Perturbation of RNA Polymerase I transcription machinery by ablation of HEATR1 triggers the RPL5/RPL11-MDM2-p53 ribosome biogenesis stress checkpoint pathway in human cells

<p>Ribosome biogenesis is an energy consuming process which takes place mainly in the nucleolus. By producing ribosomes to fuel protein synthesis, it is tightly connected with cell growth and cell cycle control. Perturbation of ribosome biogenesis leads to the activation of p53 tumor suppressor protein promoting processes like cell cycle arrest, apoptosis or senescence. This ribosome biogenesis stress pathway activates p53 through sequestration of MDM2 by a subset of ribosomal proteins (RPs), thereby stabilizing p53. Here, we identify human HEATR1, as a nucleolar protein which positively regulates ribosomal RNA (rRNA) synthesis. Downregulation of HEATR1 resulted in cell cycle arrest in a manner dependent on p53. Moreover, depletion of HEATR1 also caused disruption of nucleolar structure and activated the ribosomal biogenesis stress pathway – RPL5 / RPL11 dependent stabilization and activation of p53. These findings reveal an important role for HEATR1 in ribosome biogenesis and further support the concept that perturbation of ribosome biosynthesis results in p53-dependent cell cycle checkpoint activation, with implications for human pathologies including cancer.</p

Super-resolution microscopy of synaptonemal complexes on spreads of (B6.PWD-Chr X.1s×PWD) pachytene spermatocytes.

<p>(A) Detail of a pachytene spermatocyte of a sterile male immunostained by SYCP3 (red) and SYCP1 (green) antibodies. Properly synapsed bivalents (arrows) show two parallel threads of transverse filaments decorated by SYCP1 antibody which form the central region embedded in SYCP3 lateral elements. Unsynapsed chromosomes lack transverse filaments but display some irregular SYCP1 spots (arrowheads). (B) Example of a nonhomologous pairing and/or translocations, and asynapsis in pachynema of (B6.Chr X.1s×PWD)F1 sterile male. Bar 2000 nM.</p

Single QTL mapping of <i>Hstx2</i> on Chr X.

<p>(A) QTL analysis of testes weight in the (B6.PWD-Chr X×B6)F1×PWD cross showed a 1.5-LOD support interval between 34.6 cM to 35.3 cM on Chr X. (B) Distribution of testes weight of males carrying PWD or B6 allele of <i>DXMit87</i> marker with LOD score 30. (C) QTL analysis of sperm count in <i>ductus epididymis</i> shows the same 1.5 LOD support interval as for testes weight. (D) Distribution of sperm count of males carrying PWD or B6 allele of <i>DXMit87</i> marker (Chr X: 66.65 Mb, GRCm38) with LOD score above 20.</p

Meiotic asynapsis in female hybrids.

<p>Abbreviations of consomic strains and their hybrids: DX.1 – B6.PWD-Chr X.1; DX.1PF1 – (B6.PWD-Chr X.1×PWD)F1; DX.1sPF1 – (B6.PWD-Chr X.1s×PWD)F1; DX.1B6F1 – (B6.PWD-Chr X.1×B6)F1; DX.1sB6F1 – (B6.PWD-Chr X.1s×B6)F1; DX.1sD17F1 – (B6.PWD-Chr X.1s×B6.PWD-Chr 17)F1; D17B6F1 – (B6.PWD-Chr 17×B6)F1; B6PF1 – (B6×PWD)F1, PB6F1 – (PWD×B6)F1; PD17F1 – (PWD×B6.PWD-Chr 17)F1. (A) Chromosome synapsis in pachytene oocytes of B6 and (B6.PWD-Chr X.1s×PWD)F1 18.5–19.5 dpc female fetuses was analyzed by combination of SYCP1, SYCP3 and CREST (centromeric heterochromatin) immunostaining or by HORMAD2 and SYCP3 to detect unsynapsed chromosomes. Bar, 10 µm. (B) The frequency of oocytes showing one or more asynaptic chromosomes is similar (>40%) irrespective of <i>Hstx2</i> and <i>Prdm9/Hst1</i> genotype. (C) Although the (PWD×B6), (B6.PWD-Chr X.1s×PWD), (B6.PWD-Chr X.1×PWD) and (B6.PWD-Chr 17×PWD)F1 hybrid females do not differ in percentage of pachytene oocytes with asynapsis, the (B6.PWD-Chr 17×PWD)F1 females, conspecific for Chr 17^PWD, carry significantly less asynapsed chromosomes per cell. (D) The frequency of diplonemas in spread oocyte preparations was significantly lower (p<0.01, χ² test) in intersubspecific hybrids than in parental inbred strains..</p

Asynapsis of individual chromosomes in pachytene oocytes of intersubspecific F1 hybrids and parental controls.

a<p>Each column represents the sum of two or three independent biological replicas. Asynapsis of each Chr was measured in a separate experiment in a separate set of cells. Incidence of asynapsis of a particular Chr is shown for cells with at least one asynapsis. Value in parenthesis is an estimate of overall frequency of asynapsis of a given Chr considering the overall frequency of cells with any asynapsis (Any Chr column). n - number of cells with asynapsis. N - total number of cells examined.</p>b<p>Abbreviations: DX.1sD17F1 – (B6.PWD-Chr X.1s×B6.PWD-Chr 17)F1, DX.1sPF1 – (B6.PWD-ChrX 1s×PWD)F1, PD17F1 – (PWD×B6.PWD-Chr 17)F1.</p

Expression profiling of a cluster of MiRNA genes within the <i>Hstx1/Hstx2</i> critical region.

<p>(A) Log fold-change of expression ratio of PWD versus B6 MiRNA genes in flow-sorted testicular cells. Significant overexpression of Mir465 cluster in PWD germ cells is shown in red. (B) Validation of Mir465 overexpression in PWD primary spermatocytes by qRT PCR. Data normalized to U6 non-coding RNA (C) Log fold-change of expression ratio of MiRNA genes in (PWD×B6)F1 versus (B6×PWD)F1 (abbreviated PB6F1 and B6PF1) 14.5 d old testes. Significant upregulation in red (P<0.05). (D) qRT PCR validation of differences in Mir465 expression. Data normalized to Mir152.</p

The proposed sequence of events leading to male limited sterility of intersubspecific hybrids of house mouse.

<p>Susceptibility of heterosubspecific homologs to asynapsis is common to both sexes. <i>Prdm9/Hst1</i> and <i>Hstx2</i> hybrid sterility genes can modulate this sensitivity from 0% to >95% in spermatogenesis but not in oogenesis, depending on allelic combinations of epistatic DMIs. Multiple asynaptic autosomes provoke MSCI, contributing to hybrid male sterility. Approximately one half of unaffected oocytes ensure fertility of hybrid females. It remains to be established what is the cause of asynapsis of heterosubspecific homologs.</p

Single QTL scan for autosomal loci supporting <i>Hstx2</i> independent intrameiotic arrest of (<i>Mmd</i>×<i>Mmm</i>)F1 intersubspecific hybrids.

<p>(A) Testes weight QTLs (red) reached significance on Chrs 3 (marker UNC030163295, Chr3: 104,320,699) and 13 (JAX00357337 Chr 13:47,975,634) and QTLs for sperm count on Chrs 3, 9 (JAX00171568, Chr 9:56,491,601) and 13. Sperm count was evaluated as a binary trait (SC = 0, SC>0). (B) and (C) Additive effect of QTLs on testes weight and sperm count. For map positions and possible candidate genes see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004088#pgen.1004088.s009" target="_blank">Table S4</a>.</p

Fine mapping of <i>Hstx1</i> and <i>Hstx2</i> HS loci on Chr X using partial consomic strains.

<p>The partial consomic B6.PWD-Chr X.# females were crossed with B6 or PWD males for mapping <i>Hstx1</i> and <i>Hstx2</i>, respectively. Testes weight and sperm count were used as fertility phenotypes. The borders of introgressed PWD sequence (black) were determined by MegaMUGA genotyping for B6.PWD-Chr X.1 (abbreviated here X.1), B6.PWD-Chr X.1s (X.1s) and B6.PWD-Chr X.2 (X.2). For B6.PWD-Chr X.3 (X.3) mapping see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004088#pgen.1004088-Gregorova1" target="_blank">[20]</a>. The map positions correspond to genome assembly GRCm38, megabase scale, for details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004088#pgen.1004088.s006" target="_blank">Table S1</a>.</p

Additional file 1: of Characteristics of responders to autologous bone marrow cell therapy for no-option critical limb ischemia

Presents the dataset supporting the conclusions of this article. (XLSX 26 kb