Coding, or non-coding, that is the question.

The advent of high-throughput sequencing uncovered that our genome is pervasively transcribed into RNAs that are seemingly not translated into proteins. It was also found that non-coding RNA transcripts outnumber canonical protein-coding genes. This mindboggling discovery prompted a surge in non-coding RNA research that started unraveling the functional relevance of these new genetic units, shaking the classic definition of "gene". While the non-coding RNA revolution was still taking place, polysome/ribosome profiling and mass spectrometry analyses revealed that peptides can be translated from non-canonical open reading frames. Therefore, it is becoming evident that the coding vs non-coding dichotomy is way blurrier than anticipated. In this review, we focus on several examples in which the binary classification of coding vs non-coding genes is outdated, since the same bifunctional gene expresses both coding and non-coding products. We discuss the implications of this intricate usage of transcripts in terms of molecular mechanisms of gene expression and biological outputs, which are often concordant, but can also surprisingly be discordant. Finally, we discuss the methodological caveats that are associated with the study of bifunctional genes, and we highlight the opportunities and challenges of therapeutic exploitation of this intricacy towards the development of anticancer therapies.

Strategies for single base gene editing in an immortalized human cell line by CRISPR/Cas9 technology.

The use of CRISPR/Cas9 system has rapidly grown in the last years. Here, the optimization of gene editing of a single-nucleotide polymorphism in a human non-malignant somatic cell line of thyrocytes (Nthy-Ori) was described highlighting strategies for overcoming the problems concerning the delivery and off-targets. We employed both lentivirus and chemical lipids as delivery agents and two strategies for creating the double-strand breaks (DSB). The former induced a DSB by a classical Cas9 nuclease (standard strategy), while the second one employed a modified Cas9 creating a single-strand break (SSB). The knock-in was carried out using a single-stranded donor oligonucleotide or the HR410-PA donor vector (HR). The desired cells could be obtained by combining the double nickase system with the HR vector transfected chemically. This result could be due to the type of DSB, likely processed mainly by non-homologous end joining when blunt (standard strategy) and by HR when overhanging (double nickase). Our results showed that the double nickase is suitable for knocking-in the immortalized Nthy-Ori cell line, while the standard CRISPR/Cas9 system is suitable for gene knock-out creating in/del mutations. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03878-4.

Differential impact of BRAFV600E isoforms on tumorigenesis in a zebrafish model of melanoma.

BRAFV600E comes as two main splicing variants. The well-studied ref isoform and the recently discovered X1 isoform are co-expressed in cancer cells and differ in terms of 3'UTR length and sequence, as well as C-term protein sequence. Here, we use a melanoma model in zebrafish to study the role played by each isoform in larval pigmentation, nevi formation, and their progression into melanoma tumours. We show that both BRAFV600E-ref and BRAFV600E-X1 proteins promote larval pigmentation and nevi formation, while melanoma-free survival curves performed in adult fish indicate that BRAFV600E-ref protein is a much stronger melanoma driver that BRAFV600E-X1 protein. Crucially, we also show that the presence of the 3'UTR suppresses the effect of ref protein. Our data highlight the necessity to undertake a systematic study of BRAFV600E isoforms, in order to uncover the full spectrum of their kinase-(in)dependent and coding-(in)dependent functions, hence to develop more informed strategies for therapeutic targeting.

A cyanine-based NIR fluorescent Vemurafenib analog to probe BRAFV600E in cancer cells.

BRAF represents one of the most frequently mutated protein kinase genes and BRAFV600E mutation may be found in many types of cancer, including hairy cell leukemia (HCL), anaplastic thyroid cancer (ATC), colorectal cancer and melanoma. Herein, a fluorescent probe, based on the structure of the highly specific BRAFV600E inhibitor Vemurafenib (Vem, 1) and featuring the NIR fluorophore cyanine-5 (Cy5), was straightforwardly synthesized and characterized (Vem-L-Cy5, 3), showing promising spectroscopic properties. Biological validation in BRAFV600E-mutated cancer cells evidenced the ability of 3 to penetrate inside the cells, specifically binding to its elective target BRAFV600E with high affinity, and inhibiting MEK phosphorylation and cell growth with a potency comparable to that of native Vem 1. Taken together, these data highlight Vem-L-Cy5 3 as a useful tool to probe BRAFV600E mutation in cancer cells, and suitable to acquire precious insights for future developments of more informed BRAF inhibitors-centered therapeutic strategies.

PARP1 negatively regulates MAPK signaling by impairing BRAF-X1 translation.

In human cells BRAF oncogene is invariably expressed as a mix of two coding transcripts: BRAF-ref and BRAF-X1. These two mRNA isoforms, remarkably different in the sequence and length of their 3'UTRs, are potentially involved in distinct post-transcriptional regulatory circuits. Herein, we identify PARP1 among the mRNA Binding Proteins that specifically target the X1 3'UTR in melanoma cells. Mechanistically, PARP1 Zinc Finger domain down-regulates BRAF expression at the translational level. As a consequence, it exerts a negative impact on MAPK pathway, and sensitizes melanoma cells to BRAF and MEK inhibitors, both in vitro and in vivo. In summary, our study unveils PARP1 as a negative regulator of the highly oncogenic MAPK pathway in melanoma, through the modulation of BRAF-X1 expression.

A Network of MicroRNAs and mRNAs Involved in Melanosome Maturation and Trafficking Defines the Lower Response of Pigmentable Melanoma Cells to Targeted Therapy.

BACKGROUND: The ability to increase their degree of pigmentation is an adaptive response that confers pigmentable melanoma cells higher resistance to BRAF inhibitors (BRAFi) compared to non-pigmentable melanoma cells. METHODS: Here, we compared the miRNome and the transcriptome profile of pigmentable 501Mel and SK-Mel-5 melanoma cells vs. non-pigmentable A375 melanoma cells, following treatment with the BRAFi vemurafenib (vem). In depth bioinformatic analyses (clusterProfiler, WGCNA and SWIMmeR) allowed us to identify the miRNAs, mRNAs and biological processes (BPs) that specifically characterize the response of pigmentable melanoma cells to the drug. Such BPs were studied using appropriate assays in vitro and in vivo (xenograft in zebrafish embryos). RESULTS: Upon vem treatment, miR-192-5p, miR-211-5p, miR-374a-5p, miR-486-5p, miR-582-5p, miR-1260a and miR-7977, as well as GPR143, OCA2, RAB27A, RAB32 and TYRP1 mRNAs, are differentially expressed only in pigmentable cells. These miRNAs and mRNAs belong to BPs related to pigmentation, specifically melanosome maturation and trafficking. In fact, an increase in the number of intracellular melanosomes-due to increased maturation and/or trafficking-confers resistance to vem. CONCLUSION: We demonstrated that the ability of pigmentable cells to increase the number of intracellular melanosomes fully accounts for their higher resistance to vem compared to non-pigmentable cells. In addition, we identified a network of miRNAs and mRNAs that are involved in melanosome maturation and/or trafficking. Finally, we provide the rationale for testing BRAFi in combination with inhibitors of these biological processes, so that pigmentable melanoma cells can be turned into more sensitive non-pigmentable cells.

An Immunocompetent Environment Unravels the Proto-Oncogenic Role of miR-22.

MiR-22 was first identified as a proto-oncogenic microRNA (miRNA) due to its ability to post-transcriptionally suppress the expression of the potent PTEN (Phosphatase And Tensin Homolog) tumor suppressor gene. miR-22 tumorigenic role in cancer was subsequently supported by its ability to positively trigger lipogenesis, anabolic metabolism, and epithelial-mesenchymal transition (EMT) towards the metastatic spread. However, during the following years, the picture was complicated by the identification of targets that support a tumor-suppressive role in certain tissues or cell types. Indeed, many papers have been published where in vitro cellular assays and in vivo immunodeficient or immunosuppressed xenograft models are used. However, here we show that all the studies performed in vivo, in immunocompetent transgenic and knock-out animal models, unanimously support a proto-oncogenic role for miR-22. Since miR-22 is actively secreted from and readily exchanged between normal and tumoral cells, a functional immune dimension at play could well represent the divider that allows reconciling these contradictory findings. In addition to a critical review of this vast literature, here we provide further proof of the oncogenic role of miR-22 through the analysis of its genomic locus vis a vis the genetic landscape of human cancer.

p38 MAPK-dependent phosphorylation of transcription factor SOX2 promotes an adaptive response to BRAF inhibitors in melanoma cells.

Despite recent advances in the development of BRAF kinase inhibitors (BRAFi) for BRAF-mutant melanomas, development of resistance remains a major clinical problem. In addition to genetic alterations associated with intrinsic resistance, several adaptive response mechanisms are known to be rapidly activated to allow cell survival in response to treatment, limiting efficacy. A better understanding of the mechanisms driving resistance is urgently needed to improve the success of BRAF-targeted therapies and to make therapeutic intervention more durable. In this study, we identify the mitogen-activated protein kinase (MAPK) p38 as a novel mediator of the adaptive response of melanoma cells to BRAF-targeted therapy. Our findings demonstrate that BRAFi leads to an early increase in p38 activation, which promotes phosphorylation of the transcription factor SOX2 at Ser251, enhancing SOX2 stability, nuclear localization, and transcriptional activity. Furthermore, functional studies show that SOX2 depletion increases sensitivity of melanoma cells to BRAFi, whereas overexpression of a phosphomimetic SOX2-S251E mutant is sufficient to drive resistance and desensitize melanoma cells to BRAFi in vitro and in a zebrafish xenograft model. We also found that SOX2 phosphorylation at Ser251 confers resistance to BRAFi by binding to the promoter and increasing transcriptional activation of the ATP-binding cassette drug efflux transporter ABCG2. In summary, we unveil a p38/SOX2-mediated mechanism of adaptive response to BRAFi, which provides prosurvival signals to melanoma cells against the cytotoxic effects of BRAFi prior to acquiring resistance.

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma.

Tyr::CreER+,BrafCA/+,Ptenlox/lox genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume. The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 µm). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 µm step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement. This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-9.

We are delighted to share with you our ninth Journal Club and highlight some of the most interesting papers published recently [...].

CRISPR/Cas Technologies Applied to Pseudogenes.

Pseudogenes have been considered as nonfunctional copies of their parental genes for a long time. Indeed, they have been often defined "junk DNA" or "transcriptional noise." However, with the identification of their involvement in several biological processes, the necessity of their study is inevitably growing up. The manipulation of pseudogene expression is complicated by their high homology with parental genes and by the fact that most of them work at the transcriptional level as noncoding RNAs. With the advent of CRISPR/Cas technology, these problems can be overcome. Particularly, as we describe in this chapter, it is possible: To perform genome editing, obtaining the complete elimination of the pseudogene genomic sequence (knock-out), preventing pseudogene transcription, introducing specific mutations in the pseudogene sequence, or introducing a specific sequence (knock-in). To positively or negatively manipulate pseudogene transcription. To target pseudogene RNA and negatively regulate its expression. To edit pseudogene DNA and RNA and alter a specific sequence. Moreover, CRISPR/Cas technology can be used as an RNA Binding Protein system for molecular biology techniques (such as RNA immunoprecipitation and pull-down), as well as for transcript tracking and live imaging.

In Vivo Silencing/Overexpression of lncRNAs by CRISPR/Cas System.

Long noncoding RNAs (lncRNAs) are implicated in several biological processes and it has been observed that their expression is altered in several diseases. The generation of animal models where selective silencing or overexpression of lncRNAs can be attained is crucial for their biological characterization, since it offers the opportunity to analyze their function at the tissue specific or organismal level. CRISPR/Cas technology is a newly developed tool that allows to easily manipulate the mouse genome, in turn allowing to discover lncRNAs functions in an in vivo context. Here, we provide an overview of how CRISPR/Cas technology can be used to generate transgenic mouse models in which lncRNAs can be studied.

A eutherian-specific microRNA controls the translation of Satb2 in a model of cortical differentiation.

Cerebral cortical development is controlled by key transcription factors that specify the neuronal identities in the different layers. The mechanisms controlling their expression in distinct cells are only partially known. We investigated the expression and stability of Tbr1, Bcl11b, Fezf2, Satb2, and Cux1 mRNAs in single developing mouse cortical cells. We observe that Satb2 mRNA appears much earlier than its protein and in a set of cells broader than expected, suggesting an initial inhibition of its translation, subsequently released during development. Mechanistically, Satb2 3'UTR modulates protein translation of GFP reporters during mouse corticogenesis. We select miR-541, a eutherian-specific miRNA, and miR-92a/b as the best candidates responsible for SATB2 inhibition, being strongly expressed in early and reduced in late progenitor cells. Their inactivation triggers robust and premature SATB2 translation in both mouse and human cortical cells. Our findings indicate RNA interference as a major mechanism in timing cortical cell identities.

High-Throughput Identification of miRNA-Target Interactions in Melanoma Using miR-CATCHv2.0.

MicroRNAs (miRNAs) can regulate the expression of potentially every transcript in the cell, and the definition of miRNA-target interactions is crucial to understand their role in all biological processes. However, the identification of the miRNAs that target a specific mRNA remains a challenge. Here, we describe an innovative method called miR-CATCHv2.0 for the high-throughput identification of the miRNA species bound to an RNA of interest. We also describe how this method can overcome the limitations of the current computational and experimental methods available in this field.

Pro64His (rs4644) Polymorphism Within Galectin-3 Is a Risk Factor of Differentiated Thyroid Carcinoma and Affects the Transcriptome of Thyrocytes Engineered via CRISPR/Cas9 System.

Background: Galectin-3 (LGALS3) is an important glycoprotein involved in the malignant transformation of thyrocytes acting in the extracellular matrix, cytoplasm, and nucleus where it regulates TTF-1 and TCF4 transcription factors. Within LGALS3 gene, a common single-nucleotide polymorphism (SNP) (c.191C>A, p.Pro64His; rs4644) encoding for the variant Proline to Histidine at codon 64 has been extensively studied. However, data on rs4644 in the context of thyroid cancer are lacking. Thus, the aim of the present work was to evaluate the role of the rs4644 SNP as risk factor for differentiated thyroid cancer (DTC) and to determine the effect on the transcriptome in thyrocytes. Methods: A case/control association study in 1223 controls and 1142 unrelated consecutive DTC patients was carried out to evaluate the association between rs4644-P64H and the risk of DTC. We used the nonmalignant cell line Nthy-Ori (rs4644-C/A) and the CRISPR/Cas9 technique to generate isogenic cells carrying either the rs4644-A/A or rs4644-C/C homozygosis. Then, the transcriptome of the derivative and unmodified parental cells was analyzed by RNA-seq. Genes differentially expressed were validated by quantitative reverse transcription PCR and further tested in the parental Nthy-Ori cells after LGALS3 gene silencing, to investigate whether the expression of target genes was dependent on galectin-3 levels. Results: rs4644 AA genotype was associated with a reduced risk of DTC (compared with CC, ORadj = 0.66; 95% confidence interval = 0.46-0.93; Pass = 0.02). We found that rs4644 affects galectin-3 as a transcriptional coregulator. Among 34 genes affected by rs4644, HES1, HSPA6, SPC24, and NHS were of particular interest since their expression was rs4644-dependent (CC>AA for the first and AA>CC for the others), also in 574 thyroid tissues of Genotype-Tissue Expression (GTEx) biobank. Moreover, the expression of these genes was regulated by LGALS3-silencing. Using the proximity ligation assay in Nthy-Ori cells, we found that the TTF-1 interaction was genotype dependent. Conclusions: Our data show that in thyroid, rs4644 is a trans-expression quantitative trait locus that can modify the transcriptional expression of downstream genes, through the modulation of TTF-1.

Inducible modulation of miR-204 levels in a zebrafish melanoma model.

Here, we present miniCoopR-I, an inducible upgrade of the constitutive miniCoopR vector. We developed miniCoopR-I-sponge-204 and miniCoopR-I-pre-miR-204 vectors and we successfully tested them for their ability to achieve time- (embryo/juvenile/adult) and space- (melanocytic lineage) restricted inhibition/overexpression of miR-204, a positive modulator of pigmentation previously discovered by us. Furthermore, melanoma-free survival curves performed on induced fish at the adult stage indicate that miR-204 overexpression accelerates the development of BRAFV600E-driven melanoma. miniCoopR-I allows study of the impact that coding and non-coding modulators of pigmentation exert on melanomagenesis in adult zebrafish, uncoupling it from the impact that they exert on melanogenesis during embryonic development.This article has an associated First Person interview with the first author of the paper.

Proteomics pipeline for phosphoenrichment and its application on a human melanoma cell model.

Cell signalling is tightly regulated by post-translational modification of proteins. Among them, phosphorylation is one of the most interesting and important. Identifying phosphorylation sites on proteins is challenging and requires strategies for pre-separation and enrichment of the phosphorylated species. We applied four different methods for phospho-enrichment involving TiO2 and IMAC matrix to human melanoma cell lysates of starved A375 induced for 1 h with 1% FBS. Comparison of protocol efficiency was evaluated through peptide concentration, sulphur and phosphorus content and peptide analysis by LC-MS in the collected fractions. Our results underlined that each single method is not sufficient for a comprehensive phosphoproteome analysis. In fact, each methodology permits to identify only a fraction of the phosphoproteome contained in a whole cell lysate. The selection of the most efficient protocols and a combination of two phospho-enrichment methods allowed the assessment of this workflow able to pinpoint the main actors in the phospho-proteome cascade of A375 human melanoma cells treated with Vemurafenib.

PTENP1 is a ceRNA for PTEN: it's CRISPR clear.

Here we apply state-of-the-art CRISPR technologies to study the impact that PTENP1 pseudogene transcript has on the expression levels of its parental gene PTEN, and hence on the output of AKT signaling in cancer. Our data expand the repertoire of approaches that can be used to dissect competing endogenous RNA (ceRNA)-based interactions, while providing further experimental evidence in support of the very first one that we discovered.