造加以控制,，這些蛋白可對(duì)荷爾蒙或其它信號(hào)時(shí)作出反應(yīng)，激活各種基因,。
對(duì)于這項(xiàng)研究,，Schoenberg 及論文的共同作者,，Schoenberg實(shí)驗(yàn)室的助理研究員Yong Peng原本是想弄清楚PMR1是如何被激活并連接到信使RNA上的。他們發(fā)現(xiàn)PMR1與一種不明的酶短暫結(jié)合后,，即被激活,。 PMR1與這種酶接觸后改變了性質(zhì)，從而使其能與目標(biāo)信使RNA結(jié)合或聯(lián)接,。 Peng隨即使用單克隆抗體將PMR1及這種酶分離出來,，由于這兩種東西是聯(lián)接在一起的，結(jié)果就一起拿到了,。把這兩種東西分開后,，研究人員們鑒別出這種酶是Src，是一大類被稱作酪氨酸激酶的分子中的一種,。 這些分子的作用就象開關(guān)一樣,，能把包括PMR1在內(nèi)的其它分子打開或關(guān)閉。 “這就是這篇論文真正讓人激動(dòng)的地方,，”Schoenberg說道,。 “我們因?yàn)閷?duì)信使RNA的淍謝感興趣而沖這而來，結(jié)果我們可能偶然發(fā)現(xiàn)了癌癥的基本機(jī)理,。””
下一步,，Schoenberg及其研究助理Xiaoqian Liu及Elizabeth Murray將使用3種癌細(xì)胞系試圖找出究竟哪些信使RNA被PMR1定為目標(biāo)并加以摧毀。 “這將幫助我們了解,，Src是不是通過PMR1起作用,，從而引起癌癥。”Schoenberg說道,。
Figure 1. In Vitro and In Vivo Phosphorylation of PMR60° by c-Src
(A) Cytoplasmic extracts prepared from COS-1 cells transfected with empty vector (lanes 1 and 4) or plasmids expressing PMR60° (lanes 2 and 5) or GFP (lanes 3 and 6) with an N-terminal myc tag were immunoprecipitated with myc monoclonal antibody. The recovered proteins were assayed by western blot using a monoclonal antibody to the myc tag (left panel) or incubated in vitro with [γ-32P]ATP prior to separation on a 10% SDS-PAGE gel. Lane 7 contains recombinant c-Src that was incubated in the same manner. Radiolabeled proteins were visualized by phosphorimager. The filled circle denotes PMR60°, and the open circle denotes c-Src.
(B and C) (B) The left panel is a Coomassie blue-stained gel of recombinant PMR60 expressed in E. coli with the purified protein loaded in the last lane. The right panel is an autoradiogram of radiolabeled proteins generated by incubating increasing amounts of PMR60 (lanes 1–3) with recombinant c-Src and [γ-32P]ATP. There is no PMR60 in lane 4 to highlight the autophosphorylation of c-Src. The in vitro labeling experiment was repeated in (C) using recombinant c-Src and biotin-labeled peptides containing the tyrosine phosphorylation site of PMR60. Lane 1 contains the peptide without further modification, and lane 2 contains the peptide with phosphotyrosine substituted for the tyrosine corresponding to the phosphorylation site (Y650) of PMR60.
(D) U2OS cells were transfected with plasmids expressing myc-tagged GFP or PMR60°. Lanes 1 and 2 are western blots of input protein probed with antibody to the myc tag on each protein (upper panel) or a monoclonal antibody to endogenous c-Src (lower panel). In lanes 3 and 4, the same antibodies were used to probe western blots for recovery of these proteins by immunoprecipitation with immobilized myc antibody. The converse experiment is shown in lanes 5–8, where complexes recovered with a rabbit antibody to c-Src were probed for recovery of c-Src (lower panel, lanes 7 and 8) GFP and PMR60° (upper panel, lanes 7 and 8). The open circle in the upper panel is IgG heavy chain.
(E) U2OS cells transfected as above with myc-PMR60° were cultured without additions (lane 1), with PP2 (lanes 3 and 5), or with PP3 (lanes 2 and 4) for 30 or 60 min. Immunoprecipitated PMR60° was analyzed by western blot with antibody to the myc tag or with PY20.
原文出處：
Molecular Cell March 9, 2007: 25 (5)
c-Src Activates Endonuclease-Mediated mRNA Decay
Yong Peng and Daniel R. Schoenberg
[Summary] [Full Text] [PDF]
相關(guān)基因：
ATP2C1
Official Symbol: ATP2C1 and Name: ATPase, Ca++ transporting, type 2C, member 1 [Homo sapiens]
Other Aliases: ATP2C1A, BCPM, HHD, KIAA1347, PMR1, SPCA1, hSPCA1
Other Designations: ATP-dependent Ca(2+) pump; ATPase 2C1; ATPase, Ca(2+)-sequestering; HUSSY-28; benign chronic pemphigus (Hailey-Hailey disease); calcium-transporting ATPase 2C1; secretory pathway Ca2+/Mn2+ ATPase
Chromosome: 3; Location: 3q22.1
MIM: 604384
GeneID: 27032
SRC
Official Symbol: SRC and Name: v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) [Homo sapiens]
Other Aliases: ASV, SRC1, c-SRC, p60-Src
Other Designations: proto-oncogene tyrosine-protein kinase SRC; protooncogene SRC, Rous sarcoma; tyrosine kinase pp60c-src; tyrosine-protein kinase SRC-1
Chromosome: 20; Location: 20q12-q13
MIM: 190090
GeneID: 6714
作者簡(jiǎn)介：
Daniel R. Schoenberg Professor
Ph.D. - University of Wisconsin
Post Doctoral - Baylor College of Medicine
The overall theme of research in the Schoenberg lab is how extracellular stimuli alter gene expression through changes in the processing and metabolism of mRNAs. These concepts are addressed in project areas which study the steps involved in the activation of mRNA decay by the female sex hormone estrogen, biochemical and molecular analysis of a ribonuclease which selectively targets a specific group of mRNAs for destabilization following estrogen stimulation, analysis of an element which regulates the length of poly(A) on mRNAs targeted for degradation by the estrogen-regulated ribonuclease.
Estrogen activation of mRNA decay:>
The life of any given mRNA begins with its transcription and its co-transcriptional processing involving the removal of intervening sequences (introns) and addition of a homopolymeric poly(A) tail prior to nuclear export to the cytoplasm. Once in the cytoplasm mRNAs can be targeted to specific subcellular locations, translated, and ultimately, degraded. All of the steps in mRNA metabolism serve as potential regulatory points. Over the past 17 years we have worked to define the molecular mechanisms by which estrogen effects one of the most dramatic changes in the translational profile of any tissue thus far studied. When Xenopus laevis receive estrogen the liver ceases production of its normal complement of serum proteins, switching instead to the elaboration of large quantities of the yolk protein precursor vitellogenin. This loss in serum protein production is brought about by the destruction of all of the serum protein mRNAs, not by inhibition of their transcription. This process is dependent on the action of the nuclear estrogen receptor, but is independent of the synthesis of a new protein product. Several years ago we identified a ribonuclease activity with sequence specificity for serum protein mRNAs whose appearance on polysomes correlated with the degradation of these mRNAs. Recent work showed that this ribonuclease, termed PMR-1, exists in a latent form in an complex with its substrate mRNA bound to polysomes, and estrogen selectively activates the polysome-bound enzyme to initiate the process of mRNA degradation. We are currently working to identify the proteins that both bind to PMR-1 and constitute the mRNP complex to better understand the processes involved in endonuclease- mediated mRNA decay. Another line of research focuses on the signal transduction pathway(s) responsible for activating PMR-1 and mRNA decay using a cell line that lacks estrogen receptor but contains PMR-1. By transfecting estrogen receptor expression vectors into these cells we can activate mRNA decay upon addition of estradiol to the medium. This will provide a powerful tool for understanding the steps involved between estrogen binding to its receptor and mRNA decay.
Biochemical and genetic analysis of the messenger RNase PMR-1:
The sequence-selective Mr 60,000 RNase was purified from liver polysomes of estrogen-stimulated frogs and its cDNA cloned. Surprisingly it bears no sequence similarity to any known RNases. Rather, it is a member of the peroxidase gene family, showing the greatest sequence similarity to human myeloperoxidase. We named this enzyme PMR-1, for polysomal ribonuclease 1. Unlike enzymes of the peroxidase gene family, PMR-1 lacks both heme and N-linked oligosaccharide. This enzyme is the first vertebrate mRNA endonuclease to be cloned, thus making it a valuable tool in deciphering the processes of mRNA decay. Using purified PMR-1 and the multi-KH-domain protein vigilin we recently demonstrated for the first time the ability of an RNA-binding protein binding over an endonuclease cleavage site to specifically block cleavage by an mRNA endonuclease. Currently we are working to define the portion(s) of PMR-1 involved in its catalytic activity and to clone the human homologue. A long term goal of this work will be to apply gene array technology to identify the substrates of PMR-1 in human cells and to determine whether cell type-specific proteins guide the selection of target mRNAs by this mRNA endonuclease.
Regulated polyadenylation of nuclear pre-mRNA:
A feature all of the estrogen-destabilized serum protein mRNAs has in common is a very short, discrete poly(A) tail of 17-20 nt in length. This contrasts markedly with most somatic mRNAs, whose poly(A) tails are usually 100-200 nt long. Poly(A) addition onto nuclear pre-mRNA occurs in a two-step process in which 10+ residues are added in a slow, distributive reaction, followed by the rapid and processive addition of ~200 adenosine residues. We showed that the short poly(A) tail on albumin mRNA is also present on unprocessed albumin pre-mRNA, thus implicating either regulation of poly(A) addition, or the rapid removal of poly(A) in the nucleus as possible mechanisms for this phenomenon. We have replicated poly(A) length regulation in transfected cells, and using this approach mapped the sequence elements responsible for this (the PLE or poly(A)- limiting element) to the terminal exon of albumin pre-mRNA. The PLE is a conserved element that regulates the length of poly(A) on numerous mRNAs. We are currently working to identify the protein(s) which bind to the PLE, to determine the mechanism responsible for the regulation of poly(A) tail length, and to determine the functional consequences of regulated nuclear polyadenylation.
Recent Publications:
Peng Y and Schoenberg DR (2007) "c-Src activates endonuclease-mediated mRNA decay" Mol Cell 25, 779-87
Murray EL and Schoenberg DR (2007) "A+U-rich instability elements differentially activate 5'-3' and 3'-5' mRNA decay" Mol Cell Biol [Epub ahead of print]
Yang F, Peng Y, Murray EL, Otsuka Y, Kedersha N and Schoenberg DR (2007) "Polysome-bound endonuclease PMR1 is targeted to stress granules via stress-specific binding to TIA-1" Mol Cell Biol 26(23), 8803-13
Hartman TR, Qian S, Bolinger C, Fernandez S, Schoenberg DR and Boris-Lawrie K (2006) "RNA helicase A is necessary for translation of selected messenger RNAs" Nat Struct Mol Biol 13(6), 509-16
Ferraiuolo MA, Basak S, Dostie J, Murray EL, Schoenberg DR and Sonenberg N (2005) "A role for the eIF4E-binding protein 4E-T in p-body formation and mRNA decay" J Cell Biol 170, 913-24.
Peng J, Murray EL and Schoenberg DR (2005) "The poly(A)-limiting element enhances mRNA accumulation by increasing the efficiency of pre-mRNA 3' processing" RNA 11, 958-65.
Peng J and Schoenberg DR (2005) "RNA with a <20 nt Poly(A) tail imparted by the poly(A)-limiting element is translated as efficiently in vivo as long poly(A) mRNA" RNA 11, 1131-40.
Sellers JA, Hou L, Schoenberg DR, Batistuzzo de Medeiros SR, Wahli W and Shelness GS (2005) "Microsomal triglyceride transfer protein promotes the secretion of xenopus laevis vitellogenin A1" J Biol Chem 280(14), 13902-05.
Yang F and Schoenberg DR (2004) "Endonuclease-mediated mRNA decay involves the selective targeting of PMR1 to polyribosome-bound substrate mRNA" Mol Cell 14, 435-45.
Yang F, Peng Y and Schoenberg DR (2004) "Endonuclease-mediated mRNA decay requires tyrosine phosphorylation of polysomal ribonuclease 1 (PMR1) for the targeting and degradation of polyribosome-bound substrate mRNA" J Biol Chem 279, 48993-49002.
Schoenberg DR ed. (2004) Methods in Molecular Biology Vol. 257 - mRNA Processing and Metabolism: Methods and Protocols, Humana Press, Totowa, NJ.
Bremer KA, Stevens A and Schoenberg DR (2003) "An endonuclease activity similar to xenopus PMR1 catalyzes the degradation of normal and nonsense-containing human ß-globin mRNA in erythroid cells" RNA 9, 1157-67.
Stevens A, Zhang J, Bremer K, Hoepfner R, Wang Y, Antoniou M, Schoenberg DR and Maquat LE (2002) "Human ß-globin mRNA decay in erythroid cells: UG site-preferred endonucleolytic cleavage that is augmented by a premature termination codon" Proc Natl Acad Sci USA 99, 12741-46.
