The role of a highly conserved eubacterial ribosomal protein in translation quality control

The process of decoding is the most crucial determinant of the quality of protein synthesis. Ribosomal protein L9 was first implicated in decoding fidelity when a mutant version of L9 was found to increase the translation of a T4 phage gene. Later studies confirmed that the absence of L9 leads to increased translational bypassing, frameshifting, and stop codon readthrough. L9 is part of the large subunit of the prokaryotic ribosome and is located more than 90 Å from the site of decoding, making it difficult to envision how it might affect decoding and reading frame maintenance. Twenty years after the identification of L9\u27s putative function, there is no mechanism for how a remotely located L9 improves translation fidelity. This mystery makes our picture of translation incomplete. Despite the high conservation of L9 in eubacteria, E.coli lacking L9 does not exhibit any obvious growth defects. Thus, the evolutionary advantage conferred by L9 in bacteria is masked under laboratory conditions. In order to uncover unique L9-dependent conditions, a library of E. coli mutants was screened to isolate those that rely on L9 for fitness. Interestingly, factors found to be synergistic with L9 had no known role in fidelity. Six independent mutants were isolated, each exhibiting a severe growth defect that is partially suppressed in the presence of L9. One class of L9-dependent mutations was present in an essential ribosome biogenesis factor, Der. Der\u27s established function is in the maturation of the large ribosomal subunit. The identified mutations severely impaired the GTPase activity of Der. Interestingly, L9 did not directly compensate for the defective GTPase activity of mutant Der. The second class of L9-dependent mutations was present in EpmA and EpmB, factors required to post-translationally modify elongation factor, EF-P. EF-P\u27s established function is in the translation of poly-proline containing proteins. EF-P deficient cells were nearly inviable in the absence of L9; however, L9 did not directly influence poly-proline translation. Therefore, in each case, L9 improved cell health without altering the activity of either Der or EF-P. Remarkably, the der mutants required only the N domain of L9, whereas the absence of active EF-P required full-length, wild-type L9 for growth complementation. Thus, each mutant class needed a different aspect of L9\u27s unique architecture. In cells lacking either active EF-P or Der, there was a severe deficiency of 70S ribosomes and the indication of small subunit maturation defects, both of which worsened upon L9 depletion. These results strongly suggest that L9 plays a role in improving ribosome quality and abundance under certain conditions. Overall, the genetic screen lead to the discovery that bacteria need L9 when either of two important translation factors (Der or EF-P) is inactivated. This work has characterized the physiological requirement for L9 in each case and offers a new insight into L9\u27s assigned role in translation fidelity

Crippling the Essential GTPase Der Causes Dependence on Ribosomal Protein L9

Ribosomal protein L9 is a component of all eubacterial ribosomes, yet deletion strains display only subtle growth defects. Although L9 has been implicated in helping ribosomes maintain translation reading frame and in regulating translation bypass, no portion of the ribosome-bound protein seems capable of contacting either the peptidyltransferase center or the decoding center, so it is a mystery how L9 can influence these important processes. To reveal the physiological roles of L9 that have maintained it in evolution, we identified mutants of Escherichia coli that depend on L9 for fitness. In this report, we describe a class of L9-dependent mutants in the ribosome biogenesis GTPase Der (EngA/YphC). Purified mutant proteins were severely compromised in their GTPase activities, despite the fact that the mutations are not present in GTP hydrolysis sites. Moreover, although L9 and YihI complemented the slow-growth der phenotypes, neither factor could rescue the GTPase activities in vitro. Complementation studies revealed that the N-terminal domain of L9 is necessary and sufficient to improve the fitness of these Der mutants, suggesting that this domain may help stabilize compromised ribosomes that accumulate when Der is defective. Finally, we employed a targeted degradation system to rapidly deplete L9 from a highly compromised der mutant strain and show that the L9-dependent phenotype coincides with a cell division defect

Receptor Channel Trpc6 Is A Key Mediator Of Notch-Driven Glioblastoma Growth And Invasiveness

Glioblastoma multiforme (GBM) is the most frequent and incurable type of brain tumor of adults. Hypoxia has been shown to direct GBM toward a more aggressive and malignant state. Here we show that hypoxia increases Notch1 activation, which in turn induces the expression of transient receptor potential 6 (TRPC6) in primary samples and cell lines derived from GBM. TRPC6 is required for the development of the aggressive phenotype because knockdown of TRPC6 expression inhibits glioma growth, invasion, and angiogenesis. Functionally, TRPC6 causes a sustained elevation of intracellular calcium that is coupled to the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Pharmacologic inhibition of the calcineurin-NFAT pathway substantially reduces the development of the malignant GBM phenotypes under hypoxia. Clinically, expression of TRPC6 was elevated in GBM specimens in comparison with normal tissues. Collectively, our studies indicate that TRPC6 is a key mediator of tumor growth of GBM in vitro and in vivo and that TRPC6 may be a promising therapeutic target in the treatment of human GBM. ©2010 AACR

The Large Ribosomal Subunit Protein L9 Enables the Growth of EF-P Deficient Cells and Enhances Small Subunit Maturation

<div><p>The loss of the large ribosomal protein L9 causes a reduction in translation fidelity by an unknown mechanism. To identify pathways affected by L9, we identified mutants of <i>E</i>. <i>coli</i> that require L9 for fitness. In a prior study, we characterized L9-dependent mutations in the essential GTPase Der (EngA). Here, we describe a second class of L9-dependent mutations that either compromise or inactivate elongation factor P (EF-P, eIF5A in eukaryotes). Without L9, <i>Δefp</i> cells are practically inviable. Cell fractionation studies revealed that, in both the Der and EF-P mutant cases, L9's activity reduces immature 16S rRNA in 30S particles and partially restores the abundance of monosomes. Inspired by these findings, we discovered that L9 also enhances 16S maturation in wild-type cells. Surprisingly, although the amount of immature 16S in 30S particles was found to be elevated in <i>ΔrplI</i> cells, the amount in polysomes was low and inversely correlated with the immature 16S abundance. These findings provide an explanation for the observed fitness increases afforded by L9 in these mutants and reveal particular physiological conditions in which L9 becomes critical. Additionally, L9 may affect the partitioning of small subunits containing immature 16S rRNA.</p></div

Immature 16S rRNA is overabundant in Δ<i>rplI</i> 30S particles, but reduced in polysomes.

<p>Lysates were prepared from wild-type and Δ<i>rplI</i> cells and resolved using sucrose gradients. (<b>A</b>) 30S peak RNAs from each gradient were recovered by fractionating from the top-down, resolved in denaturing gels and stained with SYBR green II prior to densitometry. The quantified immature 16S from two experimental repeats and three measurement repeats is shown as a bar chart with the abundance reported for each of the three peak fractions. (<b>B</b>) Polysome RNA samples were collected using bottom-up fractionation. The inset shows RNA from the recovered polysomes, the immature precursor is not evident. The bar chart shows the abundance of immature lp16S (additional 115 5' nucleotides) and sp16S (additional 66 5' nucleotides) relative to total 16S determined by RT-qPCR. The amount of immature 16S is lower in the polysomes from Δ<i>rplI</i> cells despite being overabundant in the 30S particles. Error bars are standard deviations from two experimental repeats and three quantifications each.</p

The conserved L9 architecture is required to enhance the mutant growth rates.

<p>The L9-dependent mutant Δ<i>rplI::tet</i>, <i>epmB</i>-<i>W15am</i> was transformed with a battery of plasmids that express variants of L9. (<b>A</b>) A plate showing the relative colony size differences. (<b>B</b>) Liquid culture data of exponential-phase growth rates for the same strains in <i>panel A</i>. The N- and C-terminal domains failed to complement and the <i>hop-1</i>, <i>flexible</i>, and <i>bent</i> versions only marginally complemented. Error bars indicate the standard deviations of three independent exponential phase growth rate measurements. Despite discernable colony size differences, the <i>p-values</i> from Student's t-tests of the liquid culture growth rate data indicate that the growth rate advantage provided by the even most active the L9 variant (<i>flexible</i>) was not substantial.</p

Depleting L9 from Δ<i>empA</i> cells exacerbates small subunit defects.

<p>Cultures of Δ<i>epmA</i> cells expressing L9 with either a control or degradation tag were grown to early exponential phase prior to the expression of ClpXP protease to degrade <i>L9-deg</i>. Lysates were then prepared for cell fractionation studies. (<b>A</b>) A Western blot showing L9 levels before induction of the degradation system (<i>pre ind</i>.) and at the time of <i>harvest</i>. L9 was thoroughly depleted in the L9-deg culture, but not in the L9-cont culture (top panel). With L9 support (cells with the stable L9-cont), the ribosome profiles were reminiscent of those from <i>rplI+</i> Δ<i>efp</i> cells, displaying a reduction in monosomes (left panel). In the culture depleted of L9, the monosome pool was further reduced and 30S particles hyper-accumulated. (<b>B</b>) Sucrose gradients for each lysate are shown with gels of purified RNAs. The monosomes resolved as two peaks and the depletion of L9 altered their relative abundances. In addition, 30S particles became more abundant, additional immature 16S rRNA accumulated (<i>asterisk</i>), and RNA fragmentation was evident (<i>frags</i>). (<b>C</b>) The abundance of particles in Δ<i>epmA</i> cells with L9 support (L9-cont) or with L9 depleted (L9-deg) was quantified from three experiments. Both monosome peaks were integrated together and considered as "70S" for these comparisons.</p

Depleting L9 from <i>derT757I</i> cells also exacerbates a monosome deficiency.

<p>Cultures of <i>derT57I</i> cells with L9-cont or L9-deg were grown to exponential phase prior to depleting <i>L9-deg</i>. (<b>A</b>) A Western blot evaluated L9 depletion (top). With L9 support (<i>L9-cont</i>), the level of 70S particles was substantially reduced compared to <i>der+</i> cells and subunit material accumulated between the 30S and 50S peaks. L9 depletion further reduced the 70S peak. (<b>B</b>) RNA gels revealed that <i>derT57I</i> caused an increase in immature 16S rRNA (<i>asterisk</i>) and substantial 23S RNA fragmentation. Depleting L9 exacerbated both of these defects. (<b>C</b>) Particle abundances in <i>derT57I</i> cells with and without L9 support quantified from three experiments.</p

Loss of L9 leads to sensitivity to antibiotics that cause miscoding.

<p>A Δ<i>rplI</i> (L9-) strains was evaluated for its innate resistance to antibiotics and compared to the isogenic parent (L9+). Consistent with previous reports, the absence of L9 caused only a subtle reduction in growth yield in liquid cultures, but <i>ΔrplI</i> colonies are indistinguishable from wild-type. The turbidity of 100 μL cultures grown in a 96-well plate is shown for various concentrations of each drug. The error bars are standard deviations from three experiments.</p

L9 on the ribosome.

<p>A crystal structure of the <i>E</i>. <i>coli</i> ribosome is shown with large subunit RNA and proteins in <i>green</i>, small subunit RNA and proteins in <i>orange</i>, and L9 in <i>blue</i> (PDB entries 3R8S and 4GD1). Residues of the peptidyltransferase center (<i>PTC</i>) and decoding center (<i>DC</i>) are shown in <i>red</i> along with the Ser93 residue in L9 that affects decoding fidelity.</p