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An RNA G-quadruplex in the 5' UTR of the NRAS proto-oncogene modulates translation.

ABSTRACT: Guanine-rich nucleic acid sequences can adopt noncanonical four-stranded secondary structures called guanine (G)-quadruplexes. Bioinformatics analysis suggests that G-quadruplex motifs are prevalent in genomes, which raises the need to elucidate their function. There is now evidence for the existence of DNA G-quadruplexes at telomeres with associated biological function. A recent hypothesis supports the notion that gene promoter elements contain DNA G-quadruplex motifs that control gene expression at the transcriptional level. We discovered a highly conserved, thermodynamically stable RNA G-quadruplex in the 5' untranslated region (UTR) of the gene transcript of the human NRAS proto-oncogene. Using a cell-free translation system coupled to a reporter gene assay, we have demonstrated that this NRAS RNA G-quadruplex modulates translation. This is the first example of translational repression by an RNA G-quadruplex. Bioinformatics analysis has revealed 2,922 other 5' UTR RNA G-quadruplex elements in the human genome. We propose that RNA G-quadruplexes in the 5' UTR modulate gene expression at the translational level.

SUBMITTER: Kumari S

PROVIDER: S-EPMC2206252 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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An RNA G-quadruplex in the 5' UTR of the NRAS proto-oncogene modulates translation.

Kumari Sunita S Bugaut Anthony A Huppert Julian L JL Balasubramanian Shankar S

Nature chemical biology 20070225 4

Guanine-rich nucleic acid sequences can adopt noncanonical four-stranded secondary structures called guanine (G)-quadruplexes. Bioinformatics analysis suggests that G-quadruplex motifs are prevalent in genomes, which raises the need to elucidate their function. There is now evidence for the existence of DNA G-quadruplexes at telomeres with associated biological function. A recent hypothesis supports the notion that gene promoter elements contain DNA G-quadruplex motifs that control gene expressi ...[more]

PMID: 17322877

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Project description:In human and mouse, the promoter of the KRAS gene contains a nuclease hypersensitive polypurine-polypyrimidine element (NHPPE) that is essential for transcription. An interesting feature of the polypurine G-rich strand of NHPPE is its ability to assume an unusual DNA structure that, according to circular dichroism (CD) and DMS footprinting experiments, is attributed to an intramolecular parallel G-quadruplex, consisting of three G-tetrads and three loops. The human and mouse KRAS NHPPE G-rich strands display melting temperature of 64 and 73 degrees C, respectively, as well as a K+-dependent capacity to arrest DNA polymerase. Photocleavage and CD experiments showed that the cationic porphyrin TMPyP4 stacks to the external G-tetrads of the KRAS quadruplexes, increasing the T(m) by approximately 20 degrees C. These findings raise the intriguing question that the G-quadruplex formed within the NHPPE of KRAS may be involved in the regulation of transcription. Indeed, transfection experiments showed that the activity of the mouse KRAS promoter is reduced to 20% of control, in the presence of the quadruplex-stabilizing TMPyP4. In addition, we found that G-rich oligonucleotides mimicking the KRAS quadruplex, but not the corresponding 4-base mutant sequences or oligonucleotides forming quadruplexes with different structures, competed with the NHPPE duplex for binding to nuclear proteins. When vector pKRS-413, containing CAT driven by the mouse KRAS promoter, and KRAS quadruplex oligonucleotides were co-transfected in 293 cells, the expression of CAT was found to be downregulated to 40% of the control. On the basis of these data, we propose that the NHPPE of KRAS exists in equilibrium between a double-stranded form favouring transcription and a folded quadruplex form, which instead inhibits transcription. Such a mechanism, which is probably adopted by other growth-related genes, provides useful hints for the rational design of anticancer drugs against the KRAS oncogene.

Project description:Contemporary treatment of pediatric acute myeloid leukemia (AML) requires the assignment of patients to specific risk groups. To explore whether expression profiling of leukemic blasts could accurately distinguish between the known risk groups of AML, we analyzed 130 pediatric and 20 adult AML diagnostic bone marrow or peripheral blood samples using the Affymetrix U133A microarray. Class discriminating genes were identified for each of the major prognostic subtypes of pediatric AML, including t(15;17)[PML-RARalpha], t(8;21)[AML1-ETO], inv(16) [CBFbeta-MYH11], MLL chimeric fusion genes, and cases classified as FAB-M7. When subsets of these genes were used in supervised learning algorithms, an overall classification accuracy of more than 93% was achieved. Moreover, we were able to use the expression signatures generated from the pediatric samples to accurately classify adult de novo AMLs with the same genetic lesions. The class discriminating genes also provided novel insights into the molecular pathobiology of these leukemias. Finally, using a combined pediatric data set of 130 AMLs and 137 acute lymphoblastic leukemias, we identified an expression signature for cases with MLL chimeric fusion genes irrespective of lineage. Surprisingly, AMLs containing partial tandem duplications of MLL failed to cluster with MLL chimeric fusion gene cases, suggesting a significant difference in their underlying mechanism of transformation. All the gene expression arrays are available through http://www.stjuderesearch.org/site/data/AML1/ in the original study (PMID:15226186). To study the RAS gene expression in the human AML patients, a total of 104 AML cases with known KRAS and NRAS status (including 72 gene expression arrays in the original study and 32 additional arrays acquired later on), as well as 4 CD34+ normal bone marrow cases deposited in GEO GSE33315, were including in this depository. Gene expression profiling was performed on 104 single diagnosis tumor samples and 4 CD34+ normal bone marrow samples

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An RNA G-quadruplex in the 5' UTR of the NRAS proto-oncogene modulates translation.

Publications

An RNA G-quadruplex in the 5' UTR of the NRAS proto-oncogene modulates translation.

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