Aging | doi:10.18632/aging.204666. Noah Wechter, Martina Rossi, Carlos Anerillas, Dimitrios Tsitsipatis, Yulan Piao, Jinshui Fan, Jennifer L. Martindale, Supriyo De, Krystyna Mazan-Mamczarz, Myriam Gorospe
The chlorophycean taxa are distinctive from other green algae in that their mitochondrial genome sizes are reduced and their mitochondrial ribosomal RNA genes are fragmented and scrambled. To understand mitochondrial genome evolution in the Chlorophyceae, which needs more information from other taxa, and to develop a system for green algal mitochondrial studies, this work undertook the characterization of the mitochondrial genome and ribosomal RNAs of Polytomella parva. This taxon's colorless and cell wall-less features facilitate the isolation of mitochondrial-enriched fractions free of thylakoid membranes. At least two linear mitochondrial DNA (mtDNA) components with sizes of 13.5 and 3.5-kb were detected. Sequences spanning 97 and 86% of these mtDNAs, respectively, revealed that these molecules contain long, at least 1.3 kb, homologous inverted repeat sequences at their termini. The 3.5-kb mtDNA has only one coding region (nad6), the functionality of which is supported by both the relative rate at which it has accumulated non-synonymous nucleotide substitutions and its absence from the 13.5-kb mtDNA which encodes nine genes (i.e., large and small subunit rRNA genes, one tRNA gene, and six protein-coding genes). Based on DNA sequence data, it is proposed that a variant start codon, GTG, is utilized by the P. parva 13.5-kb mtDNA-encoded gene, nad5. Using the relative rate test with Chlamydomonas moewusii (= C. eugametos) as the outgroup, it is concluded that the non-synonymous nucleotide substitution rate in the mitochondrial protein-coding genes of P. parva is on average about 3.3 times that of the Chlamydomonas reinhardtii counterparts. RNA from a mitochondrial-enriched preparation was isolated, and the transcripts of the four small subunit (SSU) and eight large subunit (LSU) rRNA-coding regions were characterized by either Northern blot analysis or chemical sequencing. Secondary structure modeling of the SSU and LSU rRNA sequences was performed. The results show that (1) both the mitochondrial SSU and LSU rRNAs of P. parva are considerably shorter than their homologs from other green algae, although the main domains typical of conventional rRNAs are conserved, (2) the rRNA fragmentation pattern is different from that of other chlorophycean species but most similar to that of C. reinhardtii, among those that have been characterized, (3) three nucleotides are missing from the normally highly conserved GTPase centre of the LSU rRNA, and (4) post-transcriptional modification of the 3'-terminal region of the SSU rRNA is unusual; it has the eubacterial 3-methyluridine (corresponding to m3U at Escherichia coli 16S rRNA position 1498), but lacks the more highly conserved modifications at two adjacent A residues (corresponding to N6,N 6-dimethyladenosine at E. coli 16S rRNA positions 1518 and 1519). This research presents the first example of subgenomic mtDNAs of a green alga. The obtained data provide the necessary background for more direct functional studies of the chlorophycean mitochondrial ribosome and rRNAs, using P. parva as a model system.
A recent analysis of gene expression in renal cell carcinoma cells led to the identification of mRNAs whose translation was dependent on the presence of the von Hippel-Lindau (VHL) tumor suppressor gene product, pVHL. Here, we investigate the finding that pVHL-expressing RCC cells (VHL(+)) exhibited elevated levels of polysome-associated p53 mRNA and increased p53 protein levels compared with VHL-defective (VHL(-)) cells. Our findings indicate that p53 translation is specifically heightened in VHL(+) cells, given that (i) p53 mRNA abundance in VHL(+) and VHL(-) cells was comparable, (ii) p53 degradation did not significantly influence p53 expression, and (iii) p53 synthesis was markedly induced in VHL(+) cells. Electrophoretic mobility shift and immunoprecipitation assays to detect endogenous and radiolabeled p53 transcripts revealed that the RNA-binding protein HuR, previously shown to regulate mRNA turnover and translation, was capable of binding to the 3' untranslated region of the p53 mRNA in a VHL-dependent fashion. Interestingly, while whole-cell levels of HuR in VHL(+) and VHL(-) cells were comparable, HuR was markedly more abundant in the cytoplasmic and polysome-associated fractions of VHL(+) cells. In keeping with earlier reports, the elevated cytoplasmic HuR in VHL(+) cells was likely due to the reduced AMP-activated kinase activity in these cells. Demonstration that HuR indeed contributed to the increased expression of p53 in VHL(+) cells was obtained through use of RNA interference, which effectively reduced HuR expression and in turn caused marked decreases in p53 translation and p53 abundance. Taken together, our findings support a role for pVHL in elevating p53 expression, implicate HuR in enhancing VHL-mediated p53 translation, and suggest that VHL-mediated p53 upregulation may contribute to pVHL's tumor suppressive functions in renal cell carcinoma.
Cellular senescence is a complex biological response to sublethal damage. The RNA-binding protein HNRNPK was previously found to decrease prominently during senescence in human diploid fibroblasts. Here, analysis of the mechanisms leading to reduced HNRNPK abundance revealed that in cells undergoing senescence, HNRNPK mRNA levels declined transcriptionally and full-length HNRNPK protein was progressively lost, while the abundance of a truncated HNRNPK increased. The ensuing loss of full-length HNRNPK enhanced cell cycle arrest along with increased DNA damage. Analysis of the RNAs enriched after HNRNPK ribonucleoprotein immunoprecipitation (RIP) revealed a prominent target of HNRNPK, CDC20 mRNA, encoding a protein critical for progression through the G2/M phase of the cell division cycle. Silencing HNRNPK markedly decreased the levels of CDC20 mRNA via reduced transcription and stability of CDC20 mRNA, leading to lower CDC20 protein levels; conversely, overexpressing HNRNPK increased CDC20 production. Depletion of either HNRNPK or CDC20 impaired cell proliferation, with a concomitant reduction in the levels of CDK1, a key kinase for progression through G2/M. Given that overexpressing CDC20 in HNRNPK-silenced cells partly alleviated growth arrest, we propose that the reduction in HNRNPK levels in senescent cells contributed to inhibiting proliferation at least in part by suppressing CDC20 production.
