In vivo veritas: using yeast to probe the biological functions of G-quadruplexes

Abstract

Certain guanine-rich sequences are capable of forming higher order structures known as G-quadruplexes. Moreover, particular genomic regions in a number of highly divergent organisms are enriched for such sequences, raising the possibility that G-quadruplexes form in vivo and affect cellular processes. While G-quadruplexes have been rigorously studied in vitro, whether these structures actually form in vivo and what their roles might be in the context of the cell have remained largely unanswered questions. Recent studies suggest that G-quadruplexes participate in the regulation of such varied processes as telomere maintenance, transcriptional regulation and ribosome biogenesis. Here we review studies aimed at elucidating the in vivo functions of quadruplex structures, with a particular focus on findings in yeast. In addition, we discuss the utility of yeast model systems in the study of the cellular roles of G-quadruplexes.

Figure 1

G-quadruplex structure and ligand binding. (A) Structure of a G-quartet, with hydrogen bonds between guanines indicated by dotted lines. (B) Schematic of one type of unimolecular G-quadruplex that is composed of three planar G-quartet structures, as shown. The orientation (5’->3’) of the phosphodiester backbone is indicated by black arrows. Many other types of G-quadruplex folds are possible. (C) Binding of certain G-quadruplex interacting factors (grey circles), be they proteins or small molecular weight ligands, can promote and/or stabilize the formation of G-quadruplexes from nucleic acid strands with quadruplex-forming potential (QFP). Further, they might inhibit access of other factors to G-quadruplexes.

Figure 2

Genetic tools for analyzing G-quadruplexes in yeast. (A) Circles represent yeast colonies that might be obtained from a typical synthetic enhancement screen. The relative sizes of colonies reflect the ability of yeast cells to grow given varying genetic conditions. For example, cells in which the fictional MUT1 gene has been disrupted (mut1) grow poorly as compared with wild-type (WT) cells (indicated by the small circle, representing colony size). Combining the mut1 mutation with the mut2 mutation, which has only a very subtle growth defect, results in synthetic enhancement of the sickness displayed by the mut1 single mutant. (B) In this example of genetic suppression, another mutation (mut3) also confers a growth disadvantage to cells. When combining the mut3 mutation with a mutant allele of another gene, mut4, the latter mutation suppresses the toxicity observed in mut3 cells, allowing improved growth of the double mutant. Similarly, synthetic enhancement or suppression, caused by particular mutations, of the impaired growth caused by a G-quadruplex binding ligand could identify factors that function with G-quadruplexes. (C) Synthetic screens could also utilize transcriptional reporter constructs driven by promoters containing QFP regulatory sequences. In this example, the reporter gene encodes β-galactosidase (β-gal), which produces a blue pigment when cells are grown on the appropriate substrate (level of expression indicated by degree of shading of the circles). Mutations can be obtained that alter the effect of a quadruplex-selective ligand on expression of the reporter gene. In this case, the ligand enhances transcription of the β-gal locus downstream of the promoter quadruplex, and mutations that further upregulate β-gal expression are considered to be “enhancing”. Conversely, those that counteract the activity of the ligand, thereby repressing β-gal, are considered to be “suppressive”. (D) A screen for mutations that affect transcription from a reporter construct with promoter QFP but not from an otherwise identical construct lacking promoter QFP could be used to identify intrinsic factors that influence quadruplexes.

Figure 3

Modulation of gene expression by G-quadruplexes. (A) A model of how quadruplex formation at DNA promoter regions with QFP might modulate transcription from downstream ORFs. G-quadruplex formation might repress transcription by occluding transcriptional activators and/or creating binding sites for transcriptional repressors. Conversely, quadruplex formation might also serve to activate transcription in some cases by blocking repressors and/or recruiting activators. In either case, the state of transcription of the relevant ORF is changed from one state (cross-hatched rectangle) to another (solid black rectangle) by quadruplex formation. (B) A model of how RNA quadruplex formation in the 5’UTR (white rectangle) of mRNA transcripts might affect its translation or stability. As in (A), G-quadruplex formation could positively or negatively modulate gene expression.