Placenta specific and ribosomal RNA genes: structure and function

先前对于PLAC 1，为了确定其非常选择性的组织特异性表达的基础，我们已经表明该基因由两个启动子P1和P2表达，间隔105千碱基，并且是可变剪接的。通过克隆小鼠和人的每个启动子，定义了最小启动子区域。最小启动子区在特定位点结合核受体视黄酸X受体α（RXR-α）、LXR-β和类固醇生成因子1（SF 1）/雌激素相关受体β（ERR-），并且在它们各自的激动剂存在下，它们的结合具有刺激转录>10倍的积极作用（Placenta. 2011年11月;32（11）：877-84）。 在Oncogenesis（2013）的后续出版物中，通过建立癌细胞系的经典方法评估了癌细胞中的Plac 1表达; SV 40介导的原代细胞WI 38和IMR 90细胞的转化。我们发现，在SV 40介导的转化后，原代细胞诱导Plac 1和一系列步骤由SV 40早期区域编码的大T抗原催化，该抗原改变了通常与启动子区域结合的Tp 53阻遏物的性质，使其失去其阻遏能力，导致染色质从封闭状态变为开放状态，从而促进Plac 1的转录。然后在核受体存在下进一步刺激转录，并且如果存在额外的共激活因子NCOA 2（核受体共激活因子2），则其招募RB，导致基因的额外上调。因此，我们已经确定了基因在癌细胞中被激活的主要方式，从而提供了抑制基因活性的途径。 最近，我们已经表明，在生化水平上，Plac 1与桥粒，特别是桥粒芯糖蛋白的C-末端部分的相互作用。桥粒芯糖蛋白的C-末端被切割并作为外泌体的一部分分泌，因此可以在妊娠期间作为PLAC 1进入血流的载体，在妊娠期间以高水平检测到PLAC 1。PLAC 1的水平在母体（胎盘）的血流中分娩后显著降低。2021年7月，110：39-45）。 对于rDNA结构分析，克隆和分析问题与其他2个NIH小组合作解决。J.H. NCI的Kim和弗拉基米尔Larionov创建了一种基于转化相关重组（TAR）的先进克隆方法，该方法提供了具有多达2个rDNA重复单位的稳定克隆; NHGRI的Adam Phillippy和Alex Dilthey采用了先进的长读段测序技术（PacBio和Nanopore）以促进序列恢复和组装;我们为分析提供了注释和背景。迄今为止的主要发现是，核糖体DNA和转录区域，包括5 prime和3 prime外部转录序列，以及内部转录序列，所有这些都在组装过程中被消除，但对于成熟核糖体的形成至关重要，含有许多变体，其中一小部分深深扎根于人类进化中。13个克隆，约0.32倍的覆盖率（0.82 Mb）的染色体21 rDNA互补，揭示了以前错过了2 kb的道，几个回文结构，和超过300个变种，85个变种落在成熟的18 S/28 S rRNA序列。在独立的全基因组或RNA-Seq数据中也发现了回文断点和>80%的45 S变异等位基因，表明许多变异在人类群体中长期存在。我们已经开发了一个更新的44，838 bp rDNA参考序列注释检测到的变异，这表明了一个可能的路线，以完成分析的rDNA组成的人类基因组。大量的变异揭示了人类核糖体DNA中比以前认为的更多的、更普遍的异质性，从而打开了核糖体动力学中相应变异的可能性。此外，我们还将研究扩展到了含有人22号染色体的小鼠-人杂交细胞系中22号染色体的rDNA单元，收集了来自22号染色体的额外克隆，测序，组装，注释并提交给Genbank。这些分离株包括端粒和着丝粒边界侧翼的rDNA重复从染色体22。描述这项工作的手稿现已出版（Sci Rep 11，2997。https://doi.org/10.1038/s41598-021-82565-x) 在对人rDNABAC克隆进行分析的过程中，我们在基因间隔区发现了一个编码190个氨基酸的开放阅读框架（ORF）。 该ORF是智人所特有的，并且不存在于非人灵长类动物中。 我们现在已经证实，这是一个表达的ORF，通过提高抗体对蛋白质。 我们发现这种蛋白质在衰老和癌细胞中上调。 该蛋白在蔗糖梯度中跟踪核糖体亚基，并与核糖体加工蛋白相互作用。 这项工作目前正在处理出版。 我们现在已经将rDNA分析扩展到非人灵长类动物和小鼠。现在已经分析了来自黑猩猩、大猩猩、猩猩和恒河猴细胞系的基于TAR克隆的克隆沿着来自小鼠的53个克隆。将其组装与CHEF-凝胶大小估计一致的53个克隆中的28个与数据库序列和变体进行比较。 序列分析帮助我们完善了数据库中这些物种的rDNA基因座序列，识别和编目了新的变体。结果表明，小鼠45 S RNA含有比以前鉴定的显著量的变异，并提供了在正在进行的工作中测试这些变异体在酵母细胞中的作用的方法。 此外，我们还发现，由于重复序列之间的缺失和插入，基因间间隔区（IGS）在重复序列之间存在差异，IGS区的3端存在一个150 bp的多拷贝区域。 目前正在起草一份手稿，以供出版，其中将包括这些意见。

Previously for PLAC1, to determine the basis for its extraordinarily selective tissue-specific expression, we have shown that the gene is expressed from two promoters, P1 and P2, spaced 105 Kilobases apart and is alternatively spliced. By cloning each promoter from both mouse and human, defined the minimal promoter regions. The minimal promoter region binds nuclear receptors Retinoic Acid X Receptor alpha (RXR-alpha), LXR-beta, and Steroidogenic factor 1 (SF1)/ Estrogen related receptor beta (ERR-) at specific sites and their binding has a positive effect stimulating transcription >10 fold, in the presence of their respective agonists (Placenta. 2011 Nov;32(11):877-84). In a follow up publication, in Oncogenesis (2013), Plac1 expression in cancer cells was evaluated by a classical approach establishing cancer cell lines; SV40 mediated transformation of primary cells WI38 and IMR90 cells. We found that following SV40 mediated transformation the primary cells induced PLAC1 and a series of steps are catalyzed by Large T antigen encoded by SV40 early regions that modify Tp53 repressor properties normally bound to the promoter region such that it loses its repressive ability, bring about changes in chromatin from closed to open status facilitating Plac1 transcription. The transcription is then further stimulated in the presence of nuclear receptors and if an additional coactivator NCOA2 (nuclear receptor co-activator2) is present, it recruits RB, leading to additional up-regulation of the gene. Thus, we have defined a major way in which the gene is activated in cancer cells, which thereby provides a route to repress the gene activity. Recently, we have shown at the biochemical level that Plac1interacts with desmosomes specifically c-terminal portion of desmoglein. The c-terminal end of desmoglein is cleaved and is secreted as part of exosomes and thus could act as a carrier for PLAC1 into the blood stream during pregnancy, where it is detected at high levels during pregnancy. The levels of PLAC1 decreases dramatically after delivery in the blood stream of mother (Placenta. 2021 July, 110:39-45). For rDNA structure analysis the cloning and analysis problems were resolved with collaborations with 2 other NIH groups. J.H. Kim and Vladimir Larionov at NCI created an advanced approach to cloning based on transformation associated recombination (TAR), that provided stable clones with up to 2 repeat units of rDNA; Adam Phillippy and Alex Dilthey at NHGRI adapted advanced long-read sequencing techniques (PacBio and Nanopore) to facilitate sequence recovery and assembly; and we supplied annotation and context for the analyses. The major findings thus far are that ribosomal DNA, and transcribed regions that included 5 prime and 3 prime external transcribed sequences, and internal transcribed sequences, all of which are eliminated during assembly but are essential for formation of mature ribosomes harbor many variants, with a fraction of them deeply seated in human evolution. Thirteen clones, about 0.32-fold coverage (0.82 Mb) of the chromosome 21 rDNA complement, revealed a previously missed 2 kb tract, several palindromic structures, and over 300 variants; 85 variants fall in mature 18S/28S rRNA sequences. Palindromic breakpoints and >80% of 45S variant alleles were also found in independent whole-genome or RNA-Seq data, indicating that many variants are long established in human populations. We have developed an updated 44,838 bp rDNA reference sequence annotated with detected variants, suggesting a possible route to complete analysis of the rDNA component of the human genome. The large number of variants reveal more - and more universal - heterogeneity in human ribosomal DNA than previously considered, opening the possibility of corresponding variations in ribosome dynamics. Further, we have extended the study to rDNA units in chromosome 22 from mouse-human hybrid cell-line containing human chromosome 22, collected additional clones from chromosome 22, sequenced, assembled, annotated and submitted to Genbank. These isolates include the telomeric and centromeric borders flanking the rDNA repeats from chromosome 22. A manuscript describing this work is now published (Sci Rep 11, 2997. https://doi.org/10.1038/s41598-021-82565-x) During the analysis of human rDNA BAC clones, we identified an open reading frame (ORF) in the intergenic spacer coding for 190 amino-acid protein. This ORF is unique to Homo sapiens sapiens and is not present in non-human primates. We have now confirmed that this is an expressed ORF by raising antibody against the protein. We find that this protein is upregulated in senescent and cancer cells. The protein tracks with ribosomal subunits in a sucrose gradient and interacts with ribosomal processing proteins. This work is currently being processed for publication. We have now extended the rDNA analysis to non-human primates and mouse. TAR cloning based clones from chimp, gorilla, orangutan, and rhesus monkey cell lines along with 53 clones from mouse have been now analyzed. Twenty-eight of the 53 clones whose assemblies agreed with the CHEF-gel size estimation were compared with database sequence and variants scored. The sequence analysis has helped us to refine the current rDNA locus sequence for these species present in the database, identify and catalog new variants. The results have shown that the mouse 45S RNA contains significant amount of variation than identified before and provides a means to test the effect of these variants in yeast cells in ongoing work. Additionally, we have found that Intergenic Spacer Region (IGS) varies among repeats due to deletions and insertions between repeats and multiple copies of a 150 bp region is present at the 3end of IGS region. A manuscript is being drafted for publication to include these observations.