ashish lal - Academia.edu (original) (raw)

Papers by ashish lal

PLOS One, 2009

Background: The development of metastases involves the dissociation of cells from the primary tum... more Background: The development of metastases involves the dissociation of cells from the primary tumor to penetrate the basement membrane, invade and then exit the vasculature to seed, and colonize distant tissues. The last step, establishment of macroscopic tumors at distant sites, is the least well understood. Four isogenic mouse breast cancer cell lines (67NR, 168FARN, 4TO7, and 4T1) that differ in their ability to metastasize when implanted into the mammary fat pad are used to model the steps of metastasis. Only 4T1 forms macroscopic lung and liver metastases. Because some miRNAs are dysregulated in cancer and affect cellular transformation, tumor formation, and metastasis, we examined whether changes in miRNA expression might explain the differences in metastasis of these cells.

Breast Cancer Research, 2010

MicroRNAs (miRNAs) are a major class of small endogenous RNA molecules that post-transcriptionall... more MicroRNAs (miRNAs) are a major class of small endogenous RNA molecules that post-transcriptionally inhibit gene expression. Many miRNAs have been implicated in several human cancers, including breast cancer. Here we describe the association between altered miRNA signatures and breast cancer tumorigenesis and metastasis. The loss of several tumor suppressor miRNAs (miR-206, miR-17-5p, miR-125a, miR-125b, miR-200, let-7, miR-34 and miR-31) and the overexpression of certain oncogenic miRNAs (miR-21, miR-155, miR-10b, miR-373 and miR-520c) have been observed in many breast cancers. The gene networks orchestrated by these miRNAs are still largely unknown, although key targets have been identified that may contribute to the disease phenotype. Here we report how the observed perturbations in miRNA expression profiles may lead to disruption of key pathways involved in breast cancer.

PLOS Genetics, 2011

MicroRNAs regulate networks of genes to orchestrate cellular functions. MiR-125b, the vertebrate ... more MicroRNAs regulate networks of genes to orchestrate cellular functions. MiR-125b, the vertebrate homologue of the Caenorhabditis elegans microRNA lin-4, has been implicated in the regulation of neural and hematopoietic stem cell homeostasis, analogous to how lin-4 regulates stem cells in C. elegans. Depending on the cell context, miR-125b has been proposed to regulate both apoptosis and proliferation. Because the p53 network is a central regulator of both apoptosis and proliferation, the dual roles of miR-125b raise the question of what genes in the p53 network might be regulated by miR-125b. By using a gain-and loss-of-function screen for miR-125b targets in humans, mice, and zebrafish and by validating these targets with the luciferase assay and a novel miRNA pull-down assay, we demonstrate that miR-125b directly represses 20 novel targets in the p53 network. These targets include both apoptosis regulators like Bak1, Igfbp3, Itch, Puma, Prkra, Tp53inp1, Tp53, Zac1, and also cell-cycle regulators like cyclin C, Cdc25c, Cdkn2c, Edn1, Ppp1ca, Sel1l, in the p53 network. We found that, although each miRNA-target pair was seldom conserved, miR-125b regulation of the p53 pathway is conserved at the network level. Our results lead us to propose that miR-125b buffers and fine-tunes p53 network activity by regulating the dose of both proliferative and apoptotic regulators, with implications for tissue stem cell homeostasis and oncogenesis.

Nature Structural & Molecular Biology, 2009

Terminally differentiated cells have reduced capacity to repair double strand breaks (DSB), but t... more Terminally differentiated cells have reduced capacity to repair double strand breaks (DSB), but the molecular mechanism behind this down-regulation is unclear. Here we find that miR-24 is consistently up-regulated during post-mitotic differentiation of hematopoietic cell lines and regulates the histone variant H2AX, a key DSB repair protein that activates cell cycle checkpoint proteins and retains DSB repair factors at DSB foci. The H2AX 3'UTR contains conserved miR-24 binding sites regulated by miR-24. Both H2AX mRNA and protein are substantially reduced during hematopoietic cell terminal differentiation by miR-24 up-regulation both in in vitro differentiated cells and primary human blood cells. miR-24 suppression of H2AX renders cells hypersensitive to γ-irradiation and genotoxic drugs. Antagonizing miR-24 in differentiating cells protects them from DNA damageinduced cell death, while transfecting miR-24 mimics in dividing cells increases chromosomal breaks and unrepaired DNA damage and reduces viability in response to DNA damage. This DNA repair phenotype can be fully rescued by over-expressing miR-24-insensitive H2AX. Therefore, miR-24 up-regulation in post-replicative cells reduces H2AX and thereby renders them highly vulnerable to DNA damage. †Corresponding authors:

PLOS One, 2008

Background: Expression of the tumor suppressor p16 INK4a increases during aging and replicative s... more Background: Expression of the tumor suppressor p16 INK4a increases during aging and replicative senescence.

PLOS One, 2008

Background: Expression of the tumor suppressor p16 INK4a increases during aging and replicative s... more Background: Expression of the tumor suppressor p16 INK4a increases during aging and replicative senescence.

Nature Structural & Molecular Biology, 2010

Molecular and Cellular Biology, 2006

Stresses affecting the endoplasmic reticulum (ER) globally modulate gene expression patterns by a... more Stresses affecting the endoplasmic reticulum (ER) globally modulate gene expression patterns by altering posttranscriptional processes such as translation. Here, we use tunicamycin (Tn) to investigate ER stresstriggered changes in the translation of cytochrome c, a pivotal regulator of apoptosis. We identified two RNA-binding proteins that associate with its ϳ900-bp-long, adenine-and uridine-rich 3 untranslated region (UTR): HuR, which displayed affinity for several regions of the cytochrome c 3UTR, and T-cell-restricted intracellular antigen 1 (TIA-1), which preferentially bound the segment proximal to the coding region. HuR did not appear to influence the cytochrome c mRNA levels but instead promoted cytochrome c translation, as HuR silencing greatly diminished the levels of nascent cytochrome c protein. By contrast, TIA-1 functioned as a translational repressor of cytochrome c, with interventions to silence TIA-1 dramatically increasing cytochrome c translation. Following treatment with Tn, HuR binding to cytochrome c mRNA decreased, and both the presence of cytochrome c mRNA within actively translating polysomes and the rate of cytochrome c translation declined. Taken together, our data suggest that the translation rate of cytochrome c is determined by the opposing influences of HuR and TIA-1 upon the cytochrome c mRNA. Under unstressed conditions, cytochrome c mRNA is actively translated, but in response to ER stress agents, both HuR and TIA-1 contribute to lowering its biosynthesis rate. We propose that HuR and TIA-1 function coordinately to maintain precise levels of cytochrome c production under unstimulated conditions and to modify cytochrome c translation when damaged cells are faced with molecular decisions to follow a prosurvival or a prodeath path.

Cell Host & Microbe, 2009

A vaginal microbicide should prevent pathogen transmission without disrupting tissue barriers to ... more A vaginal microbicide should prevent pathogen transmission without disrupting tissue barriers to infection. Ideally it would not need to be applied immediately before sexual intercourse, when compliance is a problem. Intravaginal administration of small interfering RNA (siRNA) lipoplexes targeting Herpes Simplex Virus Type 2 (HSV-2) genes protects mice from HSV-2. However, protection is short-lived and the transfection lipid on its own unacceptably enhances transmission.

Proceedings of The National Academy of Sciences, 2008

Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms... more Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that DJ-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that DJ-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner.

Molecular Cell, 2009

miR-24, upregulated during terminal differentiation of multiple lineages, inhibits cell-cycle pro... more miR-24, upregulated during terminal differentiation of multiple lineages, inhibits cell-cycle progression. Antagonizing miR-24 restores postmitotic cell proliferation and enhances fibroblast proliferation, whereas overexpressing miR-24 increases the G1 compartment. The 248 mRNAs downregulated upon miR-24 overexpression are highly enriched for DNA repair and cell-cycle regulatory genes that form a direct interaction network with prominent nodes at genes that enhance (MYC, E2F2, CCNB1, and CDC2) or inhibit (p27Kip1 and VHL) cell-cycle progression. miR-24 directly regulates MYC and E2F2 and some genes that they transactivate. Enhanced proliferation from antagonizing miR-24 is abrogated by knocking down E2F2, but not MYC, and cell proliferation, inhibited by miR-24 overexpression, is rescued by miR-24-insensitive E2F2. Therefore, E2F2 is a critical miR-24 target. The E2F2 3′UTR lacks a predicted miR-24 recognition element. In fact, miR-24 regulates expression of E2F2, MYC, AURKB, CCNA2, CDC2, CDK4, and FEN1 by recognizing seedless but highly complementary sequences.

Molecular Cell, 2007

The RNA-binding protein HuR regulates the stability of many target mRNAs. Here, we report that Hu... more The RNA-binding protein HuR regulates the stability of many target mRNAs. Here, we report that HuR associated with the 3'-untranslated region of the mRNA encoding the longevity and stressresponse protein SIRT1, stabilized the SIRT1 mRNA, and increased SIRT1 expression levels. Unexpectedly, oxidative stress triggered the dissociation of the [HuR-SIRT1 mRNA] complex, in turn promoting SIRT1 mRNA decay, reducing SIRT1 abundance, and lowering cell survival. The cell cycle checkpoint kinase Chk2 was activated by H 2 O 2 , interacted with HuR, and was predicted to phosphorylate HuR at residues Ser-88, Ser-100, and Thr-118. Mutation of these residues revealed a complex pattern of HuR binding, with Ser-100 appearing important for [HuR-SIRT1 mRNA] dissociation after H 2 O 2 . Our findings demonstrate that HuR regulates SIRT1 expression, underscore functional links between the two stress-response proteins, and implicate Chk2 in these processes.

Molecular Cell, 2006

The growth arrest-and DNA damage-inducible gene GADD45a is potently upregulated in response to st... more The growth arrest-and DNA damage-inducible gene GADD45a is potently upregulated in response to stress stimuli. Here, two RNA binding proteins, the mRNA decay-promoting AUF1 and the translational suppressor TIAR, were found to interact specifically with the 3 0 untranslated region (UTR) of the GADD45a mRNA in HeLa cells. These associations were prominent in unstimulated cells, decreasing dramatically after treatment with the genotoxin methyl methanesulfonate (MMS). Analysis of both endogenous and chimeric GADD45a mRNA revealed that in untreated cells AUF1 strongly reduced GADD45a mRNA stability, whereas TIAR potently inhibited GADD45a translation. After genotoxic stress, AUF1 and TIAR dissociated from the GADD45a mRNA, thereby allowing coordinated elevations in both GADD45a mRNA half-life and translation rate, respectively. We propose that the posttranscriptional derepression of GADD45a critically contributes to its potent upregulation after DNA damage.

Molecular and Cellular Probes, 2005

We present a strategy that overcomes the high background arising during Western blotting (WB) det... more We present a strategy that overcomes the high background arising during Western blotting (WB) detection of proteins obtained through immunoprecipitation (IP). Traditional HRP-conjugated secondary antibodies, which detect the denatured heavy and light antibody chains, produce high background that often mask the signals of interest on WBs. Here, we show that HRP-conjugated Protein A and Protein G, which detect almost exclusively intact antibody molecules, can be effectively used to obtain clean and specific WB signals of target proteins.

HuR has been shown to influence the translation of several target mRNAs (Kullmann et al., 2002; M... more HuR has been shown to influence the translation of several target mRNAs (Kullmann et al., 2002; Mazan-Mamczarz et al., 2003; Lal et al., 2005; Meng et al., 2005). To test if HuR also directly modulated the rate of SIRT1 mRNA translation, nascent SIRT1 production was monitored following a brief (20-min long) incubation with L-[ 35 S]methionine and L-[ 35 S]cysteine in cells expressing either control or silenced HuR levels (Suppl. , panel A). This assay revealed that the reduced de novo translation of SIRT1 in HuRsilenced cells (one third of the translation levels seen in control cells) was comparable to the reduction in SIRT1 mRNA levels (also one third of control cells), suggesting that the reduced translation rate was simply a reflection of the reduced SIRT1 mRNA abundance ). Of note, HuR-silenced populations exhibit a reduction in total Sirt1 that is significantly greater than the reduction in mRNA levels or translation rate (compare with and with ), suggesting that additional regulatory levels, possibly at the level of proteolysis, may participate in dictating SIRT1 abundance. To further assess a possible influence of HuR on the rate of SIRT1 translation, the relative distribution of the SIRT1 mRNA on polysome gradients was examined (Suppl. . By this approach, increases in the abundance of the SIRT1 mRNA in the heavy fractions of the gradient would indicate an increased association of the SIRT1 mRNA with the translational machinery and hence an elevation in SIRT1 protein biosynthesis. However, as shown in , the relative distribution of the SIRT1 mRNA along the polysome gradients prepared from control cultures was virtually indistinguishable from that obtained using HuR siRNA cultures, further supporting the notion that HuR does not influence the translational status of the SIRT1 mRNA. In sum, HuR enhances SIRT1 mRNA stability but does not seem to influence its translation rate.

PLOS One, 2009

Background: The development of metastases involves the dissociation of cells from the primary tum... more Background: The development of metastases involves the dissociation of cells from the primary tumor to penetrate the basement membrane, invade and then exit the vasculature to seed, and colonize distant tissues. The last step, establishment of macroscopic tumors at distant sites, is the least well understood. Four isogenic mouse breast cancer cell lines (67NR, 168FARN, 4TO7, and 4T1) that differ in their ability to metastasize when implanted into the mammary fat pad are used to model the steps of metastasis. Only 4T1 forms macroscopic lung and liver metastases. Because some miRNAs are dysregulated in cancer and affect cellular transformation, tumor formation, and metastasis, we examined whether changes in miRNA expression might explain the differences in metastasis of these cells.

Breast Cancer Research, 2010

MicroRNAs (miRNAs) are a major class of small endogenous RNA molecules that post-transcriptionall... more MicroRNAs (miRNAs) are a major class of small endogenous RNA molecules that post-transcriptionally inhibit gene expression. Many miRNAs have been implicated in several human cancers, including breast cancer. Here we describe the association between altered miRNA signatures and breast cancer tumorigenesis and metastasis. The loss of several tumor suppressor miRNAs (miR-206, miR-17-5p, miR-125a, miR-125b, miR-200, let-7, miR-34 and miR-31) and the overexpression of certain oncogenic miRNAs (miR-21, miR-155, miR-10b, miR-373 and miR-520c) have been observed in many breast cancers. The gene networks orchestrated by these miRNAs are still largely unknown, although key targets have been identified that may contribute to the disease phenotype. Here we report how the observed perturbations in miRNA expression profiles may lead to disruption of key pathways involved in breast cancer.

PLOS Genetics, 2011

MicroRNAs regulate networks of genes to orchestrate cellular functions. MiR-125b, the vertebrate ... more MicroRNAs regulate networks of genes to orchestrate cellular functions. MiR-125b, the vertebrate homologue of the Caenorhabditis elegans microRNA lin-4, has been implicated in the regulation of neural and hematopoietic stem cell homeostasis, analogous to how lin-4 regulates stem cells in C. elegans. Depending on the cell context, miR-125b has been proposed to regulate both apoptosis and proliferation. Because the p53 network is a central regulator of both apoptosis and proliferation, the dual roles of miR-125b raise the question of what genes in the p53 network might be regulated by miR-125b. By using a gain-and loss-of-function screen for miR-125b targets in humans, mice, and zebrafish and by validating these targets with the luciferase assay and a novel miRNA pull-down assay, we demonstrate that miR-125b directly represses 20 novel targets in the p53 network. These targets include both apoptosis regulators like Bak1, Igfbp3, Itch, Puma, Prkra, Tp53inp1, Tp53, Zac1, and also cell-cycle regulators like cyclin C, Cdc25c, Cdkn2c, Edn1, Ppp1ca, Sel1l, in the p53 network. We found that, although each miRNA-target pair was seldom conserved, miR-125b regulation of the p53 pathway is conserved at the network level. Our results lead us to propose that miR-125b buffers and fine-tunes p53 network activity by regulating the dose of both proliferative and apoptotic regulators, with implications for tissue stem cell homeostasis and oncogenesis.

Nature Structural & Molecular Biology, 2009

Terminally differentiated cells have reduced capacity to repair double strand breaks (DSB), but t... more Terminally differentiated cells have reduced capacity to repair double strand breaks (DSB), but the molecular mechanism behind this down-regulation is unclear. Here we find that miR-24 is consistently up-regulated during post-mitotic differentiation of hematopoietic cell lines and regulates the histone variant H2AX, a key DSB repair protein that activates cell cycle checkpoint proteins and retains DSB repair factors at DSB foci. The H2AX 3'UTR contains conserved miR-24 binding sites regulated by miR-24. Both H2AX mRNA and protein are substantially reduced during hematopoietic cell terminal differentiation by miR-24 up-regulation both in in vitro differentiated cells and primary human blood cells. miR-24 suppression of H2AX renders cells hypersensitive to γ-irradiation and genotoxic drugs. Antagonizing miR-24 in differentiating cells protects them from DNA damageinduced cell death, while transfecting miR-24 mimics in dividing cells increases chromosomal breaks and unrepaired DNA damage and reduces viability in response to DNA damage. This DNA repair phenotype can be fully rescued by over-expressing miR-24-insensitive H2AX. Therefore, miR-24 up-regulation in post-replicative cells reduces H2AX and thereby renders them highly vulnerable to DNA damage. †Corresponding authors:

PLOS One, 2008

Background: Expression of the tumor suppressor p16 INK4a increases during aging and replicative s... more Background: Expression of the tumor suppressor p16 INK4a increases during aging and replicative senescence.

PLOS One, 2008

Background: Expression of the tumor suppressor p16 INK4a increases during aging and replicative s... more Background: Expression of the tumor suppressor p16 INK4a increases during aging and replicative senescence.

Nature Structural & Molecular Biology, 2010

Molecular and Cellular Biology, 2006

Stresses affecting the endoplasmic reticulum (ER) globally modulate gene expression patterns by a... more Stresses affecting the endoplasmic reticulum (ER) globally modulate gene expression patterns by altering posttranscriptional processes such as translation. Here, we use tunicamycin (Tn) to investigate ER stresstriggered changes in the translation of cytochrome c, a pivotal regulator of apoptosis. We identified two RNA-binding proteins that associate with its ϳ900-bp-long, adenine-and uridine-rich 3 untranslated region (UTR): HuR, which displayed affinity for several regions of the cytochrome c 3UTR, and T-cell-restricted intracellular antigen 1 (TIA-1), which preferentially bound the segment proximal to the coding region. HuR did not appear to influence the cytochrome c mRNA levels but instead promoted cytochrome c translation, as HuR silencing greatly diminished the levels of nascent cytochrome c protein. By contrast, TIA-1 functioned as a translational repressor of cytochrome c, with interventions to silence TIA-1 dramatically increasing cytochrome c translation. Following treatment with Tn, HuR binding to cytochrome c mRNA decreased, and both the presence of cytochrome c mRNA within actively translating polysomes and the rate of cytochrome c translation declined. Taken together, our data suggest that the translation rate of cytochrome c is determined by the opposing influences of HuR and TIA-1 upon the cytochrome c mRNA. Under unstressed conditions, cytochrome c mRNA is actively translated, but in response to ER stress agents, both HuR and TIA-1 contribute to lowering its biosynthesis rate. We propose that HuR and TIA-1 function coordinately to maintain precise levels of cytochrome c production under unstimulated conditions and to modify cytochrome c translation when damaged cells are faced with molecular decisions to follow a prosurvival or a prodeath path.

Cell Host & Microbe, 2009

A vaginal microbicide should prevent pathogen transmission without disrupting tissue barriers to ... more A vaginal microbicide should prevent pathogen transmission without disrupting tissue barriers to infection. Ideally it would not need to be applied immediately before sexual intercourse, when compliance is a problem. Intravaginal administration of small interfering RNA (siRNA) lipoplexes targeting Herpes Simplex Virus Type 2 (HSV-2) genes protects mice from HSV-2. However, protection is short-lived and the transfection lipid on its own unacceptably enhances transmission.

Proceedings of The National Academy of Sciences, 2008

Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms... more Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that DJ-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that DJ-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner.

Molecular Cell, 2009

miR-24, upregulated during terminal differentiation of multiple lineages, inhibits cell-cycle pro... more miR-24, upregulated during terminal differentiation of multiple lineages, inhibits cell-cycle progression. Antagonizing miR-24 restores postmitotic cell proliferation and enhances fibroblast proliferation, whereas overexpressing miR-24 increases the G1 compartment. The 248 mRNAs downregulated upon miR-24 overexpression are highly enriched for DNA repair and cell-cycle regulatory genes that form a direct interaction network with prominent nodes at genes that enhance (MYC, E2F2, CCNB1, and CDC2) or inhibit (p27Kip1 and VHL) cell-cycle progression. miR-24 directly regulates MYC and E2F2 and some genes that they transactivate. Enhanced proliferation from antagonizing miR-24 is abrogated by knocking down E2F2, but not MYC, and cell proliferation, inhibited by miR-24 overexpression, is rescued by miR-24-insensitive E2F2. Therefore, E2F2 is a critical miR-24 target. The E2F2 3′UTR lacks a predicted miR-24 recognition element. In fact, miR-24 regulates expression of E2F2, MYC, AURKB, CCNA2, CDC2, CDK4, and FEN1 by recognizing seedless but highly complementary sequences.

Molecular Cell, 2007

The RNA-binding protein HuR regulates the stability of many target mRNAs. Here, we report that Hu... more The RNA-binding protein HuR regulates the stability of many target mRNAs. Here, we report that HuR associated with the 3'-untranslated region of the mRNA encoding the longevity and stressresponse protein SIRT1, stabilized the SIRT1 mRNA, and increased SIRT1 expression levels. Unexpectedly, oxidative stress triggered the dissociation of the [HuR-SIRT1 mRNA] complex, in turn promoting SIRT1 mRNA decay, reducing SIRT1 abundance, and lowering cell survival. The cell cycle checkpoint kinase Chk2 was activated by H 2 O 2 , interacted with HuR, and was predicted to phosphorylate HuR at residues Ser-88, Ser-100, and Thr-118. Mutation of these residues revealed a complex pattern of HuR binding, with Ser-100 appearing important for [HuR-SIRT1 mRNA] dissociation after H 2 O 2 . Our findings demonstrate that HuR regulates SIRT1 expression, underscore functional links between the two stress-response proteins, and implicate Chk2 in these processes.

Molecular Cell, 2006

The growth arrest-and DNA damage-inducible gene GADD45a is potently upregulated in response to st... more The growth arrest-and DNA damage-inducible gene GADD45a is potently upregulated in response to stress stimuli. Here, two RNA binding proteins, the mRNA decay-promoting AUF1 and the translational suppressor TIAR, were found to interact specifically with the 3 0 untranslated region (UTR) of the GADD45a mRNA in HeLa cells. These associations were prominent in unstimulated cells, decreasing dramatically after treatment with the genotoxin methyl methanesulfonate (MMS). Analysis of both endogenous and chimeric GADD45a mRNA revealed that in untreated cells AUF1 strongly reduced GADD45a mRNA stability, whereas TIAR potently inhibited GADD45a translation. After genotoxic stress, AUF1 and TIAR dissociated from the GADD45a mRNA, thereby allowing coordinated elevations in both GADD45a mRNA half-life and translation rate, respectively. We propose that the posttranscriptional derepression of GADD45a critically contributes to its potent upregulation after DNA damage.

Molecular and Cellular Probes, 2005

We present a strategy that overcomes the high background arising during Western blotting (WB) det... more We present a strategy that overcomes the high background arising during Western blotting (WB) detection of proteins obtained through immunoprecipitation (IP). Traditional HRP-conjugated secondary antibodies, which detect the denatured heavy and light antibody chains, produce high background that often mask the signals of interest on WBs. Here, we show that HRP-conjugated Protein A and Protein G, which detect almost exclusively intact antibody molecules, can be effectively used to obtain clean and specific WB signals of target proteins.

HuR has been shown to influence the translation of several target mRNAs (Kullmann et al., 2002; M... more HuR has been shown to influence the translation of several target mRNAs (Kullmann et al., 2002; Mazan-Mamczarz et al., 2003; Lal et al., 2005; Meng et al., 2005). To test if HuR also directly modulated the rate of SIRT1 mRNA translation, nascent SIRT1 production was monitored following a brief (20-min long) incubation with L-[ 35 S]methionine and L-[ 35 S]cysteine in cells expressing either control or silenced HuR levels (Suppl. , panel A). This assay revealed that the reduced de novo translation of SIRT1 in HuRsilenced cells (one third of the translation levels seen in control cells) was comparable to the reduction in SIRT1 mRNA levels (also one third of control cells), suggesting that the reduced translation rate was simply a reflection of the reduced SIRT1 mRNA abundance ). Of note, HuR-silenced populations exhibit a reduction in total Sirt1 that is significantly greater than the reduction in mRNA levels or translation rate (compare with and with ), suggesting that additional regulatory levels, possibly at the level of proteolysis, may participate in dictating SIRT1 abundance. To further assess a possible influence of HuR on the rate of SIRT1 translation, the relative distribution of the SIRT1 mRNA on polysome gradients was examined (Suppl. . By this approach, increases in the abundance of the SIRT1 mRNA in the heavy fractions of the gradient would indicate an increased association of the SIRT1 mRNA with the translational machinery and hence an elevation in SIRT1 protein biosynthesis. However, as shown in , the relative distribution of the SIRT1 mRNA along the polysome gradients prepared from control cultures was virtually indistinguishable from that obtained using HuR siRNA cultures, further supporting the notion that HuR does not influence the translational status of the SIRT1 mRNA. In sum, HuR enhances SIRT1 mRNA stability but does not seem to influence its translation rate.