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MYC-driven B-cell lymphomas are addicted to increased levels of ribosome biogenesis (RiBi), offering the potential for therapeutic intervention. However, it is unclear whether inhibition of RiBi suppresses lymphomagenesis by decreasing translational capacity and/or by p53 activation mediated by the impaired RiBi checkpoint (IRBC). Here we generated Eμ-Myc lymphoma cells expressing inducible short hairpin RNAs to either ribosomal protein L7a (RPL7a) or RPL11, the latter an essential component of the IRBC. The loss of either protein reduced RiBi, protein synthesis, and cell proliferation to similar extents. However, only RPL7a depletion induced p53-mediated apoptosis through the selective proteasomal degradation of antiapoptotic MCL-1, indicating the critical role of the IRBC in this mechanism. Strikingly, low concentrations of the US Food and Drug Administration–approved anticancer RNA polymerase I inhibitor Actinomycin D (ActD) dramatically prolonged the survival of mice harboring Trp53+/+;Eμ-Myc but not Trp53–/–;Eμ-Myc lymphomas, which provides a rationale for treating MYC-driven B-cell lymphomas with ActD. Importantly, the molecular effects of ActD on Eμ-Myc cells were recapitulated in human B-cell lymphoma cell lines, highlighting the potential for ActD as a therapeutic avenue for p53 wild-type lymphoma.

SummaryRecently, we demonstrated that RPL5 and RPL11 act in a mutually dependent manner to inhibit Hdm2 and stabilize p53 following impaired ribosome biogenesis. Given that RPL5 and RPL11 form a preribosomal complex with noncoding 5S ribosomal RNA (rRNA) and the three have been implicated in the p53 response, we reasoned they may be part of an Hdm2-inhibitory complex. Here, we show that small interfering RNAs directed against 5S rRNA have no effect on total or nascent levels of the noncoding rRNA, though they prevent the reported Hdm4 inhibition of p53. To achieve efficient inhibition of 5S rRNA synthesis, we targeted TFIIIA, a specific RNA polymerase III cofactor, which, like depletion of either RPL5 or RPL11, did not induce p53. Instead, 5S rRNA acts in a dependent manner with RPL5 and RPL11 to inhibit Hdm2 and stabilize p53. Moreover, depletion of any one of the three components abolished the binding of the other two to Hdm2, explaining their common dependence. Finally, we demonstrate that the RPL5/RPL11/5S rRNA preribosomal complex is redirected from assembly into nascent 60S ribosomes to Hdm2 inhibition as a consequence of impaired ribosome biogenesis. Thus, the activation of the Hdm2-inhibitory complex is not a passive but a regulated event, whose potential role in tumor suppression has been recently noted.

Long INterspersed Element one (LINE-1, or L1), is a widely distributed, autonomous retrotransposon in mammalian genomes. During retrotransposition, L1 RNA functions first as a dicistronic mRNA and then as a template for cDNA synthesis. Previously, we defined internal ribosome entry sequences (IRESs) upstream of both ORFs (ORF1 and ORF2) in the dicistronic mRNA encoded by mouse L1. Here, RNA affinity chromatography was used to isolate cellular proteins that bind these regions of L1 RNA. Four proteins, the heterogeneous nuclear ribonucleoproteins (hnRNPs) R, Q and L, and nucleolin (NCL), appeared to interact specifically with the ORF2 IRES. These were depleted from HeLa cells to examine their effects on L1 IRES-mediated translation and L1 retrotransposition. NCL knockdown specifically reduced the ORF2 IRES activity, L1 and L1-assisted Alu retrotransposition without altering L1 RNA or protein abundance. These findings are consistent with NCL acting as an IRES trans-acting factor (ITAF) for ORF2 translation and hence a positive host factor for L1 retrotransposition. In contrast, hnRNPL knockdown dramatically increased L1 retrotransposition as well as L1 RNA and ORF1 protein, indicating that this cellular protein normally interferes with retrotransposition. Thus, hnRNPL joins a small, but growing list of cellular proteins that are potent negative regulators of L1 retrotransposition.

Retrotransposition amplifies LINE-1 (L1) to high copy number in mammalian genomes. The L1 protein encoded by ORF1 (ORF1p) is required for retrotransposition. This dependence on ORF1p was investigated by mutating three highly conserved residues, R238, R284 and Y318 to alanine, thereby inactivating retrotransposition. R284A and Y318A were rescued by further substituting the alanine with the appropriate conservative amino acid, e.g. lysine or phenylalanine, respectively, whereas R238K remained inactive. Quantification of the steady-state levels of L1 RNA and ORF1p failed to discriminate active from inactive variants, indicating loss of L1 retrotransposition resulted from loss of function rather than reduced expression. The two biochemical properties known for ORF1p are high-affinity RNA binding and nucleic acid chaperone activity. Only R238A/K exhibited significantly reduced RNA affinities. The nucleic acid chaperone activities of the remaining paired mutants were assessed by single-molecule DNA stretching and found to mirror retrotransposition activity. To further examine ORF1p chaperone function, their energetic barriers to DNA annealing and melting were derived from kinetic work. When plotted against each other, the ratio of these two activities distinguished functional from non-functional ORF1p variants. These findings enhance our understanding of the requirements for ORF1p in LINE-1 retrotransposition and, more generally, nucleic acid chaperone function.

A genomic PCR fragment of 4581 bp, referred to as PyRE10G, was isolated from the red alga Porphyra yezoensis. PyRE10G contained a putative open reading frame encoding gag, protease, integrase, reverse transcriptase (RT), and RNase H, but one stop codon was present in the integrase region. Southern blot analysis revealed that PyRE10G exists as a single copy in the genome. From the order of gene arrangement of polyproteins, PyRE10G appears to be a copia-like retrotransposon. Amino acid sequences of PyRE10G RT and RNase H were closely related to those of copia-like retrotransposons. In contrast, the phylogenetic tree suggested that PyRE10G integrase stands within the gypsy elements and outside the copia group. PyRE10G is the first example of a chimeric composition of copia- and gypsy-like polyprotein genes in a single element, supporting the hypothesis that long terminal repeat-containing retrotransposons have evolved by fusion of ancestral RT/RNase H and other polyprotein genes.

Reverse transcription in retroviruses and retrotransposons requires nucleic acid chaperones, which facilitate the rearrangement of nucleic acid secondary structure. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) have been extensively studied, and nucleic acid aggregation, duplex destabilization, and rapid protein binding kinetics have been identified as major components of its activity. However, the properties of other nucleic acid chaperone proteins, such as ORF1p from the retrotransposon LINE-1, are not as well understood. We used single molecule DNA stretching in combination with site-directed mutagenesis of ORF1p as a method for detailed characterization of its chaperone activity. Wild type ORF1p significantly reduces the cooperativity of the force-induced melting transition from double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA), indicating that DNA melting is more easily initiated in its presence. ORF1p also aggregates both dsDNA and ssDNA, and exhibits relatively rapid binding kinetics. Altering certain residues has dramatic effects on chaperone activity. Stretching curves in the presence of mutant R284A, which is inactive in retrotransposition assays, exhibit a cooperative melting transition and minimal DNA aggregation. Retrotransposition is partially restored with mutant R284K, which alters the melting transition and strongly aggregates ssDNA. Similarly, mutant Y318A has minimal retrotransposition activity, and stretching curves reflect only a small change in melting transition cooperativity. The Y318F mutant, which largely restores retrotransposition, alters the melting transition in a way similar to wild type ORF1p. Thus, DNA stretching results indicate that reduced cooperativity of the melting transition is associated with greater nucleic acid chaperone and retrotransposition activity of ORF1p variants.

A copia-like retrotransposon referred to as PyRE1G1 was isolated from the genome of the red alga Porphyra yezoensis. PyRE1G1 is 4807 bp in length, with 204 bp long terminal repeats (LTRs) at both ends. PyRE1G1 has an open reading frame of 1401 residues encoding gag, protease, integrase, reverse transcriptase (RT), and RNase H. From the order of gene arrangement of proteins, PyRE1G1 appears to be a copia-like retrotransposon. Genomic Southern blot analysis suggests that PyRE1G1 consists of a small gene family. From the phylogenetic tree of RT sequences, PyRE1G1 is grouped in the clade of usual copia elements and distinct from the previously isolated red algal copia-like gene PyRE10G in that the latter is closely related to a new clade of aquatic animal-specific copia-like retrotransposons.

Short interspersed element (SINE)-like sequences referred to as PySN1 and PySN2 were identified in a red alga, Porphyra yezoensis. Both elements contained an internal promoter with motifs (A box and B box) recognized by RNA polymerase III, and target site duplications at both ends. Genomic Southern blot analysis revealed that both elements were widely and abundantly distributed on the genome. 3′ and 5′ RACE suggested that PySN1 was expressed as a chimera transcript with flanking SINE-unrelated sequences and possessed the poly-A tail at the same position near the 3′ end of PySN1.

We recently demonstrated that as a consequence of impaired ribosome biogenesis, the L5/L11/5S pre-ribosomal complex is redirected to inhibit Mdm2, leading to p53 stabilization (Donati et al., 2013). In seminal studies, Eμ-Myc mice were crossed into a hypomorphic ribosomal protein (RP) L24 mutant background, which brought global translation rates to normal and extended their disease-free survival, independent of p53 (Barna et. al. 2008). However, when this mouse model harbors a single point mutation of Mdm2, which cannot bind the L5/L11/5S-MIC, mice succumb to lymphomagenesis much more rapidly than wild type Mdm2 mice (Macias et al, 2010). Moreover, inhibition of Pol I, which selectively transcribes rRNA genes, suppresses Myc driven tumors, potentially through the same L5/L11/5S-MIC (Bywater, et al, 2012). To address the role of the L5/L11/5S-MIC versus that of global translation in the Eμ-Myc B cell lymphocytes, we partially depleted mRNAs for RPL11, which in other cell types has no effect on p53 levels, or RPL7a, which is known to induce p53 stabilization in a RPL11 dependent manner. We generated stable cell lines of inducible shRNAs against RPL11 or RPL7a using a tetracycline (Tet)-regulated miR30-shRNA system, TRMPVIR retroviral vector. By titrating doxycycline, we achieved ∼50% reduction of RPL11 or RPL7a transcript levels, with Renilla shRNA (Ren) as a control. A 50% depletion of RPL11 or RPL7a mRNAs led to an equal reduction in protein synthesis and ribosome biogenesis, however, RPL11 depletion did not alter p53 levels, whereas RPL7a mRNA depletion induced p53 stabilization and Caspase-3 dependent apoptosis. RPL7A or RPL11 depletion reduced cell proliferation to half that of control cells, with more cell death observed in RPL7a depleted cells versus RPL11 depleted cells. Treatment of RPL7a depleted cells with the caspase inhibitor ZVAD, or depletion of RPL7a in a p53 negative background suppressed cell death. Expression levels of the anti-apoptotic protein Mcl-1 were elevated in Eμ-Myc cells and unchanged in RPL11 depleted cells, but reduced in RPL7a depleted cells in a p53-dependent manner. This suggests that in RPL7a depleted cells, p53 regulates Mcl-1 levels to promote cell death and tumor suppression. Finally, immunoprecipitation of Mdm2 revealed the presence of L5/L11/5S-MIC in RPL7a depleted cells, but not in RPL11 depleted cells. Although p53 was not induced in RPL11 depleted cells, reduction of RPL11 to ∼95% led to acute cell death in both p53-/- and p53 +/+ Eμ-Myc cells, likely due to the inhibition of global translation. These findings demonstrate a differential abrogation of ribosome biogenesis by partial depletion of two essential RPs, which assemble at a very similar stage in 60S ribosome. For RPL7a, it led to L5/L11/5S-MIC induced p53 mediated cell death, and this was not the case for RPL11 as it is an essential component of the complex. Citation Format: Suresh Peddigari, Carol Mercer, Sara Kozma, George Thomas. c-Myc driven B cell lymphomas: role of the RPL5/RPL11/5S rRNA-MDM2 inhibitory complex (L5/L11/5S-MIC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2024. doi:10.1158/1538-7445.AM2015-2024