Autogenous Control of 5′TOP mRNA Stability by 40S Ribosomes

Abstract

Ribosomal protein (RP) expression in higher eukaryotes is regulated translationally through the 5′TOP sequence. This mechanism evolved to more rapidly produce RPs on demand in different tissues. Here we show that 40S ribosomes, in a complex with the mRNA binding protein LARP1, selectively stabilize 5′TOP mRNAs, with disruption of this complex leading to induction of the impaired ribosome biogenesis checkpoint (IRBC) and p53 stabilization. The importance of this mechanism is underscored in 5q− syndrome, a macrocytic anemia caused by a large monoallelic deletion, which we found to also encompass the LARP1 gene. Critically, depletion of LARP1 alone in human adult CD34+ bone marrow precursor cells leads to a reduction in 5′TOP mRNAs and the induction of p53. These studies identify a 40S ribosome function independent of those in translation that, with LARP1, mediates the autogenous control of 5′TOP mRNA stability, whose disruption is implicated in the pathophysiology of 5q− syndrome.

Keywords: 40S ribosomes; 5?TOP mRNAs; 5q(?) syndrome; impaired ribosome biogenesis checkpoint; p53 stabilization; polysome profiles; ribosomal proteins.

Figure 1. Effect of LARP1 depletion on p53 stabilization and cell cycle progression

(A) Total RNA extracted from HCT116 cells transfected with siLARP1 or siNS for 48h was used for northern blot analysis and hybridized with DNA oligos complementary to either RPL5 or RPL11 mRNAs. (B) HCT116 cells were transfected with the indicated siRNAs. Whole cell lysates were analyzed by western blot analysis for the indicated proteins with the corresponding antibodies. Data is representative of four independent experiments (C) HCT116 cells treated as in panel A were subjected to propidium iodide staining and FACS analysis. Data is representative of three independent experiments. (D) HCT116 cells were transfected with siLARP1 or siNS for 24h, followed by a second transfection with siRPL7a and/or siRPL11 for an additional 24h. Data is representative of three independent experiments. α-tubulin serves as loading control.

Figure 2. Effect of LARP1 depletion on newly synthesized proteins and ribosomes

(A) HCT116 cells were transfected with siLARP1 or siNS for 48h and de novo synthesized proteins were labelled by alkyne-biotine click-iT reaction (see Experimental Procedures). The labeling efficiency of the experimental conditions was evaluated by western blotting of biotinylated proteins and hybridizing the membrane with streptavidin-IRDye800. The intensity of the signals was detected using an infrared scanner and was compared to Amido Black membrane staining. (B) Total cell extracts (INPUT) and nascent proteins from INPUTs, pulled-down using streptavidin-agarose beads (STRP-pulldown), were resolved by western blot analysis with the indicated antibodies. Data is representative of three independent experiments (C) HCT116 were transfected with a plasmid encoding for LARP1 protein. 48h after transfection cells were labeled and processed as in panel A. (D) HCT116 cells were treated with the indicated siRNAs for 48h or siS6 for 24h. Cells were pulsed with ³H-Uridine and chased with non-radioactive uridine. Total RNAs were extracted and resolved by Northern blot analysis and exposed in presence of emission enhancer. This experiment is representative of 3 independent experiments. (E) Quantification of the global protein synthesis rate by the incorporation of ³H-leucine into total protein (Data are means +/− s.e.m. n=3) *p<0.01 calculated by Student’s t test.

Figure 3. Analysis of the distribution of RPL11 and RPL23 mRNAs on polysome gradients

(A) Polysome profiles of HCT116 cells transfected with siLARP1 or siNS for 48h. The polysome profile is representative of three independent experiments. RNA analyses were carried out as described in Experimental Procedures. (A) RPL11 mRNA, (B) RPL5, (C) RPL23 and (D) HSPA1A mRNAs distribution across the gradient was evaluated in each fraction by RT-qPCR as described in Experimental Procedures. Error bars represent replicates for RT-qPCR (for RPL11 three more replicates of this experiment are shown in Fig S3A and Fig 6C) (E) HCT116 cells were transfected with the indicated siRNAs for 48h, metabolically labeled with ³⁵S-Met/Cys for 2h in DMEM 10% FBS, and chased for the indicated times with non-radioactive DMEM 10% FBS. Equal amounts of whole protein lysates in each condition were subjected to immunoprecipitation with α-L5 or α-α-tubulin antibodies. Immunocomplexes were analyzed by SDS-PAGE and visualized by autoradiography.

Figure 4. The effect of LARP1 depletion on the half-life of 5′TOP mRNA reporters

(A) Effect of rapamycin treatment on the polysomal distribution of WT-RPL32- and MU-RPL32-β-globin reporter transcripts. Following fractionation and purification, RNA was probed by northern blot with an oligo complementary to β-Globin mRNA. (B) LARP1 immunoprecipitation from cells expressing WT-RPL32- or MU-RPL32-β-Globin mRNAs. Top Panel: LARP1-immunoprecipitated complexes were RNA purified, and analyzed by RT-qPCR. The fold enrichment of β-globin cDNA was measured as compared to complexes immunoprecipitated by an IgG control antibody. Bottom panel: 20% of immunoprecipitated complexes were subjected to western blot analysis (C) Determination of WT-RPL32- and MU-RPL32-β-Globin mRNA stability upon LARP1 depletion. Tet-ON/Doxycycline-inducible WT-RPL32- or MU-RPL32-β-globin reporters stably transfected HCT116 cells were induced with doxycyline for 16h, followed by siRNA transfection for 24h. Cells were deprived of doxycycline and harvested at the indicated times. Equal amounts of total RNA were analyzed by northern blot and probed for β-globin reporter (left panel). Total RNA from biological replicates of the same samples were analyzed for β-globin mRNA by RT-qPCR and 28S rRNA (middle panel) or 18S rRNA (right panel) were used as internal controls. All time points were normalized to time 0h (right panel). P<0.05 by Student’s two-tailed t-test, mean and s.e.m. shown (n=3) (D) HCT116 Tet-ON stable clones were transfected with Doxycyline-inducible WT- or MU-RPL32-β-globin reporter plasmids, then treated with doxycycline for 24h. Total RNA was analyzed by RT-qPCR and the indicated mRNAs were measured and normalized by 28S rRNA. Data are means +/− s.e.m. n=2. * p<0.01, **p<0.05 by t Student’s.

Figure 5. 40S ribosomes control 5′TOP mRNA stability

(A) HCT116 cells were treated with siRNA targeting the indicated RPs for 48h. Equal amounts of whole cell extracts, based on total RNA, were resolved on northern blots probed for RPL11, RPL5, eEF1α and β-actin mRNAs.(B) LARP1 was immunoprecipitated from cells transfected with the indicated siRNA for 24 h and the presence of endogenous 5′TOP mRNAs, RPL23 and RPL11, or a non-TOP mRNAs, β-Actin, were determined by RT-qPCR. Data are mean +/− s.e.m. n=3 * p<0.01, **p<0.05 by t Student’s. (C) WT-RPL32 or MU-RPL32-β-globin mRNA stability was determined as in Figure 4C. β-globin mRNA was normalized by 28S rRNA in siNS and siS6 transfected cells (left panel) or by 18S rRNA in siNS and siL7a-transfected cells (right panel). p<0.05 by Student’s two-tailed t-test, mean and s.e.m. shown (n=3).

Figure 6. Native 40S ribosomes control 5′TOP mRNA stability

Polysome distribution from HCT116 cells transfected with the indicated siRNAs for 40 h A, B and C. (A) The distribution of RPL11 mRNA on a 10–50% sucrose gradient following siRNA depletion of RPS6, (B) RPL7a or (C) LARP1 after 40 h of transfection. Data are means +/− s.e.m. n=2. (D) Protein extracted from the 20% of polysome profiles fractions shown in panel A, B and C were (upper panel) subjected to dot blot analysis with an α-LARP1 antibody after staining with amido black (lower panel). Data is representative of 3 independent experiments. (E) Immunoprecipitation of LARP1 from 40S fraction isolated from growing HCT116 cells. Immunocomplexes were analyzed by western blot and probed with α-LARP1, α-S6 and α-S19 antibodies. 20% of immunoprecipitates were spiked with Luciferase mRNA external control, RNA purified and 18S rRNA and Luciferase mRNA were measured by RT-qPCR. (n=4, p<0.01) (F) Boxplot showing mRNA enrichment over inputs in the 40S peak immunoprecipitated with the LARP1 antibody and subjected to RNA-seq. Left, non-5′-TOP boxplot of 680 genes whose mRNAs are lacking a 5′-TOP. Middle, 5′-TOP boxplot of 236 genes, excluding RP genes, whose mRNAs contain a 5′TOP. Right, boxplot of 74 RP genes. T-tests on log2 fold enrichment values show all groups have significant differences, with a p-value < 2.2E-16. 5′TOP transcripts considered in the analysis were isoforms starting with a C and showing a minimum of 4 consecutive pyrimidines (See Table S1). (G) α-LARP1 immunoprecipitation from 40S fraction obtained from siNS or siL7a transfected cells, as described in E. The indicated mRNAs were measured by RT-qPCR in the 40S INPUT fractions (left panel) and in the α-LARP1 immunocomplexes enriched over IgG control IP (right panel). Data are mean −/+ s.e.m. n=4 for siNS and n=2 for siL7a. p<0.01 calculated by Student’s t test. (H) Immunoprecipitation of LARP1 form 40S fractions isolated from growing cells or cells treated with 40nM Rapamycin for 5h. RT-qPCR analysis was carried out as described in G. (I) 20% of polysomal lysates isolated in H were subjected to dot-blot analysis as in D. This data is representative of two experiments.

Figure 7. Loss of LARP1 and 5′TOP mRNAs in 5q⁻ patients and CD34+ cells

(A) Heat map representation of log₂ fold change of a 5′TOP mRNAs panel, including RPs of 5q⁻ and non 5q⁻ MDS patients versus controls (left) and a panel of non-5′TOP mRNAs for both groups (right). (+): significant fold changes; (++) those significant genes with FDR < 5%. (B) LARP1 expression levels in CD34+ cells from a cohort of 47 5q⁻ patients and 136 non 5q⁻ patients compared to 17 healthy individuals. (C) CD34+ cells from healthy donors were transduced with shControl or shLARP1 lentiviral particles and subjected to erythroid lineage differentiation protocol (see Experimental Procedures). Total RNA extracted at 7 days after transduction were analyzed by RT-qPCR. Total level of LARP1 and six RP mRNAs (left panel) and p53 target genes (right panel) were determined. (D) CD34+ cells from healthy donors treated as in C were analyzed at 7 days or 10 days after transduction by RT-qPCR. (E) HCT116 cells transfected with siLARP1 or siNS for 24 h and followed by siS14 or siS6 treatment for an additional 24h. Protein lysates were analyzed on western blots for the indicated proteins. (F) Left hand panel, LARP1 mediates the interaction between native 40S ribosomes and 5′TOP mRNAs. Middle panels, loss of either LARP1 or native 40S subunits leads to activation of the IRBC and stabilization of p53. Right hand panel, 5q- syndrome leads to a reduction of both LARP1 and native 40S ribosomes, hyperactivation of IRBC and p53, resulting in anemia.