Placenta specific and ribosomal RNA genes: structure and function

National Institute On Aging

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Abstract

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-beta) 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 PLAC1 interacts 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 ribosome 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 from Homo sapiens sapiens is present as fragmented pieces due to insertions and deletions in non-human primates, chimpanzee, orangutan and gorilla but is fully conserved in bonobo. Compared to the original ORF identified from a mouse human chromosome21 hybrid identified the sequence from CHM13 differs by one conserved amino acid change, Leucine>Valine (L>V), at position 129. Another interesting observation was that on chromosome 21 there are 38 copies of the ORF in sequential rDNA repeats 17-55 but chromosomes 13 15 and 22 contain one copy each at the most centromeric end rDNA repeat and chromosome 14 has 2 repeats. Our further, examination of rDNA sequences from 50 pangenome samples representing African, East and South Asian, American and Carribean samples showed that the ORF exists at 50 to 60 copies per haploid genome and majority of the ORF have L>V change along with the original sequence similar to the original ORF. An analysis of bonobo sequence shows additional frame preserving variations in the ORF. 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. This work is now currently submitted for publication and is under review. 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 mouse clones have been analyzed. Twenty-eight of the 53 mouse 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 describing this work is published in NAR genome and Bioinformatics.