Structural basis of NEAT1 lncRNA maturation and menRNA instability.

NEAT1 long noncoding RNA orchestrates paraspeckle assembly and impacts tumorigenesis, fertility and immunity. Its maturation requires RNase P cleavage yielding an unstable transfer RNA-like multiple endocrine neoplasia-ß tRNA-like transcript (menRNA) due to CCACCA addition. Here we report the crystal structure of human menRNA, which partially mimics tRNAs to drive RNase P and ELAC2 processing. Biophysical analyses uncover an RNA-centric, riboswitch-like mechanism whereby the nascent CCA reshapes the RNA folding landscape and propels a spontaneous conformational isomerization that directs repeat CCA addition, marking the menRNA and defective tRNAs for degradation.

Structural basis of MALAT1 RNA maturation and mascRNA biogenesis.

The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) long noncoding RNA (lncRNA) has key roles in regulating transcription, splicing, tumorigenesis, etc. Its maturation and stabilization require precise processing by RNase P, which simultaneously initiates the biogenesis of a 3' cytoplasmic MALAT1-associated small cytoplasmic RNA (mascRNA). mascRNA was proposed to fold into a transfer RNA (tRNA)-like secondary structure but lacks eight conserved linking residues required by the canonical tRNA fold. Here we report crystal structures of human mascRNA before and after processing, which reveal an ultracompact, quasi-tRNA-like structure. Despite lacking all linker residues, mascRNA faithfully recreates the characteristic 'elbow' feature of tRNAs to recruit RNase P and ElaC homolog protein 2 (ELAC2) for processing, which exhibit distinct substrate specificities. Rotation and repositioning of the D-stem and anticodon regions preclude mascRNA from aminoacylation, avoiding interference with translation. Therefore, a class of metazoan lncRNA loci uses a previously unrecognized, unusually streamlined quasi-tRNA architecture to recruit select tRNA-processing enzymes while excluding others to drive bespoke RNA biogenesis, processing and maturation.

Noncoding RNA circuitry in melanoma onset, plasticity, and therapeutic response.

Melanoma, the cancer of the melanocyte, is the deadliest form of skin cancer with an aggressive nature, propensity to metastasize and tendency to resist therapeutic intervention. Studies have identified that the re-emergence of developmental pathways in melanoma contributes to melanoma onset, plasticity, and therapeutic response. Notably, it is well known that noncoding RNAs play a critical role in the development and stress response of tissues. In this review, we focus on the noncoding RNAs, including microRNAs, long non-coding RNAs, circular RNAs, and other small RNAs, for their functions in developmental mechanisms and plasticity, which drive onset, progression, therapeutic response and resistance in melanoma. Going forward, elucidation of noncoding RNA-mediated mechanisms may provide insights that accelerate development of novel melanoma therapies.

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.

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.

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.

Pharmacoepigenomics circuits induced by a novel retinoid-polyamine conjugate in human immortalized keratinocytes.

Retinoids are widely used in diseases spanning from dermatological lesions to cancer, but exhibit severe adverse effects. A novel all-trans-Retinoic Acid (atRA)-spermine conjugate (termed RASP) has shown previously optimal in vitro and in vivo anti-inflammatory and anticancer efficacy, with undetectable teratogenic and toxic side-effects. To get insights, we treated HaCaT cells which resemble human epidermis with IC50 concentration of RASP and analyzed their miRNA expression profile. Gene ontology analysis of their predicted targets indicated dynamic networks involved in cell proliferation, signal transduction and apoptosis. Furthermore, DNA microarrays analysis verified that RASP affects the expression of the same categories of genes. A protein-protein interaction map produced using the most significant common genes, revealed hub genes of nodal functions. We conclude that RASP is a synthetic retinoid derivative with improved properties, which possess the beneficial effects of retinoids without exhibiting side-effects and with potential beneficial effects against skin diseases including skin cancer.

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.

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.

Translation regulation in skin cancer from a tRNA point of view.

Protein synthesis is a central and dynamic process, frequently deregulated in cancer through aberrant activation or expression of translation initiation factors and tRNAs. The discovery of tRNA-derived fragments, a new class of abundant and, in some cases stress-induced, small Noncoding RNAs has perplexed the epigenomics landscape and highlights the emerging regulatory role of tRNAs in translation and beyond. Skin is the biggest organ in human body, which maintains homeostasis of its multilayers through regulatory networks that induce translational reprogramming, and modulate tRNA transcription, modification and fragmentation, in response to various stress signals, like UV irradiation. In this review, we summarize recent knowledge on the role of translation regulation and tRNA biology in the alarming prevalence of skin cancer.

Expanding the repertoire of deadenylases.

Deadenylases belong to an expanding family of exoribonucleases involved mainly in mRNA stability and turnover, with the exception of PARN which has additional roles in the biogenesis of several important non-coding RNAs, including miRNAs and piRNAs. Recently, PARN in C. elegans and its homolog PNLDC1 in B. mori were reported as the elusive trimmers mediating piRNA biogenesis. In addition, characterization of mammalian PNLDC1 in comparison to PARN, showed that is specifically expressed in embryonic stem and germ cells, as well as during early embryo development. Moreover, its expression is correlated with epigenetic events mediated by the de novo DNMT3b methyltransferase and knockdown in stem cells upregulates important genes that regulate multipotency. The recent data suggest that at least some new deadenylases may have expanded roles in cell metabolism as regulators of gene expression, through mRNA deadenylation, ncRNAs biogenesis and ncRNA-mediated mRNA targeting, linking essential mechanisms that regulate epigenetic control and transition events during differentiation. The possible roles of mammalian PNLDC1 along those dynamic networks are discussed in the light of new extremely important findings.

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.