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RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer.

Wolfe AL, Singh K, Zhong Y, Drewe P, Rajasekhar VK, Sanghvi VR, Mavrakis KJ, Jiang M, Roderick JE, Van der Meulen J, Schatz JH, Rodrigo CM, Zhao C, Rondou P, de Stanchina E, Teruya-Feldstein J, Kelliher MA, Speleman F, Porco JA, Pelletier J, Rätsch G, Wendel HG - Nature (2014)

Bottom Line: Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo.Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators.Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

View Article: PubMed Central - PubMed

Affiliation: 1] Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA [2] Weill Cornell Graduate School of Medical Sciences, New York, New York 10065, USA [3].

ABSTRACT
The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5' untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

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Circular dichroism (CD) and characterization of eIF4Aa) Bar graph indicating the prevalence of each sequence motif from the rDiff data set and its predicted likelihood to form GQ structures (red); b) CD spectra scan of 9-mer motif with a 5 nt flank taken from the actual 5′UTR of the indicated genes folded with KCl; c) CD spectra scan of 12-mer motif and mutant folded in sodium phosphate buffer without KCl, note the y-axis scale; d) Relative amounts of Renilla luciferase (normalized to Firefly) expressed from the GQs (red bars) or control construct (black bars), treated with 8 nM Pateamine A (Pat. A) or 50 nM Hippuristanol (Hipp.) for 24 hours (* indicates p < 0.05, n = 3 biological replicates and n = 2 technical replicates); e) Analysis of mRNA expression of the indicated RNA helicases in normal T-cells and T-ALL cells (* indicates p < 0.05, n = 57 biological replicates)32; f) Relative amounts of Renilla luciferase expressed from the GQ construct in 3T3 cells and normalized to IRES/Firefly with either empty vector or the indicated genes, treated with Silvestrol (25 nM) for 24 hours, mean and standard deviation are shown, n = 3 biological replicates, n = 2 technical replicates.
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Figure 11: Circular dichroism (CD) and characterization of eIF4Aa) Bar graph indicating the prevalence of each sequence motif from the rDiff data set and its predicted likelihood to form GQ structures (red); b) CD spectra scan of 9-mer motif with a 5 nt flank taken from the actual 5′UTR of the indicated genes folded with KCl; c) CD spectra scan of 12-mer motif and mutant folded in sodium phosphate buffer without KCl, note the y-axis scale; d) Relative amounts of Renilla luciferase (normalized to Firefly) expressed from the GQs (red bars) or control construct (black bars), treated with 8 nM Pateamine A (Pat. A) or 50 nM Hippuristanol (Hipp.) for 24 hours (* indicates p < 0.05, n = 3 biological replicates and n = 2 technical replicates); e) Analysis of mRNA expression of the indicated RNA helicases in normal T-cells and T-ALL cells (* indicates p < 0.05, n = 57 biological replicates)32; f) Relative amounts of Renilla luciferase expressed from the GQ construct in 3T3 cells and normalized to IRES/Firefly with either empty vector or the indicated genes, treated with Silvestrol (25 nM) for 24 hours, mean and standard deviation are shown, n = 3 biological replicates, n = 2 technical replicates.

Mentions: We noticed that in many instances the 5′UTR motifs coincided with computationally predicted G-quadruplex (GQ) structures29. For example 51% of the 12-mer (CGG)4 sequences and 43% of the most common 9-mer localized precisely to the GQ structures – the ADAM10 5′UTR provides an example (Figure 4a/b, Extended Data Fig. 6a, Suppl. Table 4e–k). GQ structures form by non-Watson-Crick interactions between paired guanine nucleotides that align parallel or anti-parallel arrangements in different planes connected by at least one linker nucleotide (A or C). Accordingly, GQ structures were significantly enriched among TE down genes and 79/220 TE down transcripts harboured at least one GQ (p = 2 × 10−11) (Figure 4a–c, Suppl. Table 4e–k).


RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer.

Wolfe AL, Singh K, Zhong Y, Drewe P, Rajasekhar VK, Sanghvi VR, Mavrakis KJ, Jiang M, Roderick JE, Van der Meulen J, Schatz JH, Rodrigo CM, Zhao C, Rondou P, de Stanchina E, Teruya-Feldstein J, Kelliher MA, Speleman F, Porco JA, Pelletier J, Rätsch G, Wendel HG - Nature (2014)

Circular dichroism (CD) and characterization of eIF4Aa) Bar graph indicating the prevalence of each sequence motif from the rDiff data set and its predicted likelihood to form GQ structures (red); b) CD spectra scan of 9-mer motif with a 5 nt flank taken from the actual 5′UTR of the indicated genes folded with KCl; c) CD spectra scan of 12-mer motif and mutant folded in sodium phosphate buffer without KCl, note the y-axis scale; d) Relative amounts of Renilla luciferase (normalized to Firefly) expressed from the GQs (red bars) or control construct (black bars), treated with 8 nM Pateamine A (Pat. A) or 50 nM Hippuristanol (Hipp.) for 24 hours (* indicates p < 0.05, n = 3 biological replicates and n = 2 technical replicates); e) Analysis of mRNA expression of the indicated RNA helicases in normal T-cells and T-ALL cells (* indicates p < 0.05, n = 57 biological replicates)32; f) Relative amounts of Renilla luciferase expressed from the GQ construct in 3T3 cells and normalized to IRES/Firefly with either empty vector or the indicated genes, treated with Silvestrol (25 nM) for 24 hours, mean and standard deviation are shown, n = 3 biological replicates, n = 2 technical replicates.
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Figure 11: Circular dichroism (CD) and characterization of eIF4Aa) Bar graph indicating the prevalence of each sequence motif from the rDiff data set and its predicted likelihood to form GQ structures (red); b) CD spectra scan of 9-mer motif with a 5 nt flank taken from the actual 5′UTR of the indicated genes folded with KCl; c) CD spectra scan of 12-mer motif and mutant folded in sodium phosphate buffer without KCl, note the y-axis scale; d) Relative amounts of Renilla luciferase (normalized to Firefly) expressed from the GQs (red bars) or control construct (black bars), treated with 8 nM Pateamine A (Pat. A) or 50 nM Hippuristanol (Hipp.) for 24 hours (* indicates p < 0.05, n = 3 biological replicates and n = 2 technical replicates); e) Analysis of mRNA expression of the indicated RNA helicases in normal T-cells and T-ALL cells (* indicates p < 0.05, n = 57 biological replicates)32; f) Relative amounts of Renilla luciferase expressed from the GQ construct in 3T3 cells and normalized to IRES/Firefly with either empty vector or the indicated genes, treated with Silvestrol (25 nM) for 24 hours, mean and standard deviation are shown, n = 3 biological replicates, n = 2 technical replicates.
Mentions: We noticed that in many instances the 5′UTR motifs coincided with computationally predicted G-quadruplex (GQ) structures29. For example 51% of the 12-mer (CGG)4 sequences and 43% of the most common 9-mer localized precisely to the GQ structures – the ADAM10 5′UTR provides an example (Figure 4a/b, Extended Data Fig. 6a, Suppl. Table 4e–k). GQ structures form by non-Watson-Crick interactions between paired guanine nucleotides that align parallel or anti-parallel arrangements in different planes connected by at least one linker nucleotide (A or C). Accordingly, GQ structures were significantly enriched among TE down genes and 79/220 TE down transcripts harboured at least one GQ (p = 2 × 10−11) (Figure 4a–c, Suppl. Table 4e–k).

Bottom Line: Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo.Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators.Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

View Article: PubMed Central - PubMed

Affiliation: 1] Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA [2] Weill Cornell Graduate School of Medical Sciences, New York, New York 10065, USA [3].

ABSTRACT
The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5' untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

Show MeSH
Related in: MedlinePlus