5'-UTR G-quadruplex structures acting as translational repressors

Abstract

Given that greater than 90% of the human genome is expressed, it is logical to assume that post-transcriptional regulatory mechanisms must be the primary means of controlling the flow of information from mRNA to protein. This report describes a robust approach that includes in silico, in vitro and in cellulo experiments permitting an in-depth evaluation of the impact of G-quadruplexes as translational repressors. Sequences including potential G-quadruplexes were selected within nine distinct genes encoding proteins involved in various biological processes. Their abilities to fold into G-quadruplex structures in vitro were evaluated using circular dichroism, thermal denaturation and the novel use of in-line probing. Six sequences were observed to fold into G-quadruplex structures in vitro, all of which exhibited translational inhibition in cellulo when linked to a reporter gene. Sequence analysis, direct mutagenesis and subsequent experiments were performed in order to define the rules governing the folding of G-quadruplexes. In addition, the impact of single-nucleotide polymorphism was shown to be important in the formation of G-quadruplexes located within the 5′-untranslated region of an mRNA. In light of these results, clearly the 5′-UTR G-quadruplexes represent a class of translational repressors that is broadly distributed in the cell.

Translational Repression of the Disintegrin and Metalloprotease ADAM10 by a Stable G-quadruplex Secondary Structure in Its 5′-Untranslated Region

“…2D). Moreover, compared with the CD spectrum of the wild-type sequence, the maxima and minima were shifted slightly to higher wave lengths similar to the CD spectra of unstructured, single-stranded RNA (37,40,57). Taken together, our biophysical analysis confirms that the ADAM10GQ-WT RNA forms a highly stable, unimolecular, parallel G-quadruplex near physiological pH and salt conditions.…”

“…On the other hand, G-quadruplex signature motifs occur predominantly at the 5Ј end of the 5Ј-UTR, which suggests that G-quadruplex secondary structures are involved in translational regulation either by inhibiting the formation of the initiation complex or by inhibition of the scanning ribosome (30). In agreement with this hypothesis, a series of recent reports demonstrate that the 5Ј-UTRs of N-ras, Zic-1, membrane-type matrix metalloproteinase 3, ESR-1, Bcl-2, and others contain a G-quadruplex motif that inhibits the translation of their downstream genes (35)(36)(37)(38)(39)(40)(41)(42).…”

“…The ADAM10 G-quadruplex Motif Inhibits Translation of a Luciferase Reporter in Vivo-Recently, it was shown that G-quadruplex motifs within 5Ј-UTRs inhibit translation of their downstream gene in vitro and in vivo (35)(36)(37)(38)(39)(40)(41)(42). Moreover, artificially introduced RNA-G-quadruplex motifs near the ribosomal binding site suppress bacterial gene expression (58).…”

“…The basic structural core motif of G-quadruplexes consists of a series of G-quartet planes, each of which consists of four guanines connected by Hoogsteen-hydrogen bonds and stabilized by monovalent ions, particularly potassium ions (31-34). There is growing evidence that the formation of G-quadruplex secondary structures within 5Ј-UTRs results in translational repression of a number of different mRNAs, including N-ras, Zic-1, membrane-type matrix metalloproteinase 3, ESR-1, and Bcl-2 (35)(36)(37)(38)(39)(40)(41)(42). Similar to the IRE stem loop, the stability of G-quadruplex structures could be modulated by RNA binding proteins such as FMRP or members of the hnRNP A family (43,44).…”

The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing

“…We speculate that cytoplasmic transcripts that are not translated and enter ICM/nuage can be recognized by pICL and processed into piRNAs. This could explain why the bulk of genic piRNAs are derived from 3 ′ UTRs and the presence in pachytene piRNA precursors of G4s and other secondary structure elements, which may function to impede translation (Arora et al 2008;Beaudoin and Perreault 2010). Ultimately, identification of all components of the pICL complex and biochemical characterization with reconstitutions of its activities will be required to further address how piRNA precursors are selected to enter ICM/nuage, the role of G4 and other structural elements, and the precise molecular function of all pICL complex components in piRNA processing.…”