GAS5 suppresses c-Myc expression at protein level.

<p>(<b>A</b>) GAS5 was knocked down by siRNA in HEK-293T cells, and global protein translation was measured by [³H]-leucine incorporation assay. Bars represent mean ±SD from 3 replicates for Q-PCR and 6 replicates for [³H]-leucine incorporation assay. The experiments were performed two times. (<b>B</b>) pRF and pRF-SL plasmid were transfected into HEK-293T cells and cap-dependent protein translation efficiency was evaluated by luciferase assay. Bars represent mean ±SD from 4 replicates. The data were repeated three times. (<b>C</b>) The protein level of c-Myc (P = 0.007), Mcl1 (P = 0.88), survivin (P = 0.47) and Bcl2 (P = 0.47) was assessed by western blot (n = 3) after the cells were treated with GAS5 or control siRNA. (<b>D</b>) The mRNA level of c-Myc (P = 0.10), Mcl1 (P = 0.75), survivin (P = 0.31) and Bcl2 (P = 0.19) was quantified by Q-PCR after the cells were treated with GAS5 or control siRNA. Bars represent mean ±SD from 3 replicates. The experiment was repeated three times. (<b>E</b>) The protein level of c-Myc (P = 0.005) was assessed by western blot (n = 3) after the cells were transfected with <i>in vitro</i> transcribed GAS5 RNA. (<b>F-G</b>) The mRNA level of c-Myc (P = 0.37) (<b>F</b>) and GAS5 (<b>G</b>) was quantified by Q-PCR after the cells were transfected with <i>in vitro</i> transcribed GAS5 RNA. Bars represent mean ±SD from 3 replicates. The experiment was performed three times.</p

The Long Non-Coding RNA GAS5 Cooperates with the Eukaryotic Translation Initiation Factor 4E to Regulate c-Myc Translation

<div><p>Long noncoding RNAs (lncRNAs) are important regulators of transcription; however, their involvement in protein translation is not well known. Here we explored whether the lncRNA GAS5 is associated with translation initiation machinery and regulates translation. GAS5 was enriched with eukaryotic translation initiation factor-4E (eIF4E) in an RNA-immunoprecipitation assay using lymphoma cell lines. We identified two RNA binding motifs within eIF4E protein and the deletion of each motif inhibited the binding of GAS5 with eIF4E. To confirm the role of GAS5 in translation regulation, GAS5 siRNA and <i>in vitro</i> transcribed GAS5 RNA were used to knock down or overexpress GAS5, respectively. GAS5 siRNA had no effect on global protein translation but did specifically increase c-Myc protein level without an effect on c-Myc mRNA. The mechanism of this increase in c-Myc protein was enhanced association of c-Myc mRNA with the polysome without any effect on protein stability. In contrast, overexpression of <i>in vitro</i> transcribed GAS5 RNA suppressed c-Myc protein without affecting c-Myc mRNA. Interestingly, GAS5 was found to be bound with c-Myc mRNA, suggesting that GAS5 regulates c-Myc translation through lncRNA-mRNA interaction. Our findings have uncovered a role of GAS5 lncRNA in translation regulation through its interactions with eIF4E and c-Myc mRNA.</p></div

A schematic diagram of the GAS5 and c-Myc translation regulation.

<p>A schematic diagram of the GAS5 and c-Myc translation regulation.</p

GAS5 cooperates with eIF4E to regulate c-Myc translation.

<p>(<b>A-B</b>) eIF4E expression at mRNA (<b>A</b>) and protein level (<b>B</b>) was assessed by Q-PCR and western blot after the cells were treated with GAS5 or control siRNA. Bars represent mean ±SD from 3 replicates. The experiment was performed three times. (<b>C</b>) GAS5 expression was quantified by Q-PCR in the HEK-293T cells transfected with non-silencing or eIF4E shRNA. Bars represent mean ±SD from 3 replicates. (<b>D</b>). eIF4E protein level after the cells were stably transfected with non-silencing or eIF4E shRNA. (<b>E</b>) The protein level of c-Myc and eIF4E was assessed after the cells were transfected with GAS5/control siRNA and eIF4E plasmid.</p

GAS5 binds to eIF4E through RNA binding motifs.

<p>(<b>A</b>) RNA binding motifs, which are italic and bold, in the eIF4E protein were predicted with 2 web-based tools, BindN and PPRInt. The motif, W56, W102 and E103 for m7G binding is bold and underlined. N-terminally located sequence is motif-1 and the C-terminal one is motif-2. (<b>B-C</b>) GAS5 RNA was detected by RT-PCR after RNA-IP assay using HA antibody in the cells transfected with (<b>B</b>) RNA binding deletion mutants (HA-eIF4E^Del1, HA-eIF4E^Del2 and HA-eIF4E^Del1&2) and (<b>C</b>) cap binding mutant (HA-eIF4E^{cap mutant}). GAPDH was used as a control.</p

GAS5 interacts with eIF4E.

<p>(<b>A</b>) GAS5 mRNA was detected by RT-PCR after RNA-IP using eIF4E and IgG antibodies. The RNA-IP was repeated for two times with similar results. (<b>B</b>) The polysome and non-polysome fractions, as shown by the profile of the absorbance at 254 nm, were separated by sucrose gradient centrifugation in HEK-293T cells. The experiments were repeated three times. (<b>C</b>) The abundance of GAS5 lncRNA in the polysome and non-polysome fractions was measured by Q-PCR and normalized with total GAS5 mRNA in all the fractions. Bars represent mean ±SD from 3 replicates. Experiment was repeated three times, and a representative experiment is shown. (<b>D</b>) Protein levels of eIF4E and eIF4G in the polysome and non-polysome fractions were detected by western blot.</p

GAS5 suppresses c-Myc translation through direct binding with its mRNA.

<p>(<b>A</b>) The protein level of c-Myc was assessed by western blot after the cells were treated with GAS5 or control siRNA followed by the treatment of 100 µg/ml CHX for 1 and 2 hours. (<b>B</b>) c-Myc mRNA level associated with the polysome and non-polysome fractions of GAS5 and control siRNA transfected HEK-293T cells were evaluated by Q-PCR. Data was normalized with total mRNA (non-polysome + polysome). Bars represent mean ±SD from 3 replicates. (<b>C</b>) RNA from HEK-293T cells was pulled down by biotin-labeled GAS5 antisense DNA oligo with GAS5 sense DNA oligo as a control. The binding of c-Myc mRNA to GAS5 was evaluated by RT-PCR, in which GAPDH was used as a control.</p

A Novel Missense (M206K) STAT3 Mutation in Diffuse Large B Cell Lymphoma Deregulates STAT3 Signaling

<div><p>Persistent STAT3 activation has been found in activated B-cell like diffuse large B cell tumors (DLBCL). To investigate whether genetic mutations play a role in aberrant STAT3 signaling in DLBCL, we bi-directionally sequenced all 24 exons of the STAT3 gene in DLBCL tumors (n = 40). We identified 2 novel point mutations in 2 separate (2/40; 5%) patients at exon 7 and 24. Point mutation 2552G>A was a silent mutation in the stop codon. Another heterozygous mutation 857T>A encoded a methionine substitution by lysine at codon 206 (M206K) in the coiled-coil domain of STAT3. We performed site directed mutagenesis to mutate wild type (WT) STAT3α and STAT3β at codon 206 and constructed stable cell lines by lentiviral transfection of STAT3α^WT, STAT3α^M206K, STAT3β^WT and STAT3β^M206K plasmids. The mutation was found to increase STAT3 phosphorylation in STAT3α mutant cell lines with no effect on the STAT3β mutant cell line. Transcriptional activation was also increased in the STAT3α mutant cells compared with STAT3α WT cells as detected by a luciferase reporter assay. Moreover, STAT3α^M206K mutant cells were resistant to JAK2 pathway inhibition compared to STAT3α WT cells. These results indicate that missense mutations in STAT3 increase signaling through the JAK/STAT pathway. JAK2 inhibitors may be useful in the patient with this STAT3 mutation as well as those with pathway activation by other mechanisms.</p></div

Identification 2552G>A mutation in STAT3 in DLBCL tumors.

<p>Chromatograms of part of the patient STAT3 DNA sequence show 2552G>A mutation.</p

The effect of the STAT3 ^M206K mutation on STAT3 translocation to the nucleus, transactivation activity, and cell proliferation.

<p>(<b>A</b>) STAT3 phosphorylation (n = 3) was assessed in both nucleus and cytosol in stably transfected HEK-293T cells by mutant STAT3α^M206K and WT STAT3α plasmids. HDAC2 was used as a marker for nuclear protein, and GAPDH was used as a marker for cytoplasmic protein. (<b>B</b>) The transactivation activity of WT STAT3α^WT and mutant STAT3α^M206K was evaluated by luciferase reporter assay in Ly3 cells. Briefly, Ly3 cells were transiently transfected with plasmids STAT3α^WT and STAT3α^M206K along with a STAT3 luciferase reporter followed by IL-10 treatment for 6 hours and cells were used for luciferase assay. Bars represent mean ± SD from 3 replicates. Data was repeated three times (**P = 0.0042; ***P<0.001). The data were analyzed by the two-tailed unpaired Student's t test. (<b>C</b>) The effect of WT STAT3α^WT and mutant STAT3α^M206K on Ly3 cells proliferation by H³-thymidine incorporation assay. Bars represent mean ± SD from 4 replicates. The experiment was repeated three times (*P = 0.013). The data were analyzed by the two-tailed unpaired Student's t test.</p