Deciphering the Enigmatic Biological Functions of RNA Guanine

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Deciphering the Enigmatic Biological Functions of RNA Guanine-Quadruplex Motifs in Human Cells Munir A. Al-Zeer, and Jens Kurreck Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00904 • Publication Date (Web): 17 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

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Biochemistry

Deciphering the Enigmatic Biological Functions of RNA Guanine-Quadruplex Motifs in Human Cells Munir A. Al-Zeer, Jens Kurreck* Institute of Biotechnology, Department of Applied Biochemistry, Technische Universität Berlin, 13355 Berlin, Germany Abstract Guanine-rich sequences in nucleic acids can form non-canonical structures known as Guaninequadruplexes (G-quadruplexes), which constitute a not yet fully elucidated layer of regulatory function for central cellular processes. RNA G-quadruplexes have been shown to be involved in the modulation of translation, the regulation of (alternative) splicing, and the subcellular transport of mRNAs, among other processes. However, in living cells an equilibrium between the formation of G-quadruplex structures and their unwinding by RNA helicases is likely. The extent to which G-rich sequences adopt G-quadruplex structures in living eukaryotic cells is currently a matter of debate. Multiple lines of evidence confirm the intracellular formation of G-quadruplex structures, such as their detection by immunochemical approaches, fluorogenic probes and in vivo NMR. However, intracellular chemical probing suggests most if not all are in an unfolded state. It is therefore tempting to speculate that some G-quadruplex structures are only temporarily formed when they are required to contribute to the fine tuning of the above mentioned processes. Future research should focus on the analysis of G-quadruplex formation under physiological conditions which will allow to re-evaluate the biological function of G-quadruplex motifs in regulatory processes in their natural environment and under physiological expression levels. This will help to elucidate their significance in the regulation of central processes in molecular biology and to exploit their potential as therapeutic targets.

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The central dogma of molecular biology, as postulated by Francis Crick in the late 1950s, gives a straightforward picture of the fundamental processes in a cell: the genetic information contained in DNA flows to RNA and is finally passed into protein. The second half of the 20th century found multiple special transfers of genetic information that were not fully covered by the originally stated dogma, such as reverse transcription and RNA replication. Since the elucidation of complete genomes in the early 21st century, we realized that complexity of life is not simply based on genes and their conversion into proteins. It came as a big surprise that the human genome encodes only 20-25,000 genes or even less1 which is substantially below than original estimates of approximately 100,000 protein-coding genes. Even more staggering was the finding that the genome of Arabidopsis thaliana contains roughly 27,000 genes. Many people found it surprising that humans with their presumably sophisticated abilities to move, speak, and contemplate their own existence have a smaller number of genes than plants. A large part of the explanation of these unexpected findings will be related to the importance of regulatory mediators that control processes such as expression, alternative splicing or intracellular transport. A new light began to shine on RNA as a major player that for a long time stood in the shade of DNA and proteins as the dominant biomolecules. Small RNA molecules, such as microRNAs and long nonprotein coding RNAs, are nowadays generally accepted as important regulators of gene expression.2 Another layer of regulatory complexity whose relevance is still unclear can be found in the structure of RNA.3 A particularly interesting structure can be formed by guanine-rich sequences that assemble in a noncanonical way to form a guanine-quadruplex (G-quadruplex, also abbreviated as G4 or GQ, Figure 1).4 In this four-stranded structure, four guanines assemble to form a coplanar arrangement of a G-quartet through Hoogsteen hydrogen bonding. The complete G-quadruplex structure is then formed by -stacking of two or more G-quartets that are linked together by three loops.5 Potential G-quadruplex motifs are commonly described as Gx - N1-7 - Gx - N1-7 - Gx - N1-7 - Gx, where x is 3-6 and N denotes any nucleotide. While this definition refers to the canonical structure of G-quadruplexes, recent research revealed that non-canonical G-quadruplex structures exist beyond this definition such as those consisting of two Gtetrades only, those with additional loops connecting the tetrades or those with longer loops.6

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Figure 1. G-quadruplex structure. A) Structure of a G-tetrad. Hoogsteen hydrogen bonding is shown by the dotted lines. B) Schematic representation of a parallel, intramolecular RNA G-quadruplex structure. The arrows indicate the orientation from 5’ to 3’. G-quadruplex motifs are significantly affected by the presence of a central cation, of which potassium is the most effective monocation at stabilizing the structure.7 In contrast, Li+, with its very small ionic radius, is incapable of supporting G-quadruplex formation. Importantly, typical cellular concentrations of 140 mM K+ are sufficient to support G-quadruplex formation in vivo. In contrast to DNA G-quadruplexes, which are structurally highly diverse, RNA G-quadruplexes usually adopt the parallel conformation where all four strands are oriented in the same direction. Only recently, the first observation of an antiparallel conformation of an RNA G-quadruplex formed by human telomere RNA was reported.8 In cells, DNA is almost exclusively present as a double-stranded molecule. Thus, G-quadruplex formation of G-rich sequences has to compete with hybridization to the perfectly matching complementary strand. Although RNA normally also exists as a highly structured molecule, these interactions are often weaker as complementary sequences are interrupted by many bulges and loops so that G-quadruplex formation can be expected to be facilitated in RNA compared to DNA. In addition, RNA G-quadruplexes were reported to be more stable than their DNA counterparts.9, 10 To detect RNA G-quadruplexes on a transcriptome-wide level, an RNA G-quadruplex sequencing (rG4seq) approach was carried out which was based on the combination of reverse transcriptase stalling (RTS) by G-quadruplex structures and next-generation sequencing.11 In the presence of K+ and the G-quadruplexspecific ligand pyridostatin (PDS), an RNA G-quadruplex stabilizing ligand which increases the RTS12, 11,500 RTS sites were found in polyadenylated RNA isolated from HeLa cells which can be considered RNA Gquadruplexes. A recent review by Weldon et al. summarized new methodological developments including

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computational and experimental approaches for the detection and mapping of RNA G-quadruplexes to gain further insight into their biological functions.13 The function of DNA G-quadruplexes has been studied for several decades and they have been found to be important for the maintenance of telomeres and to have regulatory function in promoters as detailed in several excellent reviews.14-16 The study of the biological function of RNA G-quadruplexes has gained great momentum with the discovery that an RNA G-quadruplex in the 5’-UTR of the neuroblastoma RAS viral oncogene homolog (NRAS) proto-oncogene modulates translation in a cell-free translation system.17 Soon thereafter, we and others demonstrated that G-quadruplex motifs located in the 5’-UTR of a variety of mRNAs also inhibit translation in living eukaryotic cells (Figure 2).18-22 A survey of numerous studies investigating the regulatory function of G-quadruplex structures located in the 5’-UTR of mRNAs revealed that most G-quadruplexes repress translation by 30 to 70%.23 They can thus rather be considered fine regulators of gene expression than on-off switches. The role of RNA G-quadruplexes and associated transacting factors in the regulation of mRNA translation was broadly covered in the recent review by Song et al.24 Furthermore, the importance of RNA G-quadruplexes is also reflected by an increasing number of reports that link RNA G-quadruplex (dys)function to human diseases.25

Figure 2. Functions of RNA G-quadruplexes in eukaryotic cells. Among other functions, G-quadruplex motifs modulate translation, regulate (alternative) splicing, and mediate intracellular mRNA localization. For further details, see text. The moderate repressive activity of the G-quadruplexes makes them ideal candidates as regulatory control elements for the above described processes. For these purposes, dynamic behavior of the Gquadruplexes would be required, for example in response to environmental conditions or interactions with proteins or small molecules. As previously mentioned, cellular cation concentrations are in the range that 4 ACS Paragon Plus Environment

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supports formation of G-quadruplex structures. A possible mechanism that has, to the best of our knowledge, not yet been fully explored, is the modulation of RNA G-quadruplex stability by varying K+ concentrations (e.g. in neurons or cancer cells) that may influence translational activity. However, some RNA G-quadruplexes are extremely stable, even at very low K+ concentrations. It therefore remains to be clarified, to which extent RNA G-quadruplexes will be affected by physiological changes in the K+ concentration. For DNA, destabilization of G-quadruplex formation and increase of transcription has been observed during tumor progression which is associated with a decrease of intracellular K+ concentration.26 Another conceivable mechanism that modulates the stability of G-quadruplex motifs and/or mediates their regulatory effects is through binding to cellular proteins, and, consequently, several groups have investigated such interactions. Among the identified binding partners of G-quadruplexes were several heterogeneous nuclear ribonucleoproteins (hnRNPs), ribosomal proteins, and splicing factors, but also candidates that have previously not been described to interact with nucleic acids.27-29 These finding strengthen the assumption that G-quadruplexes play an important role in modulating translation and splicing. Gene ontology analysis further revealed that many of the candidate proteins are known to be tumor-related.29 Members of the hnRNP family were also reported to interact with the G-rich sequence of the telomeric repeat-containing RNA (TERRA).30 It has been reported that the fragile X mental retardation protein (FMRP) controls its own translation by a negative feedback loop when the protein binds to a Gquadruplex in the coding region of its own mRNA.31 The FMRP may also interact with G-quadruplex motifs in other mRNAs and repress their translation by various mechanisms, including the induction of the microRNA (miRNA) pathway and direct interaction with the translating ribosome.32 Furthermore, the FRAXE-associated mental retardation protein (FMR2) was shown to interact with G-quadruplex structures in mRNAs and to be involved in alterative splicing.33 RNA G-quadruplexes not only interact with proteins, but also bind small molecules. For example, anthrafurandiones and anthrathiophenediones were shown to bind to the G-quadruplex in the Kirsten Ras (KRAS) mRNA and repress translation in a dose-dependent manner.34 This interaction induced apoptosis in pancreatic cancer cells and reduced cell growth and colony formation. In another recently published study, an anionic phthalocyanine was shown to bind to the G-quadruplex in the NRAS mRNA and induce its selective cleavage upon photo-irradiation.35 This resulted in lower NRAS expression and decreased viability of cancer cells. These examples and many more suggest G-quadruplex structures can serve as targets for the therapeutic application of specific low molecular weight substances.36 While repression of translation is the most intensively investigated function of RNA G-quadruplexes, they have been reported to be involved in the regulation of further processes, such as alternative splicing, polyadenylation, and intracellular RNA transport. Alternative splicing of mRNA is an indispensable 5 ACS Paragon Plus Environment

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mechanism to increase the complexity of gene expression. Splicing factors regulate alternative splicing through binding to RNA consensus sequences.37 Additionally, RNA structures such as G-quadruplexes are emerging as important regulators in alternative splicing.38 It was shown that G-quadruplexes adjacent to splicing sites can act as cis-regulatory elements that affect the splicing machinery, for example in the c-src N1 exon.39 Further, Didot et al. demonstrated that the presence of two independent G-quadruplex motifs in exon 15 of the Fragile X mental retardation 1 (FMR1) mRNA control alternative splicing.40 Additional studies have revealed the importance of G-quadruplex structures present in introns for regulating splicing, for example in Bcl-X and CD133. 41-43 A G-quadruplex in the third intron of TP53 was shown to regulate splicing of intron 2, which resulted in differential expression of the p53 isoforms.44 In contrast, a Gquadruplex in intron 6 of human telomerase reverse transcriptase (hTERT) was proposed to silence splicing.45 A recent study suggests that RNA G-quadruplex structures promote alternative splicing via hnRNPF.28 This process inhibits an epithelial-mesenchymal transition and is thus important for tumor progression. In addition to splicing control, G-quadruplex structures are instrumental in RNA transport and localization within cells, particularly in neurons, where RNA must be sorted and transported over a long distance. Mislocation of the mRNAs may cause neurological diseases. Transport and localization of the mRNAs are important for cell polarity which was proposed to be controlled by motor proteins that interact with cis-elements usually located in the 3’-UTR of the mRNA.46. More recently, G-quadruplex structures acting as cis-elements which might interact with motor proteins, e.g. G-quadruplex structures in the 3′UTRs of the mRNAs of the key postsynaptic proteins PSD95 and CaMKIIa, have been shown to facilitate their localization in neurites.47 In addition, it was proposed that a G-quadruplex in the 3′-UTR of Annexin 2A (Anax2) mediates the localization of the Anxa2 mRNA into the axonal compartment of NSC-34 motor neuron-like cells.48 Furthermore, G-quadruplexes in the 3′-UTR of FXR1 and LRP5 mRNAs were found to increase the efficiency of alternative polyadenylation which in turn enhanced the generation of shortened transcripts.49 Over the years, multiple functions of G-quadruplexes in transcripts have been discovered in eukaryotic cells, in addition to those described above. For example, a G-quadruplex in the 3’-UTR of the Pim-1 mRNA was reported to repress translation in a similar manner as described for G-quadruplexes in 5’-UTRs.50 A Gquadruplex motif in the Vascular Endothelial Growth Factor (VEGF) mRNA was shown to be essential for cap-independent translation initiated by an internal ribosome entry site (IRES).51 G-quadruplex structures were also reported to be relevant for miRNA-mediated regulation of gene expression: When located in pri-miRNAs, they may influence their processing52, whereas a G-quadruplex motif in the 3’-UTR of an mRNA was found to prevent binding of the regulatory microRNA.53 Furthermore, G-quadruplexes were 6 ACS Paragon Plus Environment

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Biochemistry

found in viral genomes and play a regulatory role in the viral life cycle.54 For example, a G-quadruplex motif in the negative RNA strand of Hepatitis C virus was reported to regulate RNA synthesis and thereby modulate viral replication.55 Further biological functions of RNA G-quadruplex structures are constantly being reported. While more and more data accumulated on the functional diversity of RNA G-quadruplexes, a provoking paper was published in 2016, according to which RNA G-quadruplexes are globally unfolded in eukaryotic cells.56 This would, of course, question much of the functional assignments to RNA G-quadruplexes that had been made in the past. Consequently, the study was intensively discussed by the community on the biannual G-quadruplex meeting in Prague in 2017.57 In their study, Guo and Bartel used chemical probing techniques, stalling of reverse transcriptase and next generation sequencing to detect the formation of Gquadruplex structures.56 Using transcripts isolated from eukaryotic cells, they found more than 10,000 sites that form intramolecular G-quadruplexes in vitro. However, when they used chemicals that can penetrate living cells, the structures that were detected in vitro were overwhelmingly unfolded in vivo, suggesting a mechanism that resolves G-quadruplex structures in cells. Although this study is important to our understanding of the significance of RNA G-quadruplexes, the results, at face value, may be misleading and some limitations of the experimental approach have been pointed out.58 It is highly plausible that some G-quadruplexes form only temporarily and/or under certain cellular conditions and may thus have been overseen by the chemical probing procedure. Furthermore, the combined chemical probing/RNA sequencing approach requires comparatively high expression levels of transcripts to detect RNA G-quadruplexes so that the obtained results may be incomplete. The study also used unusually high concentrations of DMS for chemical probing. By doing so, weak G-quadruplexes, as well as dynamic G-quadruplexes that temporarily unfold, will be modified by DMS and may be erroneously classified as being unfolded in vivo. Finally, the experiments were based on the comparison of RNA structures found in the presence of K+, which stabilizes G-quadruplexes, to those in the presence of Na+ or Li+, which do not (or only weakly) support folding of G-quadruplexes. However, many highly stable G-quadruplex structures are known that form even in the presence of Na+ and will therefore be missed by this approach. While the study by Guo and Bartel suggests that G-quadruplex structures are largely unfolded in eukaryotic cells, several lines of evidence have been given that support their existence in living cells. In 2014, Biffi et al. demonstrated the presence of RNA G-quadruplex structures in fixed human cells using a structure-specific antibody.59 Treatment of the cells with G-quadruplex stabilizing ligands increased the intensity of the signal, which further supports the presence of RNA G-quadruplexes in cells. However, some limits of immune-based approaches were noted:58 fixation and permeabilization may lead to folding of 7 ACS Paragon Plus Environment

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structural elements that were not present in living cells. In addition, antibodies may have limited specificity due to cross-reactivity with other sequence motifs. However, another indication for the in vivo existence of RNA G-quadruplex structures was the visualization of the G-quadruplex located in the NRAS mRNA with an engineered fluorogenic hybridization probe.60 The most recent and compelling evidence came from a 19F

NMR experiment, confirming the existence of higher order RNA G-quadruplexes in living cells.61

The controversy about the presence of G-quadruplex motifs in vivo points to the importance of helicases that unwind RNA G-quadruplex structures, as an equilibrium between the thermodynamic impetus of Grich sequences to fold into G-quadruplex structures and their unfolding by helicases can be expected.62 Several helicases have been discovered that have the ability to resolve RNA G-quadruplex structures. The Rhau helicase, the gene product of DEAH-box helicase 36 (DHX36), which is also known as the G4 Resolvase 1 (G4R1) was described as representing the major RNA G-quadruplex helicase activity in HeLa cell lysates.63 Crystal structure analysis (with a DNA G-quadruplex) recently highlighted that ATP-independent structural changes of the RHAU helicase upon G-quadruplex binding are important for the unfolding process.64 However, in the previously mentioned study that reported global unfolding of RNA G-quadruplexes in eukaryotic cells, inducible deletion of DHX36 through Cre-mediated recombination in mouse embryonic fibroblasts did not induce substantial changes in RNA folding.56 The authors concluded that Rhau is dispensable for the unfolding of endogenous RNA G-quadruplex structures and further helicases must fulfill the task. It is also possible that a redundancy of the helicases exists, i.e. loss of function of one helicase may be compensated by another one. In the meantime, several additional helicases have been reported to possess RNA G-quadruplex unwinding activity: DHX9 was found to unwind RNA G-quadruplexes nearly twice as fast as the analog DNA motif65 and to facilitate translation of mRNAs with highly structured 5’-UTRs.66 Another candidate is DDX21, a DEAD-box RNA helicase with RNA G-quadruplex unwinding capacity.67 The eukaryotic initiation factor-4A (eIF4A) is another DEAD-box helicase and selected oncogenes were reported to carry Gquadruplexes in their 5’-UTRs that cause their eIF4A-dependent translation in cancer.68 Another recent study demonstrated that the CCHC-type zinc finger nucleic acid-binding protein (CNBP/ZNF9) prevents the formation of G-quadruplex structures and increases translational efficiency of its target mRNAs.69 In contrast to the above mentioned ATP-dependent helicases, CNBP is unlikely to actively unfold Gquadruplexes, but rather stailizes and traps the unfolded state. This finding indicates that proteins that are not known as classical helicases may also affect the equilibrium of the folded and unfolded state of RNA G-quadruplexes. It should also be noted that the inverse case, i.e. the potential existence of RNA chaperones that support G-quadruplex folding has not yet been seriously studied. 8 ACS Paragon Plus Environment

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A shortcoming of functional characterizations of the G-quadruplex function, as carried out in most published studies, was the standard experimental setup using reporter assays, usually based on luciferase expression driven by strong promoters.70 To gain further insight into the relevance of RNA G-quadruplexes under physiological conditions, we studied in more detail the expression of Bcl-2, a well-known inhibitor of apoptosis with high clinical relevance.71 The RNA G-quadruplex located in the 5’-UTR of Bcl-2 was reported to repress translation in reporter assays.72 In fact, expression of luciferase reporters harboring the Bcl-2 G-quadruplex motif in their 5’-UTR under control of strong promoters such as the SV40 or the CMV promoter resulted in suppression of translation relative to a mutated control.73 The inhibitory effect was more pronounced when the isolated G-quadruplex-forming sequence was cloned upstream of the luciferase reporter than for the full-length 5’-UTR. Even more surprising was the finding that the expression of Bcl-2 remained unchanged when the G-quadruplex-forming sequence was deleted at the genomic level by means of CRISPR/Cas9. Consequently, no inhibition of apoptosis was observed in cells with the disrupted G-rich sequence upstream of the Bcl-2 encoding sequence. Interestingly, the study mentioned above72, which also used the strong CMV promoter for reporter assays, reported that the extent of translational repression depends on the cell type. While the G-quadruplex motif repressed translation by approximately two-fold in MCR10A cells, a human breast epithelial cell line, the effect was reduced to 3040% in the human gastric carcinoma cell line HGC27. Together, these studies demonstrate that the extent to which a G-quadruplex suppresses translation depends on various factors, including the sequence environment and length of the 5’-UTR, the cell type and the expression level. It is thus possible that reporter assays with strong promoters oversaturate unwinding capacity of cellular helicases and, as a consequence, may be misleading and assign G-quadruplexes a prominence in cells that is overestimated. It will therefore be important to re-evaluate the biological function of G-quadruplex motifs in regulatory processes in their natural environment and under physiological expression levels. Taken together, multiple structural and functional studies have provided convincing evidence for the intracellular existence of RNA G-quadruplex structures and their contribution to the regulation of central cellular processes such as gene expression, splicing and mRNA translocation. However, the standard experimental setup used by many groups, including the authors’ own lab, may have supported overestimation of the prominence of G-quadruplexes. The paper by Guo and Bartel56 seems to place a limit on the extent of G-quadruplexes in vivo. While it is unlikely that they are pure experimental artifacts of in vitro systems, their results make it unlikely that in vivo they are the normal state. They seem to be rarer than thought before, and thus the machinery that regulates the equilibrium of folding and unwinding of these unusual, non-Watson-Crick structures needs to be further elucidated. The idea is compelling that some G-quadruplex structures are only temporarily formed, presumably even only in a subfraction of a 9 ACS Paragon Plus Environment

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given RNA molecule, which may confer upon them an important role in the fine-tuning of regulatory processes. Only with the elucidation of these features will we be able to clarify the biological function RNA G-quadruplex motifs and exploit them as therapeutic targets. Author Information Corresponding Author *E-mail: [email protected] Acknowledgement The authors are thankful to Jean-Louis Mergny, Erik Wade and Katrin Paeschke for intensive and fruitful discussions on this fascinating topic. Funding of the authors’ G-quadruplex research be the Deutsche Forschungsgemeinschaft (DFG Ku1436/7-1) is gratefully acknowledged. Notes The authors declare no competing financial interest. References [1] Ezkurdia, I., Juan, D., Rodriguez, J. M., Frankish, A., Diekhans, M., Harrow, J., Vazquez, J., Valencia, A., and Tress, M. L. (2014) Multiple evidence strands suggest that there may be as few as 19,000 human protein-coding genes, Hum. Mol. Genet. 23, 5866-5878. [2] Morris, K. V., and Mattick, J. S. (2014) The rise of regulatory RNA, Nat. Rev. Genet. 15, 423-437. [3] Leppek, K., Das, R., and Barna, M. (2018) Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them, Nat. Rev. Mol. Cell Biol. 19, 158-174. [4] Kwok, C. K., and Merrick, C. J. (2017) G-Quadruplexes: Prediction, Characterization, and Biological Application, Trends Biotechnol. 35, 997-1013. [5] Lipps, H. J., and Rhodes, D. (2009) G-quadruplex structures: in vivo evidence and function, Trends Cell Biol. 19, 414-422. [6] Bolduc, F., Garant, J. M., Allard, F., and Perreault, J. P. (2016) Irregular G-quadruplexes Found in the Untranslated Regions of Human mRNAs Influence Translation, J. Biol. Chem. 291, 21751-21760. [7] Rachwal, P. A., Findlow, I. S., Werner, J. M., Brown, T., and Fox, K. R. (2007) Intramolecular DNA quadruplexes with different arrangements of short and long loops, Nucleic Acids Res. 35, 42144222. [8] Xiao, C. D., Shibata, T., Yamamoto, Y., and Xu, Y. (2018) An intramolecular antiparallel G-quadruplex formed by human telomere RNA, Chem. Commun. 54, 3944-3946. [9] Bhattacharyya, D., Mirihana Arachchilage, G., and Basu, S. (2016) Metal Cations in G-Quadruplex Folding and Stability, Front. Chem. 4, 38. [10] Sacca, B., Lacroix, L., and Mergny, J. L. (2005) The effect of chemical modifications on the thermal stability of different G-quadruplex-forming oligonucleotides, Nucleic Acids Res. 33, 1182-1192. [11] Kwok, C. K., Marsico, G., Sahakyan, A. B., Chambers, V. S., and Balasubramanian, S. (2016) rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome, Nat. Methods 13, 841-844. 10 ACS Paragon Plus Environment

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