Borsu L, Presse F, Nahon J L
Institut de Pharmacologie Moléculaire et Cellulaire-CNRS UPR 411, 660 Route des Lucioles-Sophia-Antipolis, 06560 Valbonne, France.
J Biol Chem. 2000 Dec 22;275(51):40576-87. doi: 10.1074/jbc.M006524200.
Melanin-concentrating hormone (MCH) mRNA expression is induced by nerve growth factor and lithium in PC12 cells, whereas three large MCH RNA species are found in untreated cells. In this study, we investigated the structures, regulations of expression, and putative functions of these transcripts. Northern blot, rapid amplification of cDNA ends-polymerase chain reaction, reverse transcriptase-polymerase chain reaction, and sequencing experiments demonstrated that they are antisense RNAs complementary to the MCH gene. Two classes of antisense RNAs could be discriminated as follows: 1) non-coding unspliced RNAs that overlap mainly the coding part of the MCH gene; 2) spliced variant mRNAs complementary to the 3'-flanking end of the MCH gene and that encode putative proteins containing DNA/RNA binding domains. We named this new transcriptional unit AROM for antisense-RNA-overlapping-MCH gene. Spliced variant AROM mRNAs are expressed in a broad range of rat organs. Western blot and immunohistochemistry experiments revealed several proteins with cytoplasmic but also nuclear localization in PC12 cells. Time course studies during nerve growth factor and lithium treatment of PC12 cells indicated a reciprocal regulation of the MCH and AROM gene transcripts, reflected also at the level of AROM proteins. The major translational product is a 64-kDa protein (AROM-p64). Recombinant AROM-p64 displayed high binding to single-stranded DNA and poly(A) homopolymers suggesting that this protein could play a role in mRNA maturation/metabolism.
在PC12细胞中,神经生长因子和锂可诱导黑色素浓集激素(MCH)mRNA表达,而在未处理的细胞中可发现三种较大的MCH RNA种类。在本研究中,我们调查了这些转录本的结构、表达调控及假定功能。Northern印迹、cDNA末端快速扩增-聚合酶链反应、逆转录酶-聚合酶链反应和测序实验表明,它们是与MCH基因互补的反义RNA。可将两类反义RNA区分如下:1)主要与MCH基因编码部分重叠的非编码未剪接RNA;2)与MCH基因3'侧翼末端互补且编码含有DNA/RNA结合结构域的假定蛋白质的剪接变体mRNA。我们将这个新的转录单位命名为AROM,即反义RNA重叠MCH基因。剪接变体AROM mRNA在大鼠的多种器官中表达。蛋白质印迹和免疫组织化学实验揭示了PC12细胞中几种定位于细胞质但也定位于细胞核的蛋白质。对PC12细胞进行神经生长因子和锂处理的时间进程研究表明,MCH和AROM基因转录本相互调控,这在AROM蛋白水平也有体现。主要的翻译产物是一种64 kDa的蛋白质(AROM-p64)。重组AROM-p64对单链DNA和聚(A)均聚物显示出高结合力,表明该蛋白质可能在mRNA成熟/代谢中发挥作用。