John J. Murphy

Kalpana Surendranath

Radhakrishnan Kanagaraj

English

RNA-binding proteins (RBPs) represent a large family of proteins with an extensive array of roles that contribute to coordinating and directing multiple functions in RNA metabolism and transcription [...]

Murphy, J.

Surendranath, K.

Kanagaraj, R.

WestminsterResearch

Citation: Murphy, J.J.; Surendranath,K.; Kanagaraj, R. RNA-BindingProteins and Their Emerging Roles inCancer: Beyond the Tip of the Iceberg.Int. J. Mol. Sci. 2023, 24, 9612.https://doi.org/10.3390/ijms24119612Received: 22 May 2023Accepted: 25 May 2023Published: 1 June 2023Copyright: © 2023 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). International Journal of Molecular SciencesEditorialRNA-Binding Proteins and Their Emerging Roles in Cancer:Beyond the Tip of the IcebergJohn J. Murphy 1,*,† , Kalpana Surendranath 1,†,‡ and Radhakrishnan Kanagaraj 1,2,3,*,†1 Genome Engineering Laboratory, School of Life Sciences, University of Westminster, 115 New CavendishStreet, London W1W 6UW, UK; k.surendranath1@westminster.ac.uk2 School of Life Sciences, University of Bedfordshire, Park Square, Luton LU1 3JU, UK3 Centre for Drug Discovery and Development, Sathyabama Institute of Science and Technology,Chennai 600119, India* Correspondence: j.murphy@westminster.ac.uk (J.J.M.); k.radhakrishnan@westminster.ac.uk (R.K.)† Guest Editors of International Journal of Molecular Sciences Special Issue on RNA-Binding Proteins and TheirEmerging Roles in Cancer.‡ Correspondence may also be addressed to: k.surendranath1@westminster.ac.uk (K.S.).RNA-binding proteins (RBPs) represent a large family of proteins with an extensivearray of roles that contribute to coordinating and directing multiple functions in RNAmetabolism and transcription. Currently, there are upwards of 1500 RBPs, and unravellingtheir range of complex functions in cell biology is an ongoing research effort, where there isstill much to discover. Although RBPs primarily interact with RNA in both the nucleusand cytoplasm, these interactions often have a wider influence on cell biology, includingeffects on genomic stability and critical cell protein levels. Recent studies have shed light onspecific RBPs and their roles in cancer, but the detailed molecular mechanisms responsiblefor the reported roles of RBPs in tumorigenesis have hitherto been largely unexplored. ThisSpecial Issue, entitled “RNA-Binding Proteins and Their Emerging Roles in Cancer” inthe International Journal of Molecular Sciences (IJMS), includes seven contributions, threeoriginal articles, and four reviews covering a wide range of highly relevant topics in RBPbiology and cancer. These include cancer therapies, post-transcriptional regulation andgenome integrity, non-coding RNA regulation, RNA and DNA damage responses, andR-loop biology. A summary of the contributions that make up this recently published IJMSSpecial Issue are provided below.Nasiri-Aghdam et al. [1] provided an interesting, timely review on the pleiotropicCUGBP Elav-like family (CELF) of RBPs, which comprise six members of CELF1-6. Theseproteins are found in the nucleus and cytoplasm of cells. The major functions of theseproteins include regulating mRNA alternative splicing in the nucleus and the regulation ofmRNA stability, translation, and alternative polyadenylation in the cytoplasm by bindingto consensus sequences in the 3′UTR regions of mRNAs. Intriguingly, almost 15% of CELF1binding sites are found in intergenic regions, which primarily include non-coding RNAs(ncRNAs); therefore, the cross-regulation of CELF RBPs and ncRNAs and its potentialimportance in cancers is a focus of this review. There is currently more published infor-mation on CELF1 and CELF2 in cancers than on CELF3-6 proteins. CELF1 and CELF2often have tumour suppressor functions, but their potential oncogenic roles in certaincancers were also highlighted [1]. In the future, the pharmacological targeting of CELFproteins may be useful in novel cancer treatments [1]. Notably, CELF1 aggregation in theperinucleolar compartment (PNC) occurs more frequently in cancer cells, suggesting thatorganelle-specific drug delivery and targeting could be a potential therapeutic approachfor cancer treatments [1].Another small family of RBPs that regulate alternative splicing, such as the CELFRBPs, are the muscleblind-like (MBNL) RBPs, MBNL1, MBNL2, and MBNL3. The multi-functional roles of MBNL RBPs were evidenced in a research article by Cai et al. [2]. MBNL2Int. J. Mol. Sci. 2023, 24, 9612. https://doi.org/10.3390/ijms24119612 https://www.mdpi.com/journal/ijmsInt. J. Mol. Sci. 2023, 24, 9612 2 of 4has reported roles in tumorigenesis and the authors investigated the effect of MBNL2knockdown on the transcriptome of cancer cell lines. The authors found a strong connectionbetween MBNL2 knockdown and a reduction in cyclin-dependent kinase inhibitor 1A(p21CDKN1A) expression, which is mediated by the effects of MBNL2 on the modulationof p21 mRNA stability. They presented evidence that these effects are independent of thep53 protein [2]. MBNL2 knockdown also results in increased checkpoint kinase 1 (CHK1)S345 phosphorylation, which can be rescued by an overexpression of p21 [2]. Finally, theauthors showed that MBNL2 depletion increased DNA-damage-induced apoptosis, whileinhibiting DNA damage repair and DNA damage-induced cellular senescence [2].The review provided by Sidali et al. [3] was concerned with eight proteins, which areall well characterised as AU-rich element RBPs (AU-RBPs) and have been reported to bindto mRNAs containing AUUUA motifs in their 3′UTR, thereafter mediating either mRNAdegradation or stabilisation. These proteins mediate their post-transcriptional regulation ofgene expression effects, primarily in the cell cytoplasm; however, many of these proteinscan also be found in the nucleus. This review focused on canonical and emerging non-canonical functions, specifically in relation to their roles in maintaining genomic stability [3].AU-RBPs post-transcriptionally regulate the expression of the mRNAs involved in DNAdamage response and signalling, for instance, ZFP36 stabilises Claspin mRNA, whichis critical for the activation of CHK1 via ATR. In response to genotoxic stress, AU-RBPscan be post-translationally modified and shuttle between the cytoplasm and nucleus tocarry out specific functions. Evidence for the nuclear functions of AU-RBPs in maintaininggenomic stability is sparse, but there are some interesting avenues being explored, such asthe role of intronic AU-rich elements, which do appear to have a role in regulating mRNAexpression via the AU-RBP HuR. In addition, emerging evidence points to a role for certainAU-RBPs, for instance AUF1, in preventing the formation of R-loops after DNA damageand inhibiting the defective DNA damage response that can promote genomic instability.The reported roles for AU-RBPs in cancer are numerous; for example, AU-RBP ZFP36L1has been reported as a driver gene for human breast cancer.Löblein et al. [4] produced an interesting research article concerning post-transcriptionalregulator Musashi proteins (MSI-1 and MSI-2) in ovarian cancer. MSI RBPs functions havemostly been reported in stem cells and Löblein et al. [4] investigated whether these RBPsmaintain the cancer stem cell characteristics in ovarian cancer. Furthermore, as MSI1 andMSI2 have a high homology and overlapping functions, they also investigated whethera dual knockdown of both of these proteins might have therapeutic potential in ovariancancer. MSI expression in ovarian cancer is positively correlated with cancer stem cellrelated genes. A dual knockdown of MSI1 and MSI2 reduced cell growth and increased thesensitivity of ovarian cancer cell lines to chemotherapy, suggesting that this strategy mayhave therapeutic potential in this cancer.Dolicka et al. [5] presented a review of stress granules (SGs) and their emerging rolesin hepatocellular carcinoma (HCC). SGs form in cells in response to various cell stresssignals and are membrane-free cytosolic compartments where mRNAs are protected andtranslationally silenced. Interestingly, increased SG formation is associated with severalliver diseases, including HCC. Dolicka et al. [5] reviewed the current knowledge of SGformation and analysed the SGs proteome. Many proteins in SGs are also associated withHCC and include numerous RBPs and scaffold proteins, etc., which have been reported astumour suppressors or oncogenes in different cancers. Although an oncogenic role for SGsin HCC has not yet been firmly demonstrated, the authors reviewed the emerging evidencefor such a role and focused on the selected protein and nucleic acid components of SG thatmight have the potential to be pharmacologically targeted in future therapeutic strategiesfor HCC.Uruci et al. [6] provided a comprehensive review on R-loops, which are three-strandedstructures that arise when RNA/DNA hybrids form after an RNA strand hybridises to atranscribed DNA template strand, displacing the non-transcribed DNA template as a single-stranded DNA. They arise naturally in cells as a consequence of a number of physiologicalInt. J. Mol. Sci. 2023, 24, 9612 3 of 4processes, and these R-loops, termed “scheduled” R-loops, are mostly resolved quickly. R-loops can also have pathological functions and these “unscheduled” R-loops arise as a resultof cellular dysregulation. The formation of “unscheduled”, persistent and uncontrolled,R-loops can lead to DNA damage and genomic instability. This dual nature of R-loopformation was explored by Uruci et al. [6], who made compelling arguments for thephysiological functions of R-loop formation, including intriguing data that were consistentwith “unscheduled” R-loops having a role in DNA repair. These authors also discussedthe cellular components and mechanisms for preventing and resolving/removing R-loopsand provided details on specific “Chro-Mates” that function either as chromatin modifiersor chromatin remodelers, thus highlighting the importance of chromatin accessibility andcondensation for the presence of RNA/DNA hybrids. Readers are encouraged to read theTaneja paper to acquire new insights into the RNA/DNA damage response and the latestdevelopments in this new area of study.Nascakova et al. [7] provided a timely research article elucidating the role of thehomologous recombination factor Rad51 in regulating the R-loop formation in humancells. Using the U-2-OS-based cellular system with an inducible expression of the mutantRNaseH1 enzyme, which can recognize and bind but not cleave R-loops, the authorsshowed that the chemical inhibition of Rad51 by B02 specifically causes the formationof R-loops in the early G1 phase of the cell cycle. Furthermore, they demonstrated thatthe formation of B02-induced G1-specific R-loops requires the presence of RAD51 andRNA polymerase II transcription initiation. Finally, they presented evidence that B02treatment disrupts the pre-replication complex and promotes premature origin firing andthe initiation of DNA synthesis in the early G1 phase of the cell cycle. In addition, B02treatment was shown to accumulate R-loops near rDNA transcription start sites.A common theme that is evident in the publications in this Special Issue is that RBPsare present in both the cytoplasm and the nucleus, often shuttling between these two cellcompartments and other cytosolic compartments such as stress granules and P bodies.These observations are consistent with multiple potential regulatory functions for individ-ual RBPs in RNA transcription and metabolism. Increasing evidence is emerging that linksmutations in RBPs to tumorigenesis. There is evidence that some RBPs can regulate thestability of the RNAs involved in cell cycle control and apoptosis. Additionally, certainRBPs appear to be involved in maintaining genomic stability through direct or indirectmechanisms. At least some RBPs have roles in the regulation of R-loops, which are increas-ingly considered to be major sources of genomic instability. A picture is emerging of acomplicated interplay between the RBPs regulating multiple functions in RNA transcrip-tion and metabolism that are also crucial for maintaining genomic stability and avoidingunwanted effects on the genome, such as the generation of R-loops when RNA transcriptionconflicts with DNA replication. Interestingly, mounting data identify RNA/DNA hybridsas immunogenic species that abnormally accumulate in the cytoplasm following R-loopprocessing. Future research into the functions of RBPs in controlling these cytoplasmicRNA/DNA hybrids, the resulting innate immune activation, and its associations withmany disorders, including cancer, would be intriguing.To date, most RBPs have generally been considered as non-tractable for therapeuticpurposes. Novel approaches, such as proteolysis-targeting chimera (PROTAC) strategies,may enable selected RBPs to soon become tractable. It is likely in the future that newtherapeutic strategies coupled with more detailed analyses of tumour molecular signaturesand the growth of personalised medicine will result in selected RBPs becoming usefultherapeutic targets in cancer therapy.As Guest Editors for this IJMS Special Issue, we are pleased to present the collection ofriveting articles focused on the topic of RBPs in cancer. This collection of articles developsthe idea that RBPs have significant effects on genomic stability and cellular homeostasis,which have an impact on cancer at many levels and through various pathways. We thankthe authors for their excellent contributions that add fresh perspectives to this fascinatingand vitally important field of genome biology.Int. J. Mol. Sci. 2023, 24, 9612 4 of 4Acknowledgments: We thank all of the authors who have submitted their articles as well as all of thereviewers who have evaluated the submissions and provided helpful feedback for this Special Issue.Conflicts of Interest: The authors declare no conflict of interest.References1. Nasiri-Aghdam, M.; Garcia-Garduño, T.; Jave-Suárez, L. CELF Family Proteins in Cancer: Highlights on the RNA-BindingProtein/Noncoding RNA Regulatory Axis. Int. J. Mol. Sci. 2021, 22, 11056. [CrossRef] [PubMed]2. Cai, J.; Wang, N.; Lin, G.; Zhang, H.; Xie, W.; Zhang, Y.; Xu, N. MBNL2 Regulates DNA Damage Response via Stabilizing P21. Int.J. Mol. Sci. 2021, 22, 783. [CrossRef] [PubMed]3. Sidali, A.; Teotia, V.; Solaiman, N.S.; Bashir, N.; Kanagaraj, R.; Murphy, J.J.; Surendranath, K. AU-Rich Element RNA BindingProteins: At the Crossroads of Post-Transcriptional Regulation and Genome Integrity. Int. J. Mol. Sci. 2021, 23, 96. [CrossRef][PubMed]4. Löblein, M.T.; Falke, I.; Eich, H.T.; Greve, B.; Götte, M.; Troschel, F.M. Dual Knockdown of Musashi RNA-Binding Proteins MSI-1and MSI-2 Attenuates Putative Cancer Stem Cell Characteristics and Therapy Resistance in Ovarian Cancer Cells. Int. J. Mol. Sci.2021, 22, 11502. [CrossRef] [PubMed]5. Dolicka, D.; Foti, M.; Sobolewski, C. The Emerging Role of Stress Granules in Hepatocellular Carcinoma. Int. J. Mol. Sci. 2021,22, 9428. [CrossRef] [PubMed]6. Uruci, S.; Lo, C.S.Y.; Wheeler, D.; Taneja, N. R-Loops and Its Chro-Mates: The Strange Case of Dr. Jekyll and Mr. Hyde. Int. J. Mol.Sci. 2021, 22, 8850. [CrossRef] [PubMed]7. Nascakova, Z.; Boleslavska, B.; Urban, V.; Oravetzova, A.; Vlachova, E.; Janscak, P.; Dobrovolna, J. RAD51 Inhibition InducesR-Loop Formation in Early G1 Phase of the Cell Cycle. Int. J. Mol. Sci. 2021, 22, 3740. [CrossRef] [PubMed]Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individualauthor(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.

RNA-Binding Proteins and Their Emerging Roles in Cancer: Beyond the Tip of the Iceberg.

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RNA-Binding Proteins and Their Emerging Roles in Cancer: Beyond the Tip of the Iceberg

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