Cooperativity and Interdependency between RNA Structure and RNA–RNA Interactions

Complex RNA–RNA interactions are increasingly known to play key roles in numerous biological processes from gene expression control to ribonucleoprotein granule formation. By contrast, the nature of these interactions and characteristics of their interfaces, especially those that involve partially or wholly structured RNAs, remain elusive. Herein, we discuss different modalities of RNA–RNA interactions with an emphasis on those that depend on secondary, tertiary, or quaternary structure. We dissect recently structurally elucidated RNA–RNA complexes including RNA triplexes, riboswitches, ribozymes, and reverse transcription complexes. These analyses highlight a reciprocal relationship that intimately links RNA structure formation with RNA–RNA interactions. The interactions not only shape and sculpt RNA structures but also are enabled and modulated by the structures they create. Understanding this two-way relationship between RNA structure and interactions provides mechanistic insights into the expanding repertoire of noncoding RNA functions, and may inform the design of novel therapeutics that target RNA structures or interactions

Investigation of RNA-protein interactions and their role in protein synthesis regulation and differentiation

Accurate regulation of gene expression is controlled in many levels including mRNA’s lifecycle. mRNA stability depends mainly on their poly(A) tail length, which in turn is determined through 3'-5' exoribonucleases, known as deadenylases. Deadenylases belong to either the DEDD or the exonuclease endonuclease phosphatase (EEP) superfamily, depending on their active site residues. PNLDC1 is the most recent member of the DEDD superfamily and was reported as the trimmer of pre-piRNAs, a process which takes place in the mitochondrial surface of germ cells. hPNLDC1 is located on chromosome 6 and its expression leads to the production of two different protein isoforms through alternative splicing. On the other hand, the respective gene in M. musculus is located on chromosome 17 encoding a single isoform. We have previously shown that PNLDC1 constitutes a 3'-5' exoribonuclease acting specifically on polyadenylated substrates and was found to be highly expressed in mouse embryonic stem cells E14 and testes tissues. It has also been observed that during development Pnldc1 expression levels show a gradual decrease and in differentiated cells its expression levels were detectable only after usage of demethylating agents, such as 5'-AZA-Deoxycitidine. In order to further examine the methylation pattern of Pnldc1 we performed in silico analysis of its promoter region, which in turn revealed the presence of CpG islands. Pyrosequencing analysis revealed an increased methylation of each examined position of the promoter region during differentiation of mESCs. In a next step, after silencing of PNLDC1, NGS analysis showed that PNLDC1 regulates genes with significant role in reprogramming, cell cycle and transcriptional and translational regulation. To further investigate the absence of PNLDC1 in its physiological content, we constructed stable knockout PNLDC1 mESCs cell lines employing the CRISPR/Cas9 genome editing system. Next generation sequencing was also performed in knockout mESCs and the results are in consistency with our previous data after Pnldc1 knockdown. Further verification both in cellular and molecular level, was performed using FACS and RT-qPCR analyses which showed that differential expression of PNLDC1 affects the proliferation rate and cell cycle progression in stem cells and somatic cells. More specifically, Pnldc1’s knockdown in mESCs lead to inhibition of proliferation rate causing a cell cycle arrest during the transition from the S to G2/M phase. To the contrary, ectopic expression of PNLDC1 in HEK293T leads to increased cell proliferation. This phenotype is consistent with deregulation of expression levels of important genes and non-coding RNAs related to these processes such as Malat1, Rian, Gas5 and Neat1. To validate our observation about deregulation of the protein synthesis, polysome profiling was performed. Indeed, polysomes were more abundant in WT compared to CRISPR knockout mESCs indicating a lower translational rate after PNLDC1 deficiency. Finally, using the available structure of an hPARN isoform, molecular dynamics simulation of the active site of PNLDC1 was performed followed by a high-performance virtual screening which allowed the selection of chemical compounds that could potentially act as regulatory molecules against DEDD deadenylases. The compounds were ranked according to the calculated free energy of binding and 12 were selected for further validation. Among them 5 compounds seem to be effective. More specifically, 3 of them could affect the action of both PARN and PNLDC1, one appears specifically inhibits PNLDC1 and one increases the activity of PARN. These compounds were also tested for their effect on cell viability and physiology and subsequently next generation sequencing was performed upon treatment with two compounds indicating that could affect the expression levels of genes playing role in gene expression, signal transduction and cellular response to stress. Collectively, our results suggest that PNLDC1 an important regulator of cell proliferation, transcription and translation during early differentiation and also present chemical compounds which could be used as functional modulators of PNLDC1’s activity and its downstream effects.Η ακριβής και συντονισμένη ρύθμιση της γονιδιακής έκφρασης ελέγχεται σε πολλά επίπεδα, συμπεριλαμβανομένου του κύκλου ζωής των μορίων mRNA. Η σταθερότητα των μορίων mRNA εξαρτάται σε μεγάλο βαθμό από το μήκος της poly(A) ουράς τους, το οποίο με τη σειρά του ρυθμίζεται, μεταξύ άλλων από τη δράση 3’-5’ εξωριβονουκλεασών, γνωστές ως αποαδενυλάσες. Οι αποαδενυλάσες διακρίνονται σε δύο υπεροικογένειες, τις DEDD και EEP, με βάση τα κατάλοιπα στο ενεργό τους κέντρο. Η PNLDC1 αποτελεί το πιο πρόσφατο μέλος των DEDD αποαδενυλασών και έχει αναφερθεί ως η νουκλεάση που συμμετέχει στην επεξεργασία του 3’ άκρου των πρόδρομων μορίων piRNA σε γαμετικά κύτταρα. Το γονίδιο PNLDC1 στον άνθρωπο εδράζεται στο χρωμόσωμα 6 και η έκφρασή του οδηγεί στην παραγωγή δύο διαφορετικών πρωτεϊνικών ισομορφών μέσω εναλλακτικού ματίσματος. Αντίθετα, το συγκεκριμένο γονίδιο στο ποντίκι εδράζεται στο χρωμόσωμα 17 και κωδικοποιεί μία μόνο ισομορφή. Προηγούμενες μελέτες του εργαστηρίου μας έδειξαν ότι η PNLDC1 παρουσιάζει απόλυτη εξειδίκευση ως προς τα πολυαδενυλιωμένα υποστρώματα και εκφράζεται κυρίως σε γαμετικά και εμβρυϊκά βλαστικά κύτταρα. Τα επίπεδα έκφρασης του γονιδίου της PNLDC1 μειώνονται κατά τη διαφοροποίηση και την ανάπτυξη των κυττάρων και σε διαφοροποιημένα κύτταρα, η έκφρασή του παρατηρείται μόνο έπειτα από την επίδραση απομεθυλιωτικών παραγόντων όπως είναι το 5’-AZA-Deoxycitidine. Προκειμένου να διερευνηθεί περαιτέρω ο μηχανισμός ρύθμισης του γονιδίου της PNLDC1 στο μεταγραφικό επίπεδο πραγματοποιήθηκε in silico ανάλυση της περιοχής του προαγωγέα η οποία προέβλεψε την ύπαρξη CpG νησίδων, θέσεις οι οποίες είναι πιθανό να υπόκεινται σε μεθυλίωση. Ανάλυση pyrosequencing κατά τη διαφοροποίηση mESCs επιβεβαίωσε την αύξηση των επιπέδων μεθυλίωσης του προαγωγέα φανερώνοντας τις ακριβείς θέσεις καθώς και το ποσοστό μεθυλίωσης σε κάθε μία από αυτές ξεχωριστά. Πειράματα αποσιώπησης της έκφρασης του γονιδίου της PNLDC1 σε mESCs και αλληλούχηση νέας γενιάς στο μεταγράφωμα αυτών έδειξαν πως η PNLDC1 συμμετέχει στη ρύθμιση της έκφρασης γονιδίων που σχετίζονται τον επαναπρογραμματισμό, την εξέλιξη του κυτταρικού τους κύκλου και τη ρύθμιση της μεταγραφής και της μετάφρασής τους. Έπειτα, προκειμένου να διερευνηθεί η απουσία της PNLDC1 στις φυσιολογικές συνθήκες όπου αυτή εκφράζεται, κατασκευάστηκε σταθερή κυτταρική σειρά mESCs στην οποία πραγματοποιήθηκε απαλοιφή του γονιδίου Pnldc1 χρησιμοποιώντας το σύστημα επεξεργασίας γονιδιώματος CRISPR/Cas9. Πειράματα αλληλούχησης νέας γενιάς πραγματοποιήθηκαν εκ νέου στα κύτταρα αυτά και τα αποτελέσματα συνάδουν με αυτά έπειτα από την αποσιώπηση του συγκεκριμένου γονιδίου. Περαιτέρω μελέτη σε κυτταρικό και μοριακό επίπεδο με τη χρήση κυτταρομετρίας ροής και RT-qPCR ανάλυσης έδειξε πως η μείωση των επιπέδων έκφρασης της PNLDC1 στα mESCs οδηγεί στην αναστολή του ρυθμού πολλαπλασιασμού προκαλώντας τη διακοπή του κυτταρικού κύκλου κατά τη μετάβαση από τη φάση S στην G2/M. Το γεγονός αυτό οφείλεται στην απορρύθμιση τόσο σημαντικών γονιδίων όσο και μη κωδικών μορίων RNA όπως τα Malat1, Rian, Gas5 και Neat1 τα οποία εμπλέκονται στις παραπάνω κυτταρικές λειτουργίες. Επιπρόσθετα, ανάλυση του πολυσωμικού προφίλ έδειξε πως στα κύτταρα αγρίου τύπου απαντάται μεγαλύτερος αριθμός πολυσωμάτων σε σύγκριση με αυτά που στερούνται την PNLDC1 γεγονός που φανερώνει το χαμηλότερο ρυθμό μετάφρασης των τελευταίων. Παράλληλα, πραγματοποιήθηκε έκτοπη έκφραση της PNLDC1 σε κύτταρα HEK293T η οποία οδήγησε σε αύξηση του πολλαπλασιασμού των κυττάρων επαληθεύοντας τα παραπάνω αποτελέσματα. Τέλος, χρησιμοποιώντας τη διαθέσιμη δομή της hPARN πραγματοποιήθηκε προσομοίωση μοριακής δυναμικής του ενεργού κέντρου της PNLDC1 ακολουθούμενη από υψηλής απόδοσης εικονική αξιολόγηση η οποία επέτρεψε την επιλογή χημικών ενώσεων που εν δυνάμει μπορούν να λειτουργήσουν ως μόρια ρυθμιστές έναντι της δράσης των DEDD αποαδενυλασών. Οι ενώσεις κατατάχθηκαν με βάση την ελεύθερη ενέργεια πρόσδεσης και 12 από αυτές επιλέχθηκαν για περαιτέρω μελέτη εκ των οποίων 5 εμφάνισαν in vitro δραστικότητα. Συγκεκριμένα, 3 από αυτές αναστέλλουν τη δράση και των δύο αποαδενυλασών, μία φαίνεται να αναστέλλει αποκλειστικά την PNLDC1, ενώ μία δρα ως ενεργοποιητής της PARN. Οι ενώσεις αυτές εξετάστηκαν περαιτέρω ως προς την επίδρασή τους στη βιωσιμότητα και τη φυσιολογία των κυττάρων και επακόλουθη αλληλούχηση νέας γενιάς έδειξε πως δύο από αυτές επηρεάζουν γονίδια τα οποία εμπλέκονται σε βασικές βιολογικές λειτουργίες όπως είναι η ρύθμιση της γονιδιακής έκφρασης, η μεταγωγή σήματος και η απόκριση στο στρες. Συλλογικά, τα αποτελέσματα της παρούσας διατριβής αναδεικνύουν τη PNLDC1 ως σημαντικό ρυθμιστή της μεταγραφής, της μετάφρασης και του πολλαπλασιασμού των κυττάρων κατά πρώιμα στάδια διαφοροποίησης ενώ παράλληλα αποκαλύπτουν την ύπαρξη χημικών ενώσεων οι οποίες μπορούν να αξιοποιηθούν ως ενεργοί ρυθμιστές της δράσης του ενζύμου αυτού

The Dynamic Network of RNP RNase P Subunits

Ribonuclease P (RNase P) is an important ribonucleoprotein (RNP), responsible for the maturation of the 5′ end of precursor tRNAs (pre-tRNAs). In all organisms, the cleavage activity of a single phosphodiester bond adjacent to the first nucleotide of the acceptor stem is indispensable for cell viability and lies within an essential catalytic RNA subunit. Although RNase P is a ribozyme, its kinetic efficiency in vivo, as well as its structural variability and complexity throughout evolution, requires the presence of one protein subunit in bacteria to several protein partners in archaea and eukaryotes. Moreover, the existence of protein-only RNase P (PRORP) enzymes in several organisms and organelles suggests a more complex evolutionary timeline than previously thought. Recent detailed structures of bacterial, archaeal, human and mitochondrial RNase P complexes suggest that, although apparently dissimilar enzymes, they all recognize pre-tRNAs through conserved interactions. Interestingly, individual protein subunits of the human nuclear and mitochondrial holoenzymes have additional functions and contribute to a dynamic network of elaborate interactions and cellular processes. Herein, we summarize the role of each RNase P subunit with a focus on the human nuclear RNP and its putative role in flawless gene expression in light of recent structural studies

KRASG12C Can Either Promote or Impair Cap-Dependent Translation in Two Different Lung Adenocarcinoma Cell Lines

KRASG12C is among the most common oncogenic mutations in lung adenocarcinoma and a promising target for treatment by small-molecule inhibitors. KRAS oncogenic signaling is responsible for modulation of tumor microenvironment, with translation factors being among the most prominent deregulated targets. In the present study, we used TALENs to edit EGFRWT CL1-5 and A549 cells for integration of a Tet-inducible KRASG12C expression system. Subsequent analysis of both cell lines showed that cap-dependent translation was impaired in CL1-5 cells via involvement of mTORC2 and NF-κB. In contrast, in A549 cells, which additionally harbor the KRASG12S mutation, cap-dependent translation was favored via recruitment of mTORC1, c-MYC and the positive regulation of eIF4F complex. Downregulation of eIF1, eIF5 and eIF5B in the same cell line suggested a stringency loss of start codon selection during scanning of mRNAs. Puromycin staining and polysome profile analysis validated the enhanced translation rates in A549 cells and the impaired cap-dependent translation in CL1-5 cells. Interestingly, elevated translation rates were restored in CL1-5 cells after prolonged induction of KRASG12C through an mTORC1/p70S6K-independent way. Collectively, our results suggest that KRASG12C signaling differentially affects the regulation of the translational machinery. These differences could provide additional insights and facilitate current efforts to effectively target KRAS

KRAS^G12C Can Either Promote or Impair Cap-Dependent Translation in Two Different Lung Adenocarcinoma Cell Lines

KRASG12C is among the most common oncogenic mutations in lung adenocarcinoma and a promising target for treatment by small-molecule inhibitors. KRAS oncogenic signaling is responsible for modulation of tumor microenvironment, with translation factors being among the most prominent deregulated targets. In the present study, we used TALENs to edit EGFRWT CL1-5 and A549 cells for integration of a Tet-inducible KRASG12C expression system. Subsequent analysis of both cell lines showed that cap-dependent translation was impaired in CL1-5 cells via involvement of mTORC2 and NF-κB. In contrast, in A549 cells, which additionally harbor the KRASG12S mutation, cap-dependent translation was favored via recruitment of mTORC1, c-MYC and the positive regulation of eIF4F complex. Downregulation of eIF1, eIF5 and eIF5B in the same cell line suggested a stringency loss of start codon selection during scanning of mRNAs. Puromycin staining and polysome profile analysis validated the enhanced translation rates in A549 cells and the impaired cap-dependent translation in CL1-5 cells. Interestingly, elevated translation rates were restored in CL1-5 cells after prolonged induction of KRASG12C through an mTORC1/p70S6K-independent way. Collectively, our results suggest that KRASG12C signaling differentially affects the regulation of the translational machinery. These differences could provide additional insights and facilitate current efforts to effectively target KRAS

Revisiting miRNA Association with Melanoma Recurrence and Metastasis from a Machine Learning Point of View

The diagnostic and prognostic value of miRNAs in cutaneous melanoma (CM) has been broadly studied and supported by advanced bioinformatics tools. From early studies using miRNA arrays with several limitations, to the recent NGS-derived miRNA expression profiles, an accurate diagnostic panel of a comprehensive pre-specified set of miRNAs that could aid timely identification of specific cancer stages is still elusive, mainly because of the heterogeneity of the approaches and the samples. Herein, we summarize the existing studies that report several miRNAs as important diagnostic and prognostic biomarkers in CM. Using publicly available NGS data, we analyzed the correlation of specific miRNA expression profiles with the expression signatures of known gene targets. Combining network analytics with machine learning, we developed specific non-linear classification models that could successfully predict CM recurrence and metastasis, based on two newly identified miRNA signatures. Subsequent unbiased analyses and independent test sets (i.e., a dataset not used for training, as a validation cohort) using our prediction models resulted in 73.85% and 82.09% accuracy in predicting CM recurrence and metastasis, respectively. Overall, our approach combines detailed analysis of miRNA profiles with heuristic optimization and machine learning, which facilitates dimensionality reduction and optimization of the prediction models. Our approach provides an improved prediction strategy that could serve as an auxiliary tool towards precision treatment

Contribution of miRNAs, tRNAs and tRFs to Aberrant Signaling and Translation Deregulation in Lung Cancer

Transcriptomics profiles of miRNAs, tRNAs or tRFs are used as biomarkers, after separate examination of several cancer cell lines, blood samples or biopsies. However, the possible contribution of all three profiles on oncogenic signaling and translation as a net regulatory effect, is under investigation. The present analysis of miRNAs and tRFs from lung cancer biopsies indicated putative targets, which belong to gene networks involved in cell proliferation, transcription and translation regulation. In addition, we observed differential expression of specific tRNAs along with several tRNA-related genes with possible involvement in carcinogenesis. Transfection of lung adenocarcinoma cells with two identified tRFs and subsequent NGS analysis indicated gene targets that mediate signaling and translation regulation. Broader analysis of all major signaling and translation factors in several biopsy specimens revealed a crosstalk between the PI3K/AKT and MAPK pathways and downstream activation of eIF4E and eEF2. Subsequent polysome profile analysis and 48S pre-initiation reconstitution experiments showed increased global translation rates and indicated that aberrant expression patterns of translation initiation factors could contribute to elevated protein synthesis. Overall, our results outline the modulatory effects that possibly correlate the expression of important regulatory non-coding RNAs with aberrant signaling and translation deregulation in lung cancer

Mammalian PNLDC1 is a novel poly(A) specific exonuclease with discrete expression during early development

PNLDC1 is a homologue of poly(A) specific ribonuclease (PARN), a known deadenylase with additional role in processing of non-coding RNAs. Both enzymes were reported recently to participate in piRNA biogenesis in silkworm and C. elegans, respectively. To get insights on the role of mammalian PNLDC1, we characterized the human and mouse enzymes. PNLDC1 shows limited conservation compared to PARN and represents an evolutionary related but distinct group of enzymes. It is expressed specifically in mouse embryonic stem cells, human and mouse testes and during early mouse embryo development, while it fades during differentiation. Its expression in differentiated cells, is suppressed through methylation of its promoter by the de novo methyltransferase DNMT3B. Both enzymes are localized mainly in the ER and exhibit in vitro specificity restricted solely to 3' RNA or DNA polyadenylates. Knockdown of Pnldc1 in mESCs and subsequent NGS analysis showed that although the expression of the remaining deadenylases remains unaffected, it affects genes involved mainly in reprogramming, cell cycle and translational regulation. Mammalian PNLDC1 is a novel deadenylase expressed specifically in cell types which share regulatory mechanisms required for multipotency maintenance. Moreover, it could be involved both in posttranscriptional regulation through deadenylation and genome surveillance during early development.</p