Limits...
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.

Show MeSH

Related in: MedlinePlus

GQ structures confer eIF4A-dependent translationa) Bar graph indicating the motif prevalence and likelihood to form GQs (red); b) The ADAM10 5′UTR illustrates 12-mer and 9-mer motifs and GQs; c) Enrichment of predicted 5′UTR GQ structures in the TE down gene set; d) CD spectra scan of 12-mer motif (CGG)4, mutant oligomer (equal length and GC content), and human telomeric RNA (hTR) with known GQ structure folded in KCl, n = 5 replicates; e) CD spectra scan of 9-mer motifs with 2nt flank from the 5′UTR of indicated genes folded with KCl, n = 5 replicates; f) Melting curve for CD spectra scan at λ264nm for the 12-mer (CGG)4 and mutant oligomer, Tm = melting temperature, ΔG = free energy of unfolding; g) Calculated decrease in free energy for cellular UTRs with 1, 2, or 3+ motifs when allowed to fold into GQ structures; h) Diagram of parallel GQ conformation; i) Schematic of reporter constructs with four 12-mer motifs (GQs, red), random sequence matched for length and GC content (control, black), HCV IRES (white); j–k) Relative Renilla luciferase (normalized to IRES-Firefly) expressed from the GQ (red) or control construct (black), treated with Silvestrol (j) or Cycloheximide (k) for 24 hours (* indicates p < 0.05, n = 3 biological replicates and n = 2 technical replicates); l) Assay as above comparing empty vector and sh-eIF4A (* indicates p < 0.05, n = 3 biological replicates and n = 2 technical replicates).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492470&req=5

Figure 4: GQ structures confer eIF4A-dependent translationa) Bar graph indicating the motif prevalence and likelihood to form GQs (red); b) The ADAM10 5′UTR illustrates 12-mer and 9-mer motifs and GQs; c) Enrichment of predicted 5′UTR GQ structures in the TE down gene set; d) CD spectra scan of 12-mer motif (CGG)4, mutant oligomer (equal length and GC content), and human telomeric RNA (hTR) with known GQ structure folded in KCl, n = 5 replicates; e) CD spectra scan of 9-mer motifs with 2nt flank from the 5′UTR of indicated genes folded with KCl, n = 5 replicates; f) Melting curve for CD spectra scan at λ264nm for the 12-mer (CGG)4 and mutant oligomer, Tm = melting temperature, ΔG = free energy of unfolding; g) Calculated decrease in free energy for cellular UTRs with 1, 2, or 3+ motifs when allowed to fold into GQ structures; h) Diagram of parallel GQ conformation; i) Schematic of reporter constructs with four 12-mer motifs (GQs, red), random sequence matched for length and GC content (control, black), HCV IRES (white); j–k) Relative Renilla luciferase (normalized to IRES-Firefly) expressed from the GQ (red) or control construct (black), treated with Silvestrol (j) or Cycloheximide (k) for 24 hours (* indicates p < 0.05, n = 3 biological replicates and n = 2 technical replicates); l) Assay as above comparing empty vector and sh-eIF4A (* indicates p < 0.05, n = 3 biological replicates and 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)

GQ structures confer eIF4A-dependent translationa) Bar graph indicating the motif prevalence and likelihood to form GQs (red); b) The ADAM10 5′UTR illustrates 12-mer and 9-mer motifs and GQs; c) Enrichment of predicted 5′UTR GQ structures in the TE down gene set; d) CD spectra scan of 12-mer motif (CGG)4, mutant oligomer (equal length and GC content), and human telomeric RNA (hTR) with known GQ structure folded in KCl, n = 5 replicates; e) CD spectra scan of 9-mer motifs with 2nt flank from the 5′UTR of indicated genes folded with KCl, n = 5 replicates; f) Melting curve for CD spectra scan at λ264nm for the 12-mer (CGG)4 and mutant oligomer, Tm = melting temperature, ΔG = free energy of unfolding; g) Calculated decrease in free energy for cellular UTRs with 1, 2, or 3+ motifs when allowed to fold into GQ structures; h) Diagram of parallel GQ conformation; i) Schematic of reporter constructs with four 12-mer motifs (GQs, red), random sequence matched for length and GC content (control, black), HCV IRES (white); j–k) Relative Renilla luciferase (normalized to IRES-Firefly) expressed from the GQ (red) or control construct (black), treated with Silvestrol (j) or Cycloheximide (k) for 24 hours (* indicates p < 0.05, n = 3 biological replicates and n = 2 technical replicates); l) Assay as above comparing empty vector and sh-eIF4A (* indicates p < 0.05, n = 3 biological replicates and n = 2 technical replicates).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492470&req=5

Figure 4: GQ structures confer eIF4A-dependent translationa) Bar graph indicating the motif prevalence and likelihood to form GQs (red); b) The ADAM10 5′UTR illustrates 12-mer and 9-mer motifs and GQs; c) Enrichment of predicted 5′UTR GQ structures in the TE down gene set; d) CD spectra scan of 12-mer motif (CGG)4, mutant oligomer (equal length and GC content), and human telomeric RNA (hTR) with known GQ structure folded in KCl, n = 5 replicates; e) CD spectra scan of 9-mer motifs with 2nt flank from the 5′UTR of indicated genes folded with KCl, n = 5 replicates; f) Melting curve for CD spectra scan at λ264nm for the 12-mer (CGG)4 and mutant oligomer, Tm = melting temperature, ΔG = free energy of unfolding; g) Calculated decrease in free energy for cellular UTRs with 1, 2, or 3+ motifs when allowed to fold into GQ structures; h) Diagram of parallel GQ conformation; i) Schematic of reporter constructs with four 12-mer motifs (GQs, red), random sequence matched for length and GC content (control, black), HCV IRES (white); j–k) Relative Renilla luciferase (normalized to IRES-Firefly) expressed from the GQ (red) or control construct (black), treated with Silvestrol (j) or Cycloheximide (k) for 24 hours (* indicates p < 0.05, n = 3 biological replicates and n = 2 technical replicates); l) Assay as above comparing empty vector and sh-eIF4A (* indicates p < 0.05, n = 3 biological replicates and 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